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Welcome to the TERMIS-EU 2022,
Researchers and scientists will have an excellent opportunity to get together, network, learn and adopt innovative thinking in the exponentially growing field of tissue engineering and regenerative medicine. The TERMIS-EU 2022 relies on presenting cutting-edge science in cell biology, biomaterials, biofabrication, 3D tissues and organs development, numerical and experimental methods, clinical applications, and many more. The planned state-of-the-art plenary lectures will be complemented by invited keynotes and scientific theme-oriented sessions, also promoting the research of students and young investigators (SYIS). Scientific sessions for industrial applications and their translation in medicine will also be organized. To reflect the conference’s focus on timely and emerging hot topics, appropriate oral and poster presentations will be given.
This is the first time the TERMIS-EU conference will be held in Poland, in the second-largest and one of the oldest cities in Poland, Krakow. It is a historical and cultural center, a university city, and a hub of new technologies.
We are very enthusiastic about this Tissue Engineering and Regenerative Medicine International Society European Chapter Conference 2022. We rely on you to make it a successful event.
We look forward to meeting you in Krakow!
Prof. Wojciech Święszkowski
TERMIS-EU 2022 Conference Chair
Warsaw University of Technology
Prof. Zygmunt Pojda
TERMIS-EU 2022 Conference Co-Chair
Maria Sklodowska-Curie National Research Institute of Oncology
TBA
"Cancer is a complex disease system in which the extracellular microenvironment provides robust physicochemical cues to promote disease progression and resistance to therapy. Immunotherapies have been considered as an efficient therapeutic strategy to treat cancer and are being studied for their potential to improve prognosis and long-term survival of patients. While early clinical data shows the potential of immune therapy in treating liquid cancers, emerging evidence highlight the influence of microenvironmental factors on determining the efficacy of such therapeutic strategies to treat solid tumors. A detailed understanding of the interdependency between the microenvironment and cancer/immune cell interactions is needed to enable the efficient use of immunotherapy to treat solid cancers. Because of limitations inherent to existing model systems, development of advanced in vitro platforms including tumor microenvironment with immune cells are needed. In this talk, I will describe our efforts to create ex vivo platforms such as tumor-on-chip to study cancer cell-immune cell interactions within a tumor specific microenvironment. "
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Bone is a frequent homing site for breast cancer metastasis. Unfortunately, the majority of patients diagnosed with primary breast cancer is often affected by bone metastasis from where cancer can reach other vital organs such as the lungs. Thus, it is vital that the process of metastatic attraction and homing is modeled in vitro, harnessing tissue engineering technology, generating viable models that would be able to closely mimic the in vivo homing mechanism.
We engineered a three-dimensional breast-to-bone model using a novel microfluidic biofabrication approach by depositing human bone marrow stromal cells (hBMSCs) and a highly aggressive, invasive and poorly differentiated triple-negative breast cancer cell line (MDA-MB-231). Initially, the co-culture conditions have been optimised in order to preserve the viability of both cell types over 21-days of culture. Material inks were designed to grant the robust differentiation of HBMSCs and aid MDA-MB- 231 migration in 3D. A novel nanoclay-based material, comprising a variable ratio of alginate and gelatin, was used to encapsulate HBMSCs and investigate viability and functionality, while MDA-MB-231 were included in an RGD-based material to support cell migration and proliferation in 3D.
A metastasis-colonisation model was 3D printed and investigated for ultimate functionality. HBMSC- laden nanoclay-based ink was deposited in 3D following a lattice architecture with porosity augmented over a single planar direction, followed by the inclusion of MDA-MB-231-laden matrix on the most- top model surface, allowing the penetration and colonisation of the bone tissue.
In conclusion, we demonstrate the functionality of a 3D printed breast-to-bone model able to recapitulate the complex skeletal secondary site, illustrating the potential as a testing platform for novel therapeutic agents for metastatic process studies.
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Background and Aims: Cholangiocarcinoma (CCA) is an aggressive, heterogeneous cancer with low survival rates. Patient-derived cholangiocarcinoma organoids (CCAOs) hold potential for understanding disease progression and developing novel treatment options, based on their ability to mimic the original tumor. However, a hallmark of cancer is the disturbance of morphological cues resulting in typical dysplastic tumor architecture, an aspect not well recapitulated in CCAOs. Currently, CCAO expansion protocols focus on stimulation of the canonical WNT/β-catenin pathway, however there is growing evidence that non-canonical WNT pathways also play a crucial role in cancer progression. This project aims to establish a novel in vitro 3D model for CCA, better recapitulating the in vivo tumor, by stimulating both canonical and non-canonical WNT pathways.
Method: Branching cholangiocarcinoma organoids (BRCCAOs) (n = 3 patients) were established with a two-step protocol. First, CCAOs were initiated and cultured under standard conditions in canonical WNT stimulating expansion medium. Next, expansion medium was replaced by medium that stimulates canonical WNT-signaling (through R-spondin) and non-canonical WNT-signaling (with Dickkopf-related protein 1, DKK1) simultaneously, after which a branching-like morphology could be observed. Tumor cell behavior in BRCCAOs and CCAOs was assessed and compared through immunohistochemical stainings, bulk RNA-sequencing, and drug response studies.
Results: BRCCAOs presented a distinct branching morphology, with the formation of peripheral branches with a lumen, variable in diameter, surrounded by compact layers of cells. This morphology displayed an architecture similar to in vivo tumors, while maintaining tumorigenic potential and showing a lack of defined cellular polarity through staining of zonula occludens-1 (ZO-1). Bulk RNA-sequencing of BRCCAOs unveiled significant upregulation of cancer-associated molecular pathways, including hypoxia, compared to CCAOs and a close correlation (coefficient 0.80±0.05) to the transcriptome of the original tumor tissue. BRCCAOs also exhibited patient-specific responses to a large panel of 166 anti-cancer drugs, including a universal strong resistant phenotype to multiple drugs that have previously failed in clinical trials for CCA patients (i.e. docetaxel, palbociclib, and irinotecan). Specifically, compared to CCAOs, BRCCAOs showed an approximately 10.000-fold (p < 0.0001) increase in chemo resistance against gemcitabine and cisplatin, first-line chemotherapy drugs for CCA, independent of patient variance.This resistant phenotype mimics patient response as clinically CCA patients experience only a modest increase in overall survival when receiving gemcitabine and cisplatin combinational therapy.
Conclusion: These results demonstrate that BRCCAOs better resemble in vivo CCA tumor tissue compared to conventional CCAOs, particularly with regards to morphology, transcriptome profile, and drug responses. Gemcitabine and cisplatin combinational therapy only provides CCA patients with a modest benefit in overall survival, and BRCCAOs appear to mimic this response more closely. This fosters new possibilities for personalized medicine applications.
83767205564
Introduction: Pancreatic cancer is a devastating malignancy, and treatment options are very limited [1]. Progression of the disease and resistance to therapy are mediated by the tumour microenvironment (TME), which is composed of excessive amounts of extracellular matrix proteins, as well as stromal and immune cells, acting as a physical barrier for drug delivery [1-3]. Cell-secreted factors, such as kallikrein-related peptidases (KLKs), are important players in the TME [3]. Elevated expression of KLK6 is associated with poor survival rates in pancreatic cancer, making it an attractive target for alternative treatment strategies [3]. There is a lack of pre-clinical models of pancreatic cancer that reconstruct different elements of the TME to study disease progression and response to treatment. To address this limitation, and to explore the tumour-biological role of KLK6, we developed a TME model using a protease-sensitive star-shaped poly(ethylene glycol) (star-PEG)-heparin hydrogel system in which the mechanical and chemical properties are independently controlled [4].
Methodology: To mimic the extracellular components of pancreatic cancer, hydrogels were formed by covalently crosslinking protease-sensitive four-arm starPEG with maleimide-functionalized heparin. To allow integrin-mediated cell functions, hydrogels were functionalized with RGD peptides. Hydrogels were globally and locally characterized regarding their mechanical properties by shear rheometry and atomic force microscopy. To increase the complexity of our hydrogel model, and to include the cellular component of tumour tissues, human pancreatic cancer cells, together with cancer-associated fibroblasts and myeloid cells were grown encapsulated in hydrogels for 14 days. Cell viability was assessed by live/dead staining, and the metabolic activity was measured using the PrestoBlue assay. To study the role of KLK6, a CRISPR/Cas9 knockout (KO) approach has been applied.
Results: In order to mimic the stiffness of pancreatic tumour tissues, hydrogels with different mechanical properties ranging from ~4-15 kPa were achieved by varying the crosslinking degree between PEG and heparin. These results are consistent with reported data for patient-derived tissues [2] and were confirmed with our own, unpublished patient cohort. Our multicellular 3D cultures had a high viability and were metabolically active over the analysed timeframe. To test the clinical value of our TME model, the response towards treatment with different chemotherapeutics including gemcitabine and nab-paclitaxel, as well as stromal-targeting agents are assessed. Our CRISPR/Cas9 approach resulted in a successful KO of KLK6 gene expression in human pancreatic cancer cells. The functional consequences of this KO were analysed using our multicellular 3D cultures and an orthotopic xenograft approach.
Conclusion: Bioengineered starPEG-heparin hydrogels are a powerful 3D model to mimic the mechanical, chemical and multicellular characteristics of human tissues in the lab. Our future studies will now determine their potential as pre-clinical platforms for drug screening. Therefore, we are collecting patient-derived tumour specimens that will be included in our model to strengthen their clinical relevance.
[1] Osuna de la Pena, D. et al., Nat Commun, 12, 5623, (2021).
[2] Below, C. R. et al., Nat Mater, 21, 110-119 (2022).
[3] Candido, J. B. et al., Cancers (Basel), 13, (2021).
[4] Mahajan, V. et al., Cancers (Basel), 13, (2021).
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Nanomedicine, where therapeutic or diagnostic agents are coupled with nanoparticles (NP), offers opportunities for more efficacious treatments and accurate diagnosis of severe clinical conditions. Polymeric, lipidic, metallic or ceramic NP have been considered as carriers of these agents, but their translation into clinics requires a thorough assessment of their biocompatibility and biodistribution. However, the existing regulatory frameworks not always address the specific biocompatibility issues associated to nano-biomaterials, including cell response upon NP internalisation and accumulation within tissues. Currently available in vitro tests based on very simplistic 2D cell cultures are not suitable to mimic the clinical conditions. At the same time, in vivo animal models do not reproduce the host conditions of humans and limit the ability of tracking the NP and their interactions with cells in an accurate manner. The present paper aims to contribute to the developing of new in vitro models that can fill the gap between conventional 2D cell cultures and animal experimentation. The objective was to develop a 3D organotypic model resembling the histological features of the liver vascularised lobuli, while preserving the operator-friendly features typical of 2D cultures. Human hepatocyte carcinoma cell lines (HepG2) were co-cultured with human umbelical endothelial vascular cells (HUVEC) at a 60/40 ratio. The deriving cell suspension was seeded at a density of 105 cells/mL in a serum-free medium into tissue culture wells previously coated with a thin and completely transparent film of a synthetic biomaterial substrate, PhenoDrive-Y (Tissue Click Ltd, UK), mimicking the natural basement membrane of tissues. Lobuli-like structure formations were allowed to form over 4 days of culture in serum-free medium to limit cell proliferation, cell cycle synchronisation and cell migration. After 4 days, the cell culture was either stopped or challenged with increasing concentrations of polymeric or lipidic NP for an additional 24h. The formed lobuli-like structures were fixed in formalin and characterised for their morphology, extracellular matrix production, cytotoxicity, pro-inflammatory phenotype both in absence and presence of a challenge with NP at increasing concentrations. Uncoated tissue culture plate and Matrigel were used as controls.
Time-lapse microscopy showed that, when adhering on PhenoDrive-Y, cells gradually migrated towards each other to form lobuli-like structures throughout the well surface. Confocal microscopy and scanning electron microscopy showed that angiogenic sprouting intercalated the spheroids forming each lobulus as well as covered their surface with branching. These spheroidal formations were joined together not only by angiogenic sprouting, but also by an extracellular matrix. The incubation with relatively low concentrations of either polymeric or lipidic NP (e.g. 5 micrograms/mL) showed a preferential binding of NP to the angiogenic sprouting. When challenged by increasing concentrations of NP, these lobuli-like structures appeared to lose their architecture showing clear sign of cytotoxicity.
The present work shows that 3D organotypic cultures, using liver carcinoma cells and endothelial cells and driven by a synthetic biomimetic substrate, are able to resemble the histological features of the liver vascularised lobuli thus providing valuable data of toxicity and biodistribution of NP at cellular levels.
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INTRODUCTION:Termed as the ‘silent killer’,epithelial ovarian cancer (EOC) earns its nickname due to its high mortality rate, with 5-year survival rates of 46%,46.5% and 38% in UK, USA & Europe respectively.This has been attributed to some extent to the EOC’s high recurrence rate and resistance to currently available platinum based chemotherapeutic treatment methods. Hence,society, researchers and the clinical community are in dire need for a more in-depth study of the EOC microenvironment to design better patient specific treatments and to advance current therapeutic methods.Multiple groups have studied and reported the effect of chemotherapeutic agents on various EOC 3D in vitro models1,2,3. However,there are very few studies wherein a direct comparative study has been carried out between the different in vitro 3D models of EOC and the effect of chemotherapy within them.
Herein, we report for the first time, a direct comparative study of three different 3D in vitro platforms, namely (i) spheroids,(ii) synthetic hydrogels of various chemical configurations and (iii) polymeric scaffolds of various Extracellular Matrix (ECM) compositions on the cell growth and response to the chemotherapeutic(Cisplatin) for ovary derived(A2780) and metastatic(SK-OV-3) EOC cell lines.
METHODS: Spheroids of A2780 and SK-OV-3 cell lines were fabricated using specialized 96 well round bottom plates, provided by faCellitate (Manheim, Germany). Synthetic PeptiGels (Manchester BIOGEL,UK) were used as per manufacturer’s instructions for the hydrogel based culture while polyurethane (PU) scaffold assisted 3D cultures was carried out as per our previously published protocol4,5,6.Cultures were maintained and monitored for different time periods (10–28 days)depending on the culture platform type.Feasibility of using these models for assessment of chemotherapeutic agent (Cisplatin) was also carried out.Various in situ assays for monitoring the cell viability, cellular apoptosis and spatial organisation were also performed.
RESULTS: We report that all three 3D models can support the growth of EOC, but for different time periods (varying from 7 days to 4 weeks).We have seen that chemoresistance to Cisplatin, in vitro, observed especially for metastatic EOC cells, is platform dependent both in terms of structural as well as in terms of biochemical composition of the model/platform.Our study highlights the selection of the appropriate 3D in vitro model depends on the cell type,experimental time period and experimental question being asked as different models are appropriate even when studying the same disease.
CONCLUSION: We have shown the feasibility of using all three model (spheroids, hydrogels and polymeric scaffolds) for the culture of EOC cell lines and assessed the impact of chemotherapy on cell viability and apoptosis within those models. Our study highlights that the selection of a 3D in vitro platform depends on (i) the planned experimental/assessment time period, (ii) the type of cell to be studied, (iii) the site of cell origin in vivo and (iv) the question that needs to be answered.
REFERENCE:
1.Heredia-Soto, V. et al. Oncotarget 9(2018)
2.Raghavan, S. et al. Gynecologic oncology 138 (2015).
3.Loessner, D. et al. Biomaterials 31(2010).
4.Totti, S. et al. RSC Advances. 8(37):(2018)
5.Gupta, et al. RSC Advances. 9 (71):(2019)
6.Wishart, et al. Cancers. 13(23):(2021)
20941833117
Introduction: The assessment of bioaccessibility and bioavailability of orally-ingested compounds is key to determine their efficacy and safety. However, there is a lack of toxicological studies that combine simulated gastrointestinal (GI) digestion with subsequent intestinal absorption. Many studies expose cells directly to pristine bioactive substances, failing to consider the series of biochemical transformations that occur throughout the GI tract. Intestinal cell lines have been widely explored to build different gut-epithelial models. However, one-fits-all approaches fall short of recreating physiologically-relevant responses. Patient-derived stem-like cells are emerging as sophisticated and refined models that ultimately surpass the potential of immortalised cells.1 Organ-on-chip offers a disruptive technology to predict in vitro toxicology, presenting a reliable approach with significant advantages when compared to in vivo and cell-based models. Here we describe a two-module microfluidic device comprising the complete GI tract, where digestive and absorptive functions are combined.
Methodology: Microfluidic digestive devices were produced from rapid prototyping (CNC micromachining) of acrylic sheets, while the cell-based module was fabricated from polydimethylsiloxane using a replica moulding technique. On-chip digestion was validated using a fluorescently-labelled casein derivative and compared to the static digestion INFOGEST protocol.2 The Gut-Chip consisted of two channels separated by an in-house fabricated membrane, coated with collagen/Matrigel. Caco-2 and HT29-MTX cells were seeded on-chip at a 9:1 ratio and cultured under continuous flow (120 µL∙h-1) for 7 days. Cell morphology and the epithelial barrier formation were assessed by immunocytochemistry of occludin tight junctions and by measuring the paracellular transport of Lucifer Yellow. Intestinal crypts were isolated from human colon samples of patients undergoing tumour resection surgeries, with full patient consent and approval by Ethics Committee.3 Lgr5+ cells will be expanded as organoids to be used in human primary cell organ-on-chips.
Results: Automated on-chip digestion was in agreement with the current gold standard protocol and was able to replicate typical Michaelis-Menten kinetics. Critically, our device offers enhanced time-resolution over static methods in both gastric and intestinal digestion phases, with on-line pH and temperature sensing and actuation. On-chip Caco-2/HT29-MTX co-cultures displayed 3D villi-like structures after 7 days in culture, a significantly more relevant architecture than the one obtained with Transwell inserts. Furthermore, the on-chip intestinal barrier showed a higher permeability (3.4x10-6 cm/s) when compared to the insert culture (3.0x10-7 cm/s), closely resembling the ex vivo (4.0x10-6 cm/s). Intestinal crypts were successfully isolated from patient samples and their integration on-chip is the subject of our current goals aiming translational applications.
Conclusion: The modular microfluidic device described here shows great promise to be used as a robust tool for pharmacokinetics studies of orally-ingested compounds. The use of patient-derived organoid cultures will allow the execution of personalised studies with significant implications in both food and health applications.
Acknowledgements: We thank Joana Reis (2CA-Braga – Centro Clínico Académico de Braga) for her help with project management regarding clinical samples.
References:
1. Dutton, J. S. et al, Trends Biotechnol. 37, 744-760 (2018)
2. Brodkorb, A. et al, Nat Protoc. 14, 991-1014 (2019)
3. Sato, T. et al, Nature. 459, 262-265 (2011)
94238133006
Introduction: Bioengineered 3D cancer models allow the deconstruction of the tumour microenvironment in vitro to recreate the dynamic interactions between its extracellular and intracellular components. These bioengineered systems strongly rely on Matrigel, an undefined animal-derived matrix, to support the growth of cancer spheroids and organoids [1]. Despite its wide usage, Matrigel has poor mechanical properties and a high batch-to-batch variation, which do not capture the biomechanics of solid tumours and limit experimental reproducibility. Nanocellulose is a low-cost, sustainable and biocompatible alternative biomaterial with promising applications as hydrogel and 3D gastrointestinal organoid models [2]. In this study, collagen-nanocellulose hydrogels are used as a defined matrix of controllable stiffness to mimic elements of pancreatic tumour tissues and promote the formation of cancer spheroids.
Methodology: Nanocellulose hydrogels were synthesized by TEMPO-periodate mediated oxidation of Eucalyptus Kraft pulp suspensions and blended with bovine type I collagen solution [3]. Human pancreatic cancer cells (e.g. PANC-1, MIA PaCa-2) together with cancer-associated fibroblasts and myeloid cells were grown encapsulated in collagen-nanocellulose hydrogels for 14 days. Triple cultures were treated with the anti-cancer compound triptolide and the chemotherapeutics gemcitabine and paclitaxel. Metabolic activity and matrix stiffness were measured by Prestoblue assays and rheology.
Results: Blending of 0.2% type I collagen fibrils with 0.1% and 0.2% cellulose nanofibres formed a matrix of controllable stiffness, with a Young’s modulus ranging from 647 ± 69 to 1,189 ± 234 Pa. Pancreatic cancer cells formed spheroids of 90 ± 30 µm diameter. Cell-containing matrices reached a Young’s modulus of 3,303 ± 226 Pa, which resembles the lower profile of pancreatic cancer tissues [4]. Treatment with triptolide, gemcitabine and paclitaxel reduced the cell viability of triple cultures by 45 ± 2%. The exposure to all three drugs combined reduced the Young's modulus of the MIA PaCa-2 triple cultures by 42 ± 2%, whereas the stiffness of those containing PANC-1 cells decreased only by 8 ± 3%
Conclusion: The mechanical properties of collagen-nanocellulose matrices are controlled by varying the concentration of cellulose nanofibres. The incorporation of pancreatic cancer and stromal cells into the biomimetic hydrogels demonstrates the importance of the cellular elements for matrix stiffening. Drug treatments modulate the mechanical properties of this 3D cancer model, resulting in differential cell responses. Collagen-nanocellulose stands as an alternative matrix to Matrigel to recreate the tumour microenvironment and support the growth of cancer spheroids, as well as to screen novel or improved treatments.
References:
[1] Tomás-Bort E, Kieler M, Sharma S, et al. 3D approaches to model the tumor microenvironment of pancreatic cancer. Theranostics. 2020 10:5074–89.
[2] Curvello R, Kerr G, Micati DJ, et al. Engineered Plant-Based Nanocellulose Hydrogel for Small Intestinal Organoid Growth. Adv Sci. 2020 Nov 20;8(1):2002135.
[3] Curvello R, Kast V, Abuwarwar MH, Fletcher AL, Garnier G, Loessner D. 3D Collagen-Nanocellulose Matrices Model the Tumour Microenvironment of Pancreatic Cancer. Front Digit Health. 2021 Jul 26;3:704584.
[4] Rubiano A, Delitto D, Han S, et al. Viscoelastic properties of human pancreatic tumors and in vitro constructs to mimic mechanical properties. Acta Biomater. (2018) 67:331-340.
94238103804
The plan for tissue engineering has always been to deliver human tissue products that can repair, regenerate, and replace our organs. As far as the heart is concerned, that plan has been punched - hard - by reality (Cit. Mike Tyson). Through the lens of my post-doctoral research at Harvard, we will review advancements in heart muscle engineering and examine some of the fundamental roadblocks to its clinical translation. Specifically, we will see how simple bioengineering and image analysis tools can be used to establish unbiased and quantitative metrics of the phenotypic quality of stem cell-derived cardiac muscle cells. Further, we will see how techniques from biophysics can be used to provide a multiscale assessment of muscle contractility. Finally, we will examine how these tools can be expanded and leveraged into a new framework for cardiac tissue engineering where we stop “trying to build" a heart and we start "growing” one, instead. Round 2 with reality: ready, go!
20967802055
Elastomeric biodegradable scaffolds have been utilized as viable cardiovascular tissue surrogates in various applications, including cardiac patches, engineered vascular grafts, and heart valves. The interactions between cells and scaffold constitute an essential element in endogenous tissue growth and de novo tissue formation. Mechanical conditioning regimens are widely recognized as effective methods for facilitating extracellular matrix (ECM) accretion and improving engineered construct mechanical properties. Despite these advantageous factors, the understanding of the underlying cells - matrix interaction mechanisms remains relatively limited, hampering the development of in silico and in vitro models and the translation of engineered tissues into clinical application. In an attempt to reduce this gap in knowledge, we investigated how mechanical strain and micro-architecture impact ECM formation and elaboration. Vascular smooth muscle cells (VSMCs) have been micro-integrated into elastomeric biodegradable polyurethane scaffolds having identical microstructure. The constructs have been dynamically conditioned using a uniaxial stretch bioreactor for 21 days. Different levels of uniaxial strain, 15, 30, and 50%, have been continuously imposed at 1Hz of frequency for the entire culture period. We hypothesized that specific levels of strain and micro-architectures could be identified to enhance ECM production in quantity (collagen mass) and quality (anisotropy, stiffness). Samples were processed to evaluate ECM biosynthesis via: biochemical assay, qualitative and quantitative histological assessment, multi-photon analysis, and mechanical characterization. Experimental evaluation was coupled to a numerical model that elucidated the relationship between the scaffold micro-architecture and the strain acting on the cells. Results showed that while a 30% peak of strain level achieved maximum ECM synthesis rate, further increases in strain level led to a reduction in ECM. Likewise, micro-integrated scaffolds fabricated with different micro-architecture (i.e., different number of fibers intersections/area) have been exposed to 21 days of dynamic culture at a fixed 30% strain and a frequency of 1 Hz. Results highlighted the existence of specific micro-architectures and how topological cues are able to maximize ECM elaboration given a specific imposed macroscopic mechanical load. The improved understanding of the complex process of ECM formation in these mechanosensitive cell-scaffold models might lead to a more effective engineering and processing of cardiovascular tissue surrogates that are requested to function in highly demanding mechanical in-vivo environments.
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Introduction
Realization of tissue engineered cardiac constructs has progressed with the combination of induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and additive manufactured frameworks for guided repair[1]. Recently, we developed hexagonal 3D microfiber scaffolds with melt electrowriting (MEW). These scaffolds support contracting hiPSC-CMs and promote tissue organization and maturation[2]. However, scaling such constructs includes challenges, such as the need for nutrient and oxygen supply. We hypothesized that control over vascular patterning and deposition of cardiac cells can promote cardiac construct vascularization and native-like tissue formation. This study converges extrusion-based bioprinting and MEW to biofabricate the first anatomically-designed vascularized cardiac patch.
Methodology
Hexagonal MEW meshes were fabricated with a 3DDiscovery (REGENHU)[2]. Cell composition for myocardial and vascular components were tested using varied ratios of hiPSC-CMs, human fetal cardiac fibroblasts and human umbilical vein endothelial cells. Bioinks (myocardial and vascular) comprised of photo-crosslinkable GelMA with the addition of collagen or matrigel were optimized for both cellular performance and bioprintability by use of soluble fraction analysis, immunofluorescent staining, beating rate analysis and qPCR assessment. Viability after bioprinting was assessed using a metabolic assay and live/dead staining, comparing bioprinted to cast constructs. Finally, the myocardial and vascular bioinks were co-printed into the microfibers meshes using an anatomical design, modelled from a branch of the left anterior descending artery. The constructs were analyzed using immunofluorescent stainings.
Results
The myocardial bioink revealed enhanced native-like tissue formation using 6x107 cells/mL with a ratio of 9:1 (cardiomyocytes:fibroblasts). The vascular bioink enabled capillary-like network formations using 3x107 cells/mL with a ratio of 5:1 (endothelial cells:fibroblasts). Bioink optimization revealed that a combination of 5% GelMA with 0.8 mg/mL collagen, photocrosslinked using 0.25/2.5 mM (myocardial) and 0.5/5 mM (vascular) ruthenium/sodium persulfate had an optimal soluble graction and resulting cellular organization for both bioinks. The myocardial bioink exhibited a slowed, more synchronous beating rate pattern following 3 weeks in culture, indicating hiPSC-CM maturation. In addition, the formation of organized tissue-like structures was observed with an enhancement of troponin-T staining, compared to the other hydrogels tested. Bioprinting processes affected the vascular bioink resulting in increased metabolic output, as well as proliferation of the cells. The myocardial bioink showed a significantly reduced metabolic output 1 week following the bioprinting, however no noticeable difference in viability (live/dead assay) was observed between the bioprinted and cast groups. Converged bioprinting into the MEW mesh was achieved by ensuring the gel viscosity that allowed for integration into the hexagonal pores with all pores filled after 2 printed layers. Interfaces between the vascular and myocardial patterned components were visualized using immunofluorescent morphological stainings.
Conclusions
This study has demonstrated potential for patterning anatomically-relevant vascular pathways within a bioprinted myocardial construct, forging an opportunity for scaling up tissue engineered constructs. Our study provides an important step towards the generation of 3D in vitro cardiac models of relevant dimensions with native-like tissue architecture and function.
References
[1] Kristen & Ainsworth et al.(2019). Adv Healthcare Mater.
[2] Castilho & van Mil et al.(2018). Adv Func Mater.
20941820555
Introduction: Stem cells are being tested in clinical trials for cardiac repair. Bone marrow derived allogeneic (unrelated donor) mesenchymal stem cells and induced pluripotent stem cells (iPSC) have emerged as ideal cell types for cardiac repair and regeneration. Outcome of initial allogeneic stem cells based clinical trials was positive. There were no significant side effects observed after cell transplantation. In fact, the implanted cells were able to improve cardiac function. However, poor survival of transplanted cells in the myocardium is a major hurdle in clinical translation of stem cell-based therapies for cardiac repair.
Methods: We have previously reported that after transplantation in the ischemic heart stem cells display immunogenicity and are rejected by recipient immune system. In our ongoing studies, we have been engaged in developing immunomodulatory biomaterials-based strategies to prolong survival of transplanted stem cells in the heart. We synthesized MXene quantum dots (MQDs) with tailored surface properties to possess intrinsic immunomodulatory properties.
Results and conclusion: Our data demonstrate that MQDs were spontaneously uptaken into antigen-presenting cells and downregulated the expression of genes involved in alloantigen presentation, and consequently reduced the activation of allogeneic lymphocytes. Furthermore, MQDs are able to selectively reduce activation of CD4+IFN-γ+ T-lymphocytes and promote expansion of immunosuppressive CD4+CD25+FoxP3+ regulatory T-cells in a stimulated human lymphocyte population. Furthermore, MQDs are biocompatible with MSCs and iPSC. Next, MQDs were incorporated into a chitosan-based hydrogel to create a 3D platform for stem cell delivery to the heart. This composite immunomodulatory hydrogel-based platform improved survival of stem cells and mitigated allo-immune responses. These findings suggest that this new class of biomaterials may bridge the translational gap in stem cells and biomaterials-based strategies for cardiac tissue engineering.
20941815786
Modeling the cardiac pathological traits would be of paramount utility to elucidate possible targets of new therapeutics for unmet pathology (e.g., Dilated Cardiomyopathy (DCM [1])). Traditional in vitro systems lack the complexity of human physiological conditions, resulting poorly reliable for tissue engineering studies. Organs-on-chip (OoC) have been shown to be a promising alternative, offering the ease to integrate stimulation and sensors [2]. In previously developed beating heart-on-chip [3]–[5], despite advanced tissue maturation through mechanical stimulation have been demonstrated, the coupling with electrical readout allowing for a broader electrophysiological recording space and high throughput analyses is still missing.
To overcome these issues, we developed µPEA, an electrode array system coupled with the beating-heart-on-chip [5] able to provide 3D microtissues with mechanical stimulation and electrical readouts, using noninvasive 2D electrodes. We validated µPEA-Heart-on-Chip by assessing electrophysiology of neonatal rat cells and we exploited it to study the effect of mechanical stimulation on human DCM models.
Methodology:
Electrodes were designed using CAD and developed in glass substrate using physical vapor deposition (Cr/Au, 100nm). Electrodes arrays consist of five recordings, two references and one ground electrodes for measuring field potential. Chips were connected to a platform interface with a multiplexing system followed by an amplifier coupled with a bandpass filter (0.5-100Hz).
Electrodes characterizations were done by measuring impedance spectroscopy and by filling the OoC with PBS. iPSC-CM were generated by following well-established differentiation protocols of iPSC derived from DCM patients [6]. Neonatal rat cardiomyocytes, DCM iPSC-CMs, and isogenic controls were embedded in fibrin gel at 80-120 · 106 cells/mL and cultured for 5-10 days in static or mechanically active (i.e. 10% uniaxial strain at 1Hz) environment. After maturation, electrophysiological signals were measured from different electrodes.
Results:
The µPEA-Heart-on-Chip was successfully assembled by integrating and aligning the innovative patterned surface with the mechanically active OoC (the electrodes are positioned along the cardiac microtissue). Electrodes impedance measurements were 124.5 ± 3.1 KΩ at 1Khz, in line with literature values [7]. Neonatal rat cardiomyocytes microtissues started to spontaneously beat after 4 days in culture. Field potentials were successfully measured through all five different electrodes and key parameters were evaluated. DCM iPSC-CM were efficiently differentiated in 2D, as evidenced by immunofluorescence staining of cardiac Troponin T. The DCM diseased model was established within the µPEA-Heart-on-Chip, with cells that organized and interconnected within the 3D environment. Electrophysiological and contractility changes of the DCM model in response to the mechanical stimulation is currently under evaluation.
In Conclusion, here we described the development of the µPEA-Heart-on-Chip capable to integrate a mechanical stimulation with an electrophysiological activity recording system. The platform represents an unprecedented tool to investigate electrical cardiac alterations in healthy or diseased model in response to mechanical stimulation.
References
1.B. J. Maron et al, Circulation, 2006.
2.G. A. Clarke et al, Sensors, 2021.
3.A. Marsano et al., Lab Chip, 2016.
4.R. Visone et al, APL Bioeng., 2018.
5.R. Visone et al., Biofabrication, 2021.
6.N. Sun et al., Sci. Transl. Med., 2012.
7. H. Cui et al., Biomed. Eng. Online, 2019.
94238124605
Restoration of cardiac functionality after myocardial infarction represents a major clinical challenge1. Recently, we found that transient transfection with a microRNA combination (miRcombo: miR-1, miR-133, miR-208 and 499) is able to trigger direct reprogramming of adult human cardiac fibroblasts (AHCFs) into induced cardiomyocyte (iCMs) in vitro2. However, achieving efficient direct reprogramming still remains a challenge. Direct reprogramming of human fibroblasts could be enhanced by culturing miRcombo-transfected cells in a three-dimensional (3D) environment with biomimetic biophysical and biochemical cues3,4. Herein, the ability of cardiac-like extracellular matrix (called Biomatrix, BM) to enhance miRcombo-mediated direct cell reprogramming was studied. Then, miRcombo-transfected cells were cultured in a biomimetic microenvironment consisting of a 3D fibrin hydrogel containing BM.
Methodology
BM was produced by in vitro culture of AHCFs for 21 days, followed by decellularization. AHCFs were transfected with miRcombo and then cultured for 2 weeks on the surface of: uncoated and BM-coated polystyrene (PS) dishes, on the top of fibrin hydrogels (2D hydrogel), or embedded into 3D fibrin hydrogels without (3D hydrogel) or with BM (3D BM hydrogels). The expression of cardiac markers and cell maturation was analysed by ddPCR, immunofluorescence and calcium transient analysis.
Results
Culture of miRcombo-transfected cells on BM-coated vs. uncoated PS dishes enhanced direct reprogramming efficiency, enhancing TNNT2, ACTC1 and CACNA1C expression and increasing the percentage of cardiac troponin T (cTnT)-positive cells at 15 days culture time. Culture in 3D hydrogel after miRcombo transfection significantly improved direct reprogramming efficiency respect to uncoated PS and 2D hydrogel, increasing TNNT2, SCN5A and MYL7 expression. Finally, miRcombo-transfected cells were cultured in 3D BM hydrogel providing cardiac tissue-mimetic biophysical and biochemical cues. The expressions of cardiomyocyte genes and cTnT were significantly enhanced in cells cultured for 15 days in 3D BM compared to 3D hydrogels. Calcium transient was enhanced in 3D BM compared to 3D hydrogels in miRcombo cells, showing higher slope of calcium upstroke.
Conclusions
Overall results demonstrated that a biomimetic 3D culture microenvironment can enhance the direct reprogramming efficiency of miRcombo-transfected human adult cardiac fibroblasts into iCMs. Future investigations will elucidate which molecular barriers to direct reprogramming can be overcome by the use of 3D biomimetic cell culture substrates, paving the way to the research for more efficient strategies for direct cardiac reprogramming.
References
1. Jayawardena, T. M. et al. Circ. Res. 110, 1465–1473 (2012).
2. Paoletti, C. et al. Front. Bioeng. Biotechnol. (2020).
3. Li, Y. et al. Sci. Rep. 6, (2016).
4. Paoletti, C. & Chiono. Front. Cardiovasc. Med. 8, (2021).
62825449926
INTRODUCTION: Cardiovascular diseases (CVDs) remain the leading cause of death worldwide, contributing a huge burden on healthcare providers. Myocardial infarction (MI) is one of the most fatal results of CVDs as it can lead to ultimate heart failure. Available treatments are used to mitigate many of the symptoms of MI, however they are not designed to repair the damaged tissue. A proposed solution to this lack of a regenerative treatment is a tissue engineered myocardial patch which would deliver healthy cells to repopulate the infarct area. In this research, natural and biocompatible materials, a polyhydroxyalkanoate (PHA)1-3 and alginate3, are used with the aim of producing a cellular multimaterial myocardial patch for this purpose. The patch would incorporate human induced pluripotent cardiomyocytes (hiPSC-CMs) and endothelial cells (hiPSC-ECs)
METHODS: Bacterial fermentation of Pseudomonas species was carried out to produce the PHA poly(3-hydroxyocatnoate-co-3-hydroxydecanoate), P(3HO-co-3HD), and this was purified and extracted from the bacteria using Soxhlet extraction. Resazurin assays with a C2C12 myoblast cell line were used to test the biocompatibility of P(3HO-co-3HD) and alginate hydrogel, the polymers were 3D printed (fused deposition modelling) to produce a multimaterial patch, with C2C12 cells encapsulated in the alginate and 3D-bioprinted. hiPSC-CMs and hiPSC-ECs were produced from (hiPSCs), seeded onto P(3HO-co-3HD) films, live/dead stained, and functionally analysed. Multimaterial patches were also tested in vivo in a rat model with an induced infarct.
RESULTS: P(3HO-co-3HD) has been successfully produced and characterised to confirm its chemical structure and 3HO:3HD molar ratio; mechanical properties which show that it is highly elastomeric, making it a suitable polymer for myocardial applications; and thermal properties, including a melting point of around 54oC making it easy to 3D print. P(3HO-co-3HD) and alginate were shown to both be non-cytotoxic via the resazurin assay. 3D printing was carried out with these materials, with C2C12 cells encapsulated in the alginate, and successfully produced cellular multimaterial patches at a high resolution, while maintaining cell viability. To improve the cell types included in the patch, hiPSC-CMs and hiPSC-ECs were produced, with initial results showing that they adhere to P(3HO-co-3HD) with good viability, and retain functionality through beating and calcium handling, as seen with Fluo-4 imaging.
CONCLUSIONS: Multimaterial patch production and successful encapsulation and printing of cells shows promise for the development of a functional cellular multimaterial patch. Future aims in this project are to include hiPSC-ECs and hiPSC-CMs in the multimaterial patch before carrying out in vitro and in vivo experiments in a rodent MI model.
REFERENCES:
Majid QA, et al. Natural biomaterials for cardiac tissue engineering: a highly biocompatible solution. Frontiers in Cardiovascular Medicine 7, 192, 2020
Bagdadi AV, et al. Poly(3-hydroxyoctanoate), a promising new material for cardiac tissue engineering. Journal of Tissue Engineering and Regenerative Medicine 12, 495, 2018
Rai R, et al. Medium chain length polyhydroxyalkanoates, promising new biomedical materials for the future. Materials Science and Engineering: R: Reports 72, 29, 2011
41883671128
"Bioactive glasses (BGs) are being increasingly investigated as antibacterial materials for developing scaffolds for bone tissue engineering (TE). Such applications are based on the biochemical reactions occurring on the BG surface in contact with the biological environment, which involve the (controlled) release of biologically active ions during BG scaffold degradation. Such ions are capable of stimulating specific cellular responses involved in bone tissue growth [1]. In addition, BGs can induce immunomodulatory effects to trigger bone regeneration and wound healing [2]. Expressing a further functionality of BGs, specific compositions are designed to release ions with antibacterial effect (Ag, Cu, Zn, Ga among other).
In this presentation, ion doped BGs will be discussed in the broad context of their use in bone tissue engineering. In particular, results will be presented to illustrate the osteogenic, angiogenic and antibacterial effect of a series of silicate and borate BGs. Specific concentrations of such ions enhance the secretion of vascular endothelial growth factor and the different mechanisms by which different ions induce angiogenesis will be discussed. The results of vitro studies will be presented that show the effect of ion concentration (following BG dissolution) on stem cell behavior. In an attempt to enhance the mechanical and biological performance of BG scaffolds, the polymer coating approach will be discussed. Such coatings (using synthetic or natural polymers) provide additional functionalities [3], for example, incorporating mesoporous BG nanoparticles as therapeutic drug carriers. Thus, the dual, simultaneous and tuned release of biologically active ions and therapeutic drugs (both antibiotics and growth factors) from BG scaffolds can be exploited to induce synergistic antibacterial, osteogenic and angiogenic effects.
References
[1] A. Hoppe, et al., A review of the biological response to ionic dissolution products from bioactive glasses and glass-ceramics, Biomaterials 32 (2011) 2757-2774.
[2] K. Zheng, et al., Immunomodulatory bioactive glasses for tissue regeneration, Acta Biomater.133 (2021) 168-186.
[3] T. Reiter; et al. Bioactive glass based scaffolds coated with gelatin for the sustained release of icariin. Bioactive Mater. 4 (2019) 1-7. "
62903406568
Introduction
Bone tumour removal, traumas with large defects or infections, and degenerative diseases are the main catastrophic events impeding complete bone healing. Autologous and allogenic bone grafting, and biologically inert metallic devices have limitations such as non-availability of autogenous bone, risk of infectious disease transmission, subsequent surgical removal, and bacterial infections [1]. Therefore, to overcome the limitations, we fabricated composite scaffolds based on a combination of Poly(3-hydroxybutyrate) [P(3HB)], a natural biocompatible and bioresorbable polymer of bacterial origin, and a Borosilicate-based bioactive glass doped with Zinc (BS-Zn). Moreover, the Zinc-doped bioglass is used to provide antibacterial activity, as Zn2+ is known for its strong anti-inflammatory and bactericidal properties. [2]
Methodology
P(3HB) was produced by bacterial fermentation of B. sacchari in the presence of excess of carbon and nitrogen limitation. Gas chromatography-mass spectrometry (GC-MS), Fourier-transform Infrared spectroscopy (FTIR), Nuclear Magnetic Resonance spectroscopy (NMR), and Differential Scanning Calorimetry (DSC) have been conducted to characterise the resulting polymer. Solvent casting has been used to produce composite films and X-Ray Diffraction (XRD), Scanning Electron Microscopy (SEM), High-resolution X-ray CT and tensile testing were conducted to determine their properties.
MG63 human osteoblast cell line was cultured on the composite and neat polymer films to assess biocompatibility, quantified using the resazurin assay. Subsequently, Live/Dead assay was performed using calcein green and ethidium bromide. In order to evaluate the antimicrobial activity, Minimum Inhibitory (MIC) and Minimum Bactericidal Concentration (MBC) were determined using ISO20776 against E. coli 8739, S. aureus 2569 and S. aureus 6538P for P(3HB)/BS films loaded with Gentamicin and P(3HB)/BS-Zn. The HALO Test was also carried out for the composite films.
Results
Chemical analysis confirmed that the polymer produced was Poly(3-hydroxybutyrate). The thermal properties of the extracted polymer analysed by DSC were found to be very similar to previous studies and commercially available products [3]. XRD analysis resulted in profiles which indicated the amorphous nature of the bioactive glasses. The addition of nanosized bioglasses induced a nanostructured topography on the surface of the composites, as visible by SEM images. Moreover, X-ray CT confirmed the homogeneous distribution of the bioglass filler in the polymeric matrix. The resazurin assay demonstrated the biocompatibility of P(3HB) and the related composites. The MIC/MBC test and the HALO test confirmed the antimicrobial activity of the composites.
Conclusions
In this study, a short-chain length PHA was produced by bacterial fermentation and its properties were investigated. Composite films have been synthesised using Borosilicate-based bioactive glasses. These have been further characterised to confirm the properties of the composite materials. The biocompatibility of the films was confirmed using the MG63 human osteoblast cell line. Also, the antimicrobial activity of P(3HB)/BS composite films loaded with gentamicin and P(3HB)/BS-Zn were investigated. The final aim of this project is the development of a promising material with antimicrobial activity for bone regeneration.
References
1. Koons, G.L. et al., Nat. Rev. Mater., vol. 5, n. 8, 584-603, 2020
2. Schuhladen, K. et al., Journal of Non-Crystalline Solids, n. 502, 22-34, 2018
3. Misra, S.K. et al., Biomacromolecules, n. 8, 2112-2119, 2007
83767228705
Introduction:
Implant infection, due to bacterial contamination, is a significant problem that represents one of the main causes of implant loss over time. In addition, the incidence of antibiotic resistance is steadily increasing, and alternative ways to fight or prevent infection have become the subject of biomedical research, and several surface modification and coating techniques have recently been developed for antibacterial applications.
In this work, three different molecules: alpha tocopherol, its water-soluble version alpha-tocopheryl phosphate, and a synthesized peptoid (GN-2-Npm9) were studied and chosen to create surfaces with antibacterial and anti-inflammatory properties.
Experimental Method:
Titanium Ti6Al4V alloy samples (ASTM B348, Gr5, Titanium Consulting and Trading, 10 mm diameter discs) were ground (up to 400 grit), then washed in acetone and deionized water. The discs were chemically treated to increase nanoscale roughness, to expose OH groups, and to make the surface more suitable for the grafting. The treated samples were irradiated with UV light to reduce carbon contamination,.
The modifications of titanium surfaces are explored as a coating or through functionalization and a procedure for proper characterization of this substrates was investigated. Physical and chemical characterization was performed through specific measurement techniques such as FTIR-ATR, Z-potential, reflectance spectroscopy, contact angle and release tests.
Biological characterization was performed through cellular and antibacterial assays.
Results and discussion:
The modified surfaces are compared through FTIR-ATR and reflectance spectroscopy, z-Potential titration curves, contact angle measurements, release test, and tape test. The aim is to verify the effective presence of the molecules on the surfaces, the chemical stability over time, mechanical adhesion properties and hydrophobic behaviour for all three grafted molecules. The biological outcome is tested by the cellular and antibacterial tests. According to the results, both grafting and coating can be effectively performed and the biological response can be modulated from anti-adhesive to tissue integration by properly selecting the grafted biomolecules according to the final application (temporary or permanent implants). The surfaces show interesting antibacterial and anti-inflammatory properties.
Conclusion:
This work highlights a promising new application of these biomolecules as possible candidates for bone implant surfaces that reduce the risk of an excessive pro-inflammatory response, the risk of implant-associated infections, and allow for the disincentive of antibiotic use associated with the post-operative period.
73296319608
Introduction
Since the discovery of bioactive glasses (BG) in the late 60s [1], the research in this area has significantly increased to obtain compositions with multiple functionalities not only from the materials aspect but also to provide a favorable biological response for tissue regeneration. BGs have the ability to react with the surrounding environment and bond to hard tissue. This property is characterized by the formation of a hydroxycarbonate apatite (HCA) layer on the material with a similar composition to the inorganic component of bones. Simultaneously, the dissolution of BG takes place leading to the release of biologically active ions, which stimulate the formation of new tissue at a cellular level. The incorporation of metallic ions in the BGs composition has been considered to obtain glasses able to stimulate the formation of bones, support biological processes such as angiogenesis and provide an antibacterial effect on the implantation site [2].
Methodology
Silicate-based BG glasses were produced with the melt-quench method. The well-known 45S5 BG composition was considered as reference material and new compositions were obtained incorporating zinc, boron and strontium. The bioactive behavior was assessed in simulated body fluid and characterized via FTIR, SEM and XRD. In vitro cell studies were carried out using pre-osteoblast cells MC3T3-E1. Moreover, the antibacterial effect of BGs was determined via indirect and direct experiments with Gram-positive and Gram-negative bacteria, S. aureus and E. coli, respectively. Turbidity measurements, counting colony-forming units and metabolic assays were used.
Results
The formation of an HCA layer on the material’s surface was characterized by the detection of adsorption bands in FTIR spectra attributed to calcium phosphate and crystalline reflections of HCA. Which were also detected with XRD. The incorporation of the studied metallic ions delayed the formation of HCA compared to the non-doped 45S5 BG, which exhibited faster bioactivity after 1 day compared to 3 days for Sr-doped BGs and 7 days for B- and Zn-BGs. In terms of cell viability, the glasses containing boron and strontium outperformed the reference BG at all tested concentrations, whereas Zn-doped BG presented the lowest cell viability, particularly, at the highest BG concentration. The minimum inhibitory concentration of the dissolution products of BGs required to kill bacteria was lower for the Sr-doped BG and the reference 45S5 BG compared to the other glasses. However, all materials exhibited an antibacterial effect compared to the control sample (bacteria without BGs).
Conclusion
All studied BG compositions exhibited beneficial properties in terms of bioactivity and biological response. Although the bioactivity was slightly delayed, the in vitro biological assays showed a superior biological effect of Sr-doped BGs compared to control cells, control bacteria and to the other BGs. These results suggest a potential application of ion-doped BGs for bone regeneration.
References
[1] Hench L.L., J. Mater. Sci. Mater. Med. 17, 967–978 (2006)
[2] Hoppe, A., et al., Biomaterials. 32, 2757-2774 (2011)
62825450346
Introduction: In the last years, partly given to the changes in the age structure of the population, there has been a skyrocketing increase of the number of orthopaedic surgeries [1]. With the rising number of implantations, the absolute number of complications is inevitably increasing at the same pace, causing not only distress for the patients but also a significant economic burden [2]. One of the major causes of implant failure is biofilm-associated infections, which represent a huge challenge given their high tolerance to antibiotic therapy. Drop on demand technology has proven to be a valuable tool to develop antimicrobial coatings to avoid bacterial attachment and biofilm development onto the implant surface. This technique enables the production of complex drug release profiles allowing a sustained release of antibiotics and biofilm inhibitors [3]. In this work, we developed PLGA antimicrobial coatings on titanium discs using drop on demand technology. N-(abiet-8,11,13-trien-18-oyl) cyclohexyl-L-alanine (DHA1) was used as a biofilm inhibitor.
Methodology: PLGA (PDLG 5004A, Corbion) and PEG (35 kDa, Sigma) were dissolved in organic solvents. DHA1 was added to the solution at different concentrations (10, 20 and 30% w/w). The viscosity of ink formulations was studied with a viscometer (DV-III Ultra, Brookfield). The printability of the different formulations was studied using a 3D Discovery (regenHU) inkjet microvalve-based 3D printer. The printing parameters were optimized, studying the pressure, opening valve time (OT), distance nozzle-sample, and nozzle diameter. The antimicrobial properties of this coating were studied by assessing its capacity preventing Staphylococcus aureus ATCC 25923 adhesion to the titanium surface.
Results: The viscosity and printability of different ink formulations were studied. A small range of viscosity was observed to obtain a good droplet formation and printability. A too high viscosity produced the clog of the nozzle, while a too low viscosity produced splashes and formation of satellites. The thickness of the coatings was in the range of 30-40 mm and 3 mg×cm-2 per layer. The presence of DHA1 in the coatings was confirmed by FTIR. The coatings loaded with different concentrations of DHA1 (10, 20 and 30% DHA1) reduced bacterial adherence up to 4-logs when compared with the titanium coated with PLGA-PEG with no load. Moreover, the released eluates from the coating with 30% DHA1 load managed to inhibit biofilm formation up to 24 h.
Conclusions: Drop on demand is a suitable technique for the development of antimicrobial coatings. The coating developed here proved high capacity preventing S. aureus biofilm formation. As far as we know, this is one of the first drop on demand coatings incorporating a biofilm inhibitor.
References
[1] S Veerachamy et al., Proceedings of the Institution of Mechanical Engineers. Part H, Journal of engineering in medicine 228 (10), 1083 (2014).
[2] SM Kurtz et al., The Journal of arthroplasty 27 (8 Suppl), 61 (2012).
[3] CL Ventola, P T 39 (10), 704 (2014).
[4] I Reigada et al., Microorganisms 8 (3) (2020).
[5] R Perez-Tanoira et al., J. Biomed. Mater. Res. Part A 105 (1), 62 (2017).
73296375448
"Introduction
Implantation of biomedical devices is followed by immune response to the implant, as well as occasionally bacterial and yeast/fungi infections (1-3). In this context, new implant materials and coatings that deal with medical device-associated complications are required. Antibacterial and anti-inflammatory materials are also required for wound healing applications, especially in diabetic patients with chronic infected wounds. Such wounds are associated with high levels of pro-inflammatory cytokine secretion and iNOS production, contributing to non-healing phenotype.
Methodology
We have previously described thin films made of hyaluronic acid (HA) and polyarginine (PAR) that can be applied to all kind of medical devices. Such films constructed by layer-by-layer assembly demonstrated antimicrobial and anti-inflammatory properties (4-6). Recently, we presented antibacterial HA-based hydrogels cross-linked with 1,4-butanediol diglycidyl ether (BDDE). In HA hydrogels cross-linked with BDDE, carboxylic groups are preserved, and can be used for complexation with positively-charged antibacterial polymers such as PAR (7). Now, we used this system for multifunctional HA-based hydrogels.
Results
For the first time, we fabricated PAR/miRNA-loaded HA hydrogels with antibacterial and anti-inflammatory properties, which simultaneously act as miRNA delivery system (article submitted).
We demonstrate that PAR decreases inflammatory response of LPS-stimulated macrophages and accelerates fibroblast migration in macrophage/fibroblast co-culture system, suggesting a positive effect on wound healing. Furthermore, PAR allows to load miRNA into HA hydrogels, and then to deliver them into the cells. Potentially any miRNA can be used, making the system highly versatile. For instance, the hydrogels can be associated to different functional miRNAs, such as anti-inflammatory or angiogenic, to increase anti-inflammatory properties or to induce revascularization at the wound site.
Conclusion
To our knowledge, this study is the first describing miRNA-loaded hydrogels with antibacterial effect and anti-inflammatory features, making this system promising for infection treatment and foreign body response modulation. We believe that our system can become useful for the treatment of infected wounds such as diabetic ulcers, that are extremely difficult to heal and usually end up with amputations.
References
1. Morais, J. M. et al. AAPS J. 12, 188-196 (2010).
2. VanEpps, J. S. et al. Shock 46, 597-608 (2016).
3. Shen, P. et al. Acta Biomater. 126, 31-44 (2021).
4. Özçelik, H. et al. Adv. Healthc. Mater. 4, 2026-2036 (2015).
5. Mutschler, A. et al., Chem. Mater .28, 8700-8709 (2016).
6. Mutschler, A. et al., Chem. Mater. 29, 3195-3201 (2017).
7. Gribova, V. et al., ACS Appl. Mater. Interfaces 12, 19258-19267 (2020)."
41883661749
INTRODUCTION: Bacterial antibiotic resistance increases every year, creating an urgent need to develop new antibacterial materials. Gallium has been studied since the 1970s as an effective treatment for bone diseases and has recently shown antibacterial activity against different bacterial strains. Therefore, gallium doped hydroxyapatite (GaHAp) could be used as an antibacterial agent. The aim of the study was to investigate the effect of gallium on hydroxyapatite properties and P. aeruginosa growth.
METHODS: GaHAp was synthesized with a wet chemical precipitation method with a concentration of gallium 2wt%, 4wt%, 6.3wt% and 8wt%. CaO, H3PO4 and Ga(NO3)3*xH2O were used as raw materials. Synthesis was performed at 45 °C and final pH was 6.95±0.05. GaHAp was characterised with X-ray Diffraction analysis (XRD), Fourier Transform Infrared Spectroscopy (FTIR) and Specific Surface Area (SSA) was measured with N2 adsorption system BET method. Ion release in Dulbecco’s Modified Eagle Medium (DMEM) was measured for 18 days by Inductively Coupled Plasma Mass Spectrometry (ICP-MS). P. aeruginosa growth was conducted in Tryptic soya broth (TSB) with GaHAp concentrations of 1, 2 and 4 mg/mL (dry sample). The growth curves were obtained by monitoring the absorbance (optical density) at 600 nm (OD600) for 18 hours and subtracting starting OD600-t=0 from all measurements.
RESULTS: The obtained GaHAp showed characteristic apatite peaks on XRD with decreased crystallinity. Additional gallium compound phases were not observed. The obtained GaHAp compared to HAp has a higher specific surface area, indicating a decrease in particle size. Ga3+ ions were constantly released over 18 days with a release range of ~ 40%. The antibacterial test showed that 4 mg/mL of GaHAp suspension induced a total inhibition of P. aeruginosa.
CONCLUSIONS: Gallium ions inhibit hydroxyapatite crystal formation. GaHAp allows a sustained release kinetic of Ga3+ ions. Also, GaHAp has the potential of P. aeruginosa bacteria growth inhibition.
ACKNOWLEDGEMENTS: This research is funded by the EuroNanoMed III project “NANO delivery system for one-shot regenerative therapy of peri-implantitis” (ImlpantNano).
Authors acknowledge financial support from the European Union’s Horizon 2020 research and innovation programme under the grant agreement No 857287.
52354582899
Introduction
Bone infections (osteomyelitis) are difficult to treat due to the formation of biofilms, antibiotic resistance, and limited penetration of systemic antibiotics to infection site(1). Highly-porous collagen-hydroxyapatite (C-HA) scaffolds have proven capacity to regenerate critical-sized bone defects in vivo and human clinical trials(2). However, they still lack appropriate mechanical properties to support larger defects and weight-bearing applications(2). The objective of this study is to develop a reinforced non-antibiotic antimicrobial-doped hydroxyapatite scaffold to overcome issues surrounding antibiotic resistance, while providing a suitable environment for bone regeneration. The aims are to (i) optimise the delivery of metal-based antimicrobials from the scaffold without impeding pro-osteogenic properties in vitro, (ii) reinforce the metal-based antimicrobial scaffold using 3D printing to enhance the mechanical properties, and (iii) evaluate the reinforced scaffold system in a weight bearing rat femoral defect in vivo.
Methods
A range of metal-based antimicrobial doped hydroxyapatite (MBA-HA) doses (formulations not disclosed due to IP restrictions) were incorporated into type-1 collagen matrix to fabricate collagen-MBA-HA (C-MBA-HA) scaffolds. The C-MBA-HA scaffolds were evaluated for cell viability and osteogenesis using rat mesenchymal stromal cells in vitro up to 28 days. The scaffold’s antibacterial properties against S. aureus were assessed in vitro. A 3D-printed polymer framework was combined with the collagen matrix for reinforcement. The compressive modulus, porosity, and microarchitecture were assessed. The reinforced scaffolds were further evaluated in vitro to ensure there were no negative affect of reinforcement on the osteogenic and antimicrobial capacity. Ongoing evaluation of the bone healing potential of C-MBA-HA is being assessed in a 5mm long critical-sized rat femoral defect and will be compared against C-HA and empty defects at 2,4, and 8 weeks using micro-computerisation tomography (μCT) and histology at 8 weeks.
Results
Biomimetic C-HA scaffolds were successfully functionalized with MBA-HA to achieve antimicrobial properties while continuing to support osteogenesis. Specifically, the C-MBA-HA scaffolds resulted in equivalent calcium deposition to the C-HA scaffolds at 28 days, which was further validated with the homogenous distribution of alizarin red staining, i.e., revealing cell-mediated mineralization throughout the scaffolds. The C-MBA-HA scaffolds achieved a 50% reduction of S. aureus, as well as the development of inhibition zones on S. aureus agar plates after 24 hours. The 3D-printed polymer framework was successfully integrated into the C-MBA-HA scaffold to significantly enhance its mechanical properties to better mimic cancellous bone, while maintaining high porosity and an microarchitectural structure favourable for cellular infiltration and bone formation. In vitro cellular and microbial assessment of the reinforced C-MBA-HA scaffolds demonstrated no reduction in beneficial properties when compared to the non-reinforced scaffold.
Conclusion
The successful development of this non-antibiotic antimicrobial and osteoconductive scaffold with enhanced mechanical properties for treatment of weight bearing large defects has the potential to be a one-step local treatment for osteomyelitis.
Funding through Science Foundation Ireland (grant 12/RC/2278 and 17/SP/4721). Co-funded by Johnson & Johnson 3D Printing Innovation & Customer Solutions, Johnson & Johnson Services Inc.
20941802728
"Polysaccharides belong to the most abundant biomaterials on earth. They form structural elements of plant and animal tissues, but many of them have also important regulatory functions towards cells, tissues and organs. Glycosaminoglycans (GAG) and other carboxylated and sulfated polysaccharides possess a bioactivity that is due to their high affinity to a plethora of proteins that belong to insoluble and soluble components of cell environments, but also by direct interaction of these carbohydrates with cell receptors. Hence, making surface coatings or hydrogels from natural or semisynthetic GAG for implants that guide cell behavior towards differentiation to bone or cartilage might be an promising approach ti mimik the natural environment of cells in tissues.
Here, covalent binding mechanisms based on oxidation of pendant hydroxyl groups or introduction of reactive side groups like thiols in monosaccharide subunits permit direct coupling to surfaces or cross-linking mechanisms using Schiff´s base or disulfide bond formation, respectively. Physical immobilization can exploit the inherent charge of these molecules that permits formation of multilayers on surfaces kept together by ion pairing, but also intrinsic cross-linking of activated GAG. Both mechanism can be also used to form 3D structures as in situ gelling hydrogels that permit embedding of growth factors and cells.
Native and oxidized GAGs have been used to prepare multilayer coatings that could promote either osteogenic or chrondrogenic differentiation using chondroitin sulfate (CS)and hyaluronan as polyanions, respectively. Multilayer systems with oxidized CS were also useful as system for controlled presentation of osteogenic growth factor BMP-2. Thiolated CS and chitosan could be also used for redox-sensitive multilayer coatings that change their cell adhesion properties in dependence on disulfide bond formation, which functions as stimuli-responsive system for cell culture. Apart from making bioactive surface coatings both cross-linking mechanisms can be also used to form 3D structures as in situ gelling hydrogels that permit embedding of growth factors and cells.
In conclusion of our studies we could fabricate 2D and 3D systems that are instructive in controlling cell spreading, growth and differentiation of mesenchymal stem and other cells, which will be shown with examples of chondrogenic and osteogenic differentiation of cells."
94355102884
"Many biomaterials have been proposed to produce porous scaffolds, nanofibers and nanoparticles for different medical treatments and applications. Systems combining natural polymers and synthetic biodegradable polymers offer particular properties adequate for those demanding applications. Those biomaterial systems can be tailored with enhanced mechanical properties, processability, cell-friendly surfaces and tunable biodegradability. Those biomaterials can beprocessed by melting or solvent routes into devices with wide range of applications such as biodegradable scaffolds, films or particles and adaptable to many other high performance biomedical applications.
Non-woven meshes of polymeric ultrafine fibers with fiber diameters in the nanometer range can be produced by electrospinning. Those meshes are highly porous and have a high surface area-to-volume ratio. Furthermore, they can mimic the fibrous structure of the extracellular matrix of human tissues and can be used as scaffolds for Tissue Engineering (TE). There is a great interest in developing also nanoparticles and hydrogels from those polymeric systems for injectable treatment modalities. All those structures can be used as substrates for specific surface functionalization having fine-tuned bioactivity and biological performance. This strategy enables developing highly controlled devices for exposure, capture and, whenever needed, inactivation of biological biomolecules. Those high-performance devices offer the specificity and local bioactivity that enable to design novel treatment modalities in various disease conditions.
This talk will review our latest developments biomaterials, nanoparticles and nanofibre meshes in the context of novel therapeutic applications."
20967807208
"Osteoarthritis, a degenerative disease of the cartilage and subchondral bone, is becoming more prevalent due to an aging world population. Although some clinical interventions are available, the solutions are often temporary due to fibrocartilage formation in the long term, which has mismatching mechanical properties. Tissue engineers have been designing osteochondral (OC) scaffolds to induce functional, sustainable regeneration of the articular cartilage. An important characteristic of the OC unit is the presence of continuous gradients such as biochemical or mechanical ones1. Additive manufacturing is preferably used to create porous fibrous architectures to host cells for tissue engineering. To introduce continuous property gradients in these scaffolds, it seems straightforward to continuously extrude an incremental ratio of two biomaterials during the printing process. Although successfully extruded continuous gradients have been achieved with hydrogels2, it remains challenging for solid polymers. Therefore, solid constructs often contain discontinuous gradients with ‘hard’ interfaces, prone to delamination, and moreover a lower degree of biomimicry3. Thus, we aim to design a construct with a continuous material gradient containing specifically addressable groups on the surface to anchor differentiation-inducing peptides. We finally want to create a peptide gradient to gradually direct human mesenchymal stromal cell (hMSC) differentiation, from a chondrogenic towards an osteogenic phenotype, across a construct.
To this end, we synthesized poly(caprolactone) with terminal azide and maleimide groups, which we mixed in a continuous fashion via an in-house developed printhead4. We visualized the continuous material gradient in our constructs with a dye and show control over the gradient distribution. Additionally, mechanical analysis showed that, when comparing a discrete and a continuous gradient, the brittleness of our PCLA is lost in the continuously extruded scaffolds. Furthermore, we successfully modified the surface by reacting complementary dyes on the surface with a density in the 102 pmol/cm2 regime. After a three week cell differentiation study, we observed a strong effect of the differentiation media on hMSC fate. Interestingly, in basic media with our chondrogenic peptide, we observed enhanced (hypertrophic) chondrogenic differentiation in absence of the differentiation factors. Finally, our materials showed good biocompatibility, without severe toxic effects, and abundant tissue regeneration in a subcutaneous rat model.
Overall, we have successfully manufactured a construct with a continuous material gradient via an extrusion-based approach using an in-house developed printhead. Moreover, the continuous gradient construct has more resilient mechanical properties and a modifiable surface, making this fabrication method also interesting for applications beyond OC ones. Our hMSC differentiation study shows promise to influence cell fate in the basic media, but further investigation on the active parameters to achieve larger cell fate differences is required, before moving towards gradual differentiation.
31412761817
"[Introduction] In recent years, biomolecules have been used to functionalize the surface of scaffold materials to support tissue engineering applications. For the functionalized surface for enhancing the cell culture efficiency, peptide coating has been one of the major strategies to provide surface property mimicking the extracellular matrix (ECM). In the past days, the cell adhesion triggered by such surface modification molecules had been mainly understood through the ligand-receptor interactions, such as integrin recognition mechanisms. On the other side, recent mechanobiology studies have revealed that cells are attracted and recognize more varieties of scaffold surface parameters, such as physicochemical properties. Proteoglycan-associated cell interactions are one of such cell-surface interactions. However, compared to the integrin-mediated adhesions, such cell-surface interactions triggered by physicochemical properties have not yet been clarified, since there are too many parameters to investigate. In order to understand the complex combinational effect of physicochemical parameters on the cell adhesion surface, we focused to examine the effect of peptide-coated surfaces, since peptides are functionalization molecules that can be designed in a combinatorial manner. Our research group has successfully obtained several cell-selective adhesion peptides and osteogenesis-promoting peptides by peptide array screening and has been carrying out research on peptide-based materials with high functionality [1,2]. In addition, we have previously reported that amino acids on the surface of materials can change cell adhesion [3]. In this study, we investigated to evaluate the combinatorial effect of peptide and amino acid-coated surfaces by the introduction of high-content image analysis.
[Methods] By combining laboratory automation technology, image processing, and peptide surface modification techniques using DOPA, we developed a new image-based profiling platform to evaluate the delicate differences of cell adhesion profiles. On the multi-well plate, we immobilized RGD peptide with and without the coating of single amino acids (20 variations) and created the surface conditions in which the neighboring physicochemical atmospheres are different with the same RGD coated surface. On such combinatorial property design surfaces, we evaluated the cell adhesion and cell extension rates by fluorescent image processing capturing cytoskeleton responses.
[Results and conclusions] From our data, we found that the surface physicochemical properties created by amino acid coating drastically changed the RGD peptide functionalization effect, and therefore lead us to find the optimum surface property condition to maximize the peptide effect for cell adhesion. Our results suggest that the control of physicochemical property design can empower and stabilize the surface functionalization of cell culture scaffolds.
62825453767
INTRODUCTION: Pelvic Organ Prolapse (POP) is characterized by the descendance of the pelvic organs due to weakening of the pelvic floor. Up to 20% of the women get recurrence after surgery, implying that surgical outcomes are poor due to suboptimal wound healing[1]. Tissue engineering has shown great potential in stimulating regeneration by combining cells, biomaterials and biochemical cues. Polyisocyanopeptide (PIC) hydrogels are synthetic, thermosensitive and highly biomimetic, displaying stress-stiffening behavior similar to other biomacromolecules[2]. Furthermore, the PIC hydrogel can be functionalized with growth factors, like basic fibroblast growth factor (bFGF) to further promote cell proliferation and extracellular matrix (ECM) remodeling[3]. In this study, we developed a bFGF-functionalized hydrogel and investigate its wound healing capabilities in vitro.
Methodology: PIC polymers were synthesized and conjugated with a cell-adhesive peptide GRGDS as previously reported[4]. bFGF was reacted with DBCO-PEG4-NHS and Alexa647-NHS at 3 and 1.5 equivalent respectively in PBS. Next, the bFGF-DBCO was purified over a 10 kDa spin filter and conjugated to the PIC polymer overnight at 4°C. The bioactivity of PIC-bFGF was validated on 3T3 fibroblasts and human adipose-derived stem cells (ADSCs) using the CellTiter-Glo® assay. To assess its wound healing capabilities in vitro, the PIC-bFGF (50 ng/mL), encapsulated with ADSCs was evaluated at day 1, 7, 14 and 28. Cell viability was visualized with a LIVE/DEAD staining. ECM deposition was evaluated by quantifying (Sirius red staining) and visualizing (CNA-OG488) collagen and quantification of elastin (FastinTM Elastin assay).
RESULTS: Dose-response curves were generated to validate the bioactivity of PIC-bFGF (EC50 = 18.3 ng/mL), showing a 3-fold induction in proliferation which is in line with the positive control whereby bFGF is added soluble to the PIC-RGD hydrogel (EC50 = 3.7 ng/mL). ADSCs (EC50 = 17.9 ng/mL) are highly viable in the PIC-bFGF. Furthermore, total collagen amount significantly increases up to day 7 (p<0.001; data not shown) resulting in mature collagen network at day 14. Preliminary data shows no significant increase in total collagen and elastin in the PIC-bFGF versus PIC-RGD yet.
CONCLUSION: PIC-bFGF is bioactive in both 3T3 and ADSCs. Therefore, the PIC-bFGF is very promising and will be studied to investigate whether wound healing can be promoted in vivo. Further research is ongoing to provide more insight in the promoting effect of PIC-bFGF versus PIC-RGD in ADSCs regarding ECM metabolism in vitro.
REFERENCES: 1 Ismail, S. et al., Int Urogynaecol J. 27, 1619-1632 (2016). 2 Das, R. et al., Nat. Mater. 15, 318-325 (2016). 3 van Velthoven, M.J.J. et al., manuscript in preparation (2022). 4Liu, K. et al., ACS. Appl. Mater. Interfaces. 212, 56723-56730 (2020).
83767201967
Parkinson's disease (PD) is a neurodegenerative disease clinically characterized by motor disabilities. Current therapies are not being fully effective. Remarkably, the neuroregulatory molecules secreted by mesenchymal stem cells (MSCs) have been suggested as an alternative therapy. However, direct injection into the brain is the delivery approach that has been used in pre-clinical models. Thus, the main goal of this work was to develop brain-targeting liposomes to deliver the secretome of MSCs and allow a systemic delivery treatment. For that, liposomes were functionalized with lactoferrin (Lf), whose receptors are overexpressed in endothelial cells present at blood brain barrier, as well as in dopaminergic neurons at substantia nigra, in both animal models and PD patients. The particle size distribution, polydispersity index (PDI) and zeta potential of liposomes were assessed by Dynamic Light Scattering (DLS). An in vitro release profile study was performed to predict the in vivo bioperformance of MSC secretome-loaded liposomes. To determine the effect of the liposomes on cell viability, MTS assay was performed using SH-SY5Y cells. The DLS results presented hydrodynamic diameters around 100 nanometers and relatively low PDI values. Liposomes were able to encapsulate the MSC secretome, allowing a sustained release profile. MTS assay demonstrated that liposomes did not induce alterations on viability of SH-SY5Y cells. SH-SY5Y cells were differentiated into a mature dopaminergic neuronal phenotype and exposed to the neurotoxin 6-hydroxidopamine to reproduce an in vitro cell model of PD. Lf-modified MSC secretome-loaded liposomes were able to protect the viability of these cells after neurotoxin exposure. A biodistribution study was performed in mice using Texas Red-labelled liposomes, 5 hours after intravenous administration. The study showed that Lf-modified liposomes were detected on the brain of mice in a higher concentration, when compared to the liposomes that were not functionalized. Remarkably, intravenous treatment of Lf-modified MSC secretome-loaded liposomes were able to improve motor disabilities in a mice model of PD. These results highlight the potential of MSC secretome-loaded liposomes to function as a brain-targeting delivery system therapy for PD.
73296320484
Introduction
Current tissue engineering treatment strategies for end-stage articular cartilage fail to produce long term functional cartilage tissue. Here, melt electrowriting (MEW) is used to fabricate 3D scaffolds with micro-resolution to mimic the structural properties of the native cartilage extracellular matrix[1]. These scaffolds are activated using atmospheric-pressure plasma jet (APPJ), allowing for covalent immobilization of transforming growth factor beta 1 (TGF), an important cytokine for the production and maintenance of cartilage[2], onto the scaffold’s microfibers. It is hypothesized these biofunctionalized scaffolds will support differentiation of mesenchymal stromal cells (MSCs) into the chondrogenic lineage and subsequent cartilage-like matrix deposition.
Methodology
Poly-e-caprolactone MEW scaffolds were fabricated using a 3DDiscovery printer (REGENHU), then activated using a computer-controlled APPJ device[3]. Wettability and x-ray photoelectron spectroscopy were used to assess surface chemistry changes. TGF was subsequently immobilized onto the MEW scaffolds by submersion in solution (1 µg/mL). Characterization of protein immobilization was performed using enzyme-linked immunosorbent assay (ELISA) and immunofluorescence detection. In silico modelling was performed to investigate the potential benefit of immobilizing TGF rather than supplying the TGF in the medium. In vitro experiments were performed by seeding equine-derived MSCs into the scaffolds and then culturing for 28 days. The groups investigated included APPJ-treated constructs with (+APPJ +TGF) and without (+APPJ -TGF) immobilized TGF, as well as untreated constructs with (-APPJ +TGF) and without (-APPJ -TGF) TGF supplied through the culture medium. Cartilage-like formation was quantified with dimethyl methylene blue/picogreen assays for glycosaminoglycan (GAG) production and confirmed with histological analysis, including safranin-O and collagen type I & II immunohistochemistry. Matrix deposition was additionally analyzed using compressive testing.
Results
ELISA results confirmed covalent TGF concentration on the APPJ-functionalized scaffolds while immunofluorescently-labelled TGF was detected visually in microfiber scaffolds (following 0.1% Tween20 washing). The APPJ treatment caused increased hydrophilicity of the scaffolds, resulting in efficient cellular infiltration. In vitro analysis demonstrated that GAG production was significantly enhanced in both the immobilized TGF (+APPJ +TGF) and TGF (-APPJ +TGF) in medium groups, compared to the control groups without TGF supplementation (-APPJ +/-TGF). This finding was further validated by the heightened production of GAGs and collagen type II, observed in histological sections. In addition, in vitro and in silico analysis revealed that immobilized TGF on the scaffolds was more advantageous than TGF supplied directly through the medium. Following the 28-day culture period, the immobilized TGF (+APPJ +TGF) construct group exhibited increased compressive modulus (>3 fold) and GAG production (>5 fold) when compared to the TGF in medium (-APPJ +TGF) construct group.
Conclusions
APPJ-surface treatment facilitated covalent immobilization of TGF onto MEW scaffolds. Immobilized TGF retained bioactivity and promoted the differentiation of MSCs into the chondrogenic lineage. Our results also demonstrate that the new constructs with immobilized TGF support cartilage-like-tissue formation. These findings drive new perspectives for reagent-free, growth factor-functionalized constructs with controllable, high-resolution geometries for guided tissue regeneration.
References
[1] Castilho et al. 2019. Acta Biomaterialia.
[2] Wang, Rigueur & Lyons. 2014. Birth Defects Res C Embryo Today.
[3] Alavi et al. 2020. ACS Applied Materials & Interfaces.
31412712444
"Quantum Sensing for measuring free radical generation in living cells
Romana Schirhagl
Groningen UniversityUniversity Medical Center Groningen, Antonius Deusinglaan 1, 9713AW Groningen, the Netherlands
Free radical generation plays a key role in many biological processes including cell communication, immune responses and maturation. However, radical formation is also a hallmark of ageing and often elevated when cells experience stress. As a result, they are important in many different diseases including cancer, cardiovascular diseases or viral and bacterial infections. However, free radicals are short lived and reactive and thus difficult to measure for the state of the art. Also, since they occur in low concentrations they are difficult to localise. We have circumvented this problem by using quantum sensing which allows nanoscale MRI. Here I would like to show our work in immune cells[1,2]. In these cells we were able to target nanodiamonds to single mitochondria and measure the metabolic activity of the organelles as well as their stress response[1]. We were able to conduct a similar study also in primary cells which where harvested from donors[2]. Despite these donors all being healthy there were drastic differences in how aggressive their dendritic cells reacted towards a stressor. With these measurements we could confirm that these differences that were also evident in other metabolic parameters also were obvious when observing free radical generation.
References
"
31451701155
Introduction
Bone defects above a critical size exhaust the self-healing capacity of bone and in these circumstances intervention is needed to promote regeneration. In recent decades, an increasing number of tissue engineered bone grafts have been developed. However, expensive and laborious screenings in vivo are necessary to assess their safety and efficacy. Rodents are the first choice for initial in vivo screens but their size limits the dimensions and number of bone grafts that can be tested in orthotopic locations. Here, we report the development of a refined subcutaneous semi-orthotopic bone defect model coupled with a semi-automated longitudinal in vivo micro-CT registration method1.
Methodology
The model is based on four bovine bone implants, which are subcutaneously implanted on the back of immunodeficient mice, where bone healing potential of grafts can be evaluated in an artificially created 4mm wide defect in vivo. Crucially, these defects are “critical size” and unable to heal within the timeframe of the study without intervention. Various grafts were prepared to modulate bone regeneration inside the defect; cortical bone chips, tissue engineered constructs consisting of MSC pellets, collagen scaffolds or a combination, and cartilage grafts. Animals were scanned by micro-CT after implantation and then every two weeks until sacrifice at week 8. For analysis of architectural changes in bone structure, a semi-automatic algorithm for longitudinal micro-CT imaging was developed. Micro-CT scans were segmented into binary datasets and afterwards a co-registration method was performed to assess bone morphometric parameters of the implanted defect over time, followed by histological assessment.
Results
Firstly it was assessed if a graft composed of cortical bone chips, the current gold-standard, would increase bone regeneration in the defect of our model. After 8 weeks, empty defects filled their mineralised bone volume, analysed by micro-CT, by 4±3%, while the defects implanted with bone chips showed a net bone volume increase of 26±8%. Histological analysis confirmed that bone formation was stimulated by bone chips. In this study we demonstrated that bone regeneration can be assessed and that osteogenic performance of grafts composed of solely biomaterial, cells or a combination can be studied effectively. Additionally, with the use of image registration a method to analyse the testing pocket only was developed, which was combined with bone morphometric analysis to monitor defect healing longitudinally in the same animal, thus limiting the number of animals needed .
Conclusion(s)
Our novel semi-orthotopic in vivo model suggests that it is possible to overcome some of the current limitations that rodent bone defect models pose, in particular regarding number and size, since in previous mouse critical-size defect models defects with a size of 3mm3 were reported, while in our model each of the four grafts contains a 50mm3 defect. With the semi-automated micro-CT method we have developed a quantitative technique for assessing the testing pocket of our bone defect model. Our data supports that the semi-orthotopic model combined with the novel micro-CT registration method provides an excellent approach for assessment of new biomaterials or tissue engineered constructs for large bone defect repair.
31412714049
Introduction
Extracellular matrix communicates to the nuclear environment by external stimuli that affect Lamina organization and chromatin distribution [1]. It is known that integrins transmit mechanical stimuli to the actin filaments in turn connected to the LINC complex consisting of Nesprin and SUN proteins [2]. SUN proteins are supposed to link the nuclear Lamina [2], that rearranges according to external impulse. Despite the relevance of SUN1-Lamin A/C in nuclear reshaping, their binding domains have not been identified yet.
We here designed computational protocol to study the SUN1-Lamin A/C binding domain that plays key role in lamina sensitivity to external load.
Methodology
Computational protocol was designed to reconstruct the SUN1 nuclear domain (1-315aa according to Uniprot server). The secondary structure of SUN1 was predicted via a consensus study of eight different servers while I-TASSER server was used for tertiary structure. To stabilize the 3D selected model, molecular dynamic annealing test was implemented via NAMD software (from 300K to 500K deltat=5ns, deltaT=5K explicit water box; Charmrun force field). To predict SUN1-Lamin A/C interaction, Haddock server was used to perform molecular docking analysis between SUN1 predicted model and all the X-ray solved domains of Lamin A/C in order to cover its full length. Combining affinity energy values with cluster dimension we selected the most reliable 3D structure of SUN1-Lamin A/C complex. H-bonds analysis was performed with VMD server.
Results
The secondary structure of SUN1 N-terminal domain was estimated with 87% of confidence value and it was used to predict 3D model. 3D model reliability was supported by 65.4% of similarity with expected secondary structure and its high stability during annealing simulation (90.2% of similarity). Testing SUN1 domain affinity to all the solved Lamin A/C domains we identified the Ig-fold domain (1IFR) as the most affine one due to its high energy interaction as supported by 4 H-bonds (aa 295,303,140,141 and 456,490,496 for SUN1 and Lamin, respectively).
Conclusion
We here estimated SUN1-Lamin A/C interaction structure required to elucidate its key role in force transmission from extra cellular matrix to the nucleus. The reliability of the developed protocol supported by the consistency between our data and the literature ones, introduces our strategy as new promising tool for 3D reconstruction of proteins [3]. Moreover, the SUN1-Lamin A/C complex reconstruction represents the first step in deepen external force effects on nuclear shape. Considering the high occurrence of laminopathies-related single point mutations in Lamin A/C Ig-fold domain, we suggest that these mutations may alter SUN1-Lamin A/C interaction with consequences on nuclear sensitivity and thus gene activation. Further computational analyses could verify this hypothesis and X-ray technique will be used to validate the SUN1-Lamin complex.
References
[1] Remuzzi A et al. Cells. 2020;9(8):1873. doi:10.3390/cells9081873
[2] Donnaloja F, et al. Cells. 2020: 24;9(5):1306. doi: 10.3390/cells9051306
[3] Haque F, et al. J Biol Chem. 2010 29;285(5):3487-98. doi: 10.1074/jbc.M109.071910.
31412714589
Introduction: Liver fibrosis is caused by progressive accumulation of extracellular matrix (ECM) coupled with chronic inflammation. Advanced liver fibrosis results in increased risk of liver cancer, cirrhosis, portal hypertension and liver failure, resulting in the need for liver transplantation. Studies of the mechanisms that promote fibrosis are necessary to understand this multi-faceted disease and for the development of novel therapeutic targets. Traditional cell culture models often lack the immune cell compartment and the ECM, failing to recapitulate the complex fibrotic microenvironment, which is highly immune-mediated. Bioengineering allows for the development of disease models to study complex diseases, such as liver fibrosis, where both cellular and extracellular microenvironment components contribute to the pathology. Here, we describe two novel bioengineered models which incorporate the dynamic co-culture of circulating immune cells in decellularised liver ECM-scaffolds, supported by two custom-made perfusion bioreactors, and we demonstrate how these models allow to explore immune responses to fibrotic liver ECMs.
Methodology: We developed two custom-made bioreactors for whole rat livers or human tissue segment culture (WL and HuTS bioreactor respectively). Decellularised normal and fibrotic rat and human liver tissues were obtained following established detergent and enzymatic treatment protocols. Human peripheral blood mononuclear cells (PBMCs) from healthy donors, were perfused in WL or HuTS bioreactors in absence (baseline characterization) or presence of decellularised normal or fibrotic liver ECM-scaffolds. Circulating cell viability, phenotype, and cytokine production were assessed (via FACS and Luminex) in comparison to static culture conditions. The gene expression and phenotype of PBMCs present inside the scaffolds were examined via qPCR and immunofluorescence.
Results: The custom-made bioreactors supported perfusion of PBMCs for up to 7-days without altering cell viability and phenotype in comparison to conventional static culture conditions. Bioreactor culture also improved cell distribution inside the scaffolds compared to static cultures, suggesting that perfusion culture better promotes cell-ECM interactions.
FACS analysis of circulating PBMCs showed that co-culture with both healthy or fibrotic liver matrix-induced an increase in the relative proportion of NKT cells and B cells and that this increase was greater when cultured with fibrotic livers. When cultured with fibrotic scaffolds, the number of circulating T cells decreased and interestingly, monocytes sub-populations changed in response to healthy or fibrotic liver matrices.
Immunofluorescent staining on sections of perfused ECM-matrices revelated that immune cells infiltrated the ECM-scaffolds. These were mostly composed of monocytes and macrophages, with higher relative proportion in fibrotic livers, indicating fibrotic ECM-induced homing of monocyte-derived macrophages, an event that recapitulates the in vivo fibrotic microenvironment.
Cytokine analysis revealed that the ECM triggers the release of innate and adaptive immune system cytokines and those related to pro-regenerative immune responses.
Conclusion: Here we show the validation of two innovative bioreactor-based systems which allow for the perfusion of immune cells through decellularised liver matrices. This perfusion system proved to be suitable for the study of immune cell interaction with normal or fibrotic ECM, and more in general, with bioengineered multi-cellular liver constructs, and showed that mechanisms of chronic inflammation observed in fibrosis can be replicated in vitro.
41883621066
Introduction:
Regenerative medicine is focusing the attention on immune engineering strategies taking advantage of biomaterials to influence stem cells fate and to stimulate the production of immune modulatory factors to be employed in cells free treatment (1). The Graphene Oxide (GO) was proved to be able to affect stem cells behaviour and to modulate their immune response (2). According to this evidence, the present project was designed to produce secretome with immune modulatory potential by combining GO and/or LPS with Ovine Amniotic Epithelial Stem Cells (oAECs), which exhibited teno-regenerative and high immunomodulatory properties in vitro and in vivo (3), to be used in tendon regenerative medicine.
Methodology:
AECs isolated from the amniotic membrane of ovine fetuses (4) were seeded in standard condition (AEC) or with 25µM of progesterone (AEC+P4) (4) or on GO functionalized cover slides (GO). At 70% of confluence, the cells were treated with 1 μg/ml LPS (4) for 1h (AEC+LPS; AEC+P4+LPS, GO+LPS), and incubated for 24h in the Serum Free media. At the end of the experiment, the Epithelial Mesenchymal Transition (EMT) process was assessed within the different tested group by analysing Cytokeratin 8 (CYTO8) and Vimentin (VIM), epithelial and mesenchymal markers respectively, protein expressions and by evaluating the involvement of pSMAD2 and SMAD2/3 pathways. The collected conditioned media (CMs) were used to analyse the profile expression of 40 immunomodulatory cytokines by Inflammation Antibody Array Membrane.
Results:
The results demonstrated that GO alone or with LPS (GO+LPS) induces AEC morphology changes shifting the protein patterns expression toward the mesenchymal phenotype, as showed by negativity to CYTO8 and positivity to VIM. This data suggests the GO accelerated EMT process in AEC and AEC+LPS. In contrast, P4 was able to maintain the epithelial phenotype with higher CYTO8 expression in AEC (AEC+P4) (4) also after inflammatory stimulus (AEC+P4+LPS). Moreover, the upregulation of pSMAD2/SMAD2 proteins ratio was observed in GO group and especially in GO+LPS samples compared to those present in AEC, AEC+LPS, AEC+P4 and AEC+P4+LPS (p<0.001) supporting the EMT transition GO-dependent. Furthermore, preliminary inflammatory array assay results on cells CMs suggested that GO was able to modify the cytokines expression profile in cells secretome, by reducing in particular the anti-inflammatory IL10, IL11, IL13 TIMP2 (tissue metalloproteinase inhibitor 2) and CXCL9 (recruiter of leukocytes) chemokine release highly induced in AEC after LPS treatment.
Conclusions:
These preliminary data demonstrated that GO accelerates the EMT process in AEC altering their immune response and affecting the release of immune factors. In order to employ these secretomes in regenerative medicine, more experiments are needed to deepen the knowledge of the link between EMT and immunomodulation and to evaluate their biological effects on immune cells.
Acknowledgement: This research was founded by Perspective for Future Innovation in Tendon Repair H2020MSCA-ITN-EJD-P4 FIT- Grant Agreement ID: 955685.
Bibliography:
1. Rahmati, M et al., Adv Exp Med Biol. 1119:1-19. (2018)
2. Maleki, M et al., BioMol Concepts; 11: 182–200. (2020)
3. Barboni, B. et al., Cell Transplant. 21.11: 2377-2395. (2012)
4. Canciello, A. et al., Sci. 7.1: 1-14. (2017)
94238120655
Cell-extracellular communication in granular systems might be explored for tissue engineering and to understand and mimic physiological responses. Particulate systems can be designed as attractive platforms with free movement controlled mainly by intracellular forces and cell migration. We here explore the possibility that the size of the particles composing these systems might have a role in cell response in a sense that a successful long-term cell-particle adhesion might require a minimum traction force for the bond reinforcement. In this work, commercial polystyrene microspheres (type I-collagen coated) are freely assembled and loosely packed as a quasi-3D granular system in a liquid environment. Three size ranges of microspheres (14-20 μm, 38-45 μm, 85-105 μm) were chosen to evaluate the response of human mesenchymal stem cells derived from the adipose tissue (hASC) in these spherical substrates. Cellular characterization was evaluated from 4 hours to a week via metabolic activity, cell adhesion and morphology. Experimental data indicates that objects with increasing diameters (from ~40 μm to ~100 μm) are able to sustain cell adhesion and promote proliferation within seven days of culture. On the other hand, the less explored size comprising 14-20 μm microparticles is more susceptible to cell-mediated mobility, arresting a cell-ECM reinforcement causing early cell detachment. Mechanistic experimental controls through particle sintering allowed to overcome particle mobility and promote cell adhesion in small particles (14-20 μm) as well as increased viability. Weakening cell contractility in larger microspheres (85-105 μm) difficulted the adhesion reinforcement contributing to cell detachment in an otherwise favourable substrate for long-term cell maintenance. Furthermore, an in-silico model addressing pertinent mechanisms of cell attachment to particle beds was developed, corroborating particle free and fixed scenarios. Combining such models with biological assessments could ease the understanding and design of innovative platforms for healthcare-associated problematics.
The authors acknowledge the financial support given by the Portuguese Foundation for Science and Technology (FCT) with the project “CellFi” (PTDC/BTM-ORG/3215/2020), and the European Research Council for the project “ATLAS” (ERC-2014-AdG-669858). This work was developed within the scope of the project CICECO-Aveiro Institute of Materials, UIDB/50011/2020, UIDP/50011/2020 & LA/P/0006/2020, financed by national funds through the FCT/MEC (PIDDAC) and when appropriate co-financed by FEDER under the PT2020 Partnership Agreement. We also acknowledge financial support from the Portuguese Foundation for Science and Technology (FCT) under Contracts no. PTDC/FIS-MAC/28146/2017 (LISBOA-01-0145-FEDER-028146), PTDC/FIS-MAC/5689/2020, UIDB/00618/2020, UIDP/00618/2020, CEECIND/00586/2017, CEECIND/03605/2017, 2021.04817.BD and SFRH/BD/143955/2019.
94238145927
Smart materials, that react in a controllable and reversible way to external stimuli by varying a specific physical or chemical quantity, show great potential for the development of advanced biomedical strategies, including biosensing, tissue regeneration and repair, immuno- and cancer therapy.1 Among the different types of smart materials, piezoelectric ones, that convert mechanical solicitations into electrical potential variations and vice versa, represent suitable candidates to induce specific cell behaviors through electric cues, mainly by improving biomimicry of the cell microenvironment.2
Although the presence of piezoelectric properties in different tissues is known, their specific influence on cells is still unknown. Mesenchymal stem cells (MSCs) have been found to show increased formation of focal adhesions when cultured on negatively charged substrate.3 Triggering the piezoelectric effect, MSCs show increased differentiation on positively and negatively charged surfaces compared to those cultured on neutral substrates, exposing the importance of combined electrical and mechanical stimulation for bone-regeneration.4,5
One of the main obstacles for translating these materials into clinical practice is the lack of proper understanding of the mechanisms controlling the cellular response to them as well as to the fact that biomaterial-cell interactions are often mediated though proteins, fact that has not been properly addressed in the case of electroactive biomaterials. In fact, the behavior of cells is strongly influenced by conformation of extracellular-matrix proteins,6 and the electrostatic forces of the surfaces to which these proteins adhere, determine their conformation. Thus, we hypothesize that the impact of electrical stimulation on cells can be also tuned by the effect of the physicochemical properties of the biomaterials on the proteins that cover them. However, the influence of electrical stimulation on the material-protein interface remains largely undescribed.
In the present work, we explore the effect of the electric cues on the deposition of extracellular proteins on piezoelectric poly(vinylidene fluoride), PVDF, surfaces with distinct net surface charge, and how these differences affects MSC fate. Using microscopic, spectroscopic, and biochemical techniques, we have uncovered large differences in the deposition dynamics, surface coverage and supramolecular organization of collagen and fibronectin as a function of the electrical charge of the surface to which they adhere. Specifically, positively and negatively charged PVDF surfaces promote proteins adsorption, showing higher amount of protein immobilized on the surface compared to neutral PVDF. Regarding cell response, our semi-automatic analysis of fluorescently-stained MSCs, revealed significant differences not only in MSC spreading and nuclear area, but also in the focal adhesion density.
The presented results allow to regulate the structural features of the deposited extracellular proteins through the control of the surface charge, in order to guide cellular behavior and to obtain specific responses.
References:
1.Langer, R. et al., Nature, 428, 487-492 (2004).
2. Kapat, K. et al., Adv. Funct. Mater., 30, 1909045-1909067 (2020).
3. Cavalcanti-Adam, E. A. et al., Biophys. J. 92, 2964–2974 (2007).
4. Sobreiro-Almeida, R. et al., Int. J. Mol. Sci. 18, 2391-2408 (2017).
5. Ribeiro, C. et al., J. Biomed. Mater. Res. A. 103, 2172–2175 (2015).
6. Trappmann, B. et al., Nat. Mat., 11, 642-649 (2012).
20941830804
Introduction: Among scaffold preparation approaches, biofabrication can provide tridimensional scaffolds with precise and defined architectures that can promote tissue integration and neoangiogenesis after implantation (1). Therefore, the evaluation of tissue ingrowth and vascularization of 3D printed scaffolds is a crucial step in establishing a functional scaffold that could direct the successful tissue regeneration (2). The hen chorioallantoic membrane (CAM) has been proposed as a biorelevant alternative to animal studies for the assessment of implantable scaffolds in vivo. However, current approaches to quantify integration and vascularization, such as histology, lack of spatial resolution. As an alternative, microCT can be helpful to study the extent of tissue integration and vessel architecture within porous implants (3). In this work, we developed a simple and non-destructive method to evaluate tissue ingrowth and neoangiogenesis in 3D printed scaffolds using a CAM model, and further validated in two distinct porous architectures.
Methodology: PLA scaffolds (10 mm in diameter, 4 mm height) with 500 µm pore size designed with/without open lateral porosity were prepared by 3D printing. Sterilized scaffolds were placed on the CAM of fertilized eggs previously incubated for 7 days (Coren, Spain) and incubated at 37 °C for 5 or 7 days. In order to quantify the scaffold integration and vascularization, the CAM tissue surrounding the scaffolds (25 mm in diameter) was fixed in paraformaldehyde, extracted using a scalpel, and immersed in lugol 0.1% for 4 h. Then, samples were washed in PBS, water excess removed and then, scanned at 5 µm voxel resolution using a Skyscan 1272 microCT (Bruker, Belgium). Tissue ingrowth was obtained from the structural changes of structure volume before and after incubation. Finally, the structure and porosity of the reconstructed samples and the new vessels developed within the structure of the scaffolds were analyzed using CTAn software (Bruker, Belgium). Histological analysis was carried out in paraffin-embedded samples after microCT analysis to further quantify new tissue ingrowth and vessel formation and dimensions.
Results: scaffolds designed with open lateral porosity showed a significant higher integration with the CAM since tissue migrated towards the inner regions of the scaffolds not only from the underneath but also from the side surface of the scaffolds. The incubation with lugol allowed for the precise visualization of vessels and new tissue growing within the scaffolds due to the distinct contrast-enhanced radiopacity of vessels, tissue and scaffold components, as validated by histological and histomorphometrical analysis.
Conclusion: The developed methodology enabled the precise evaluation of tissue integration and vascularization in an embryonic in vivo model. As a non-destructive technique, the developed microCT method is a robust approach for initial in vivo screening of novel porous biomaterials, while complying with the principles of 3Rs.
References:
1. Agarwal, R. and Garcia, A.J. Adv Drug Deliv Rev 94, 53-62 (2015).
2. Velasco, M.A. et al., BioMed Res Int, 729076 (2015)
3. Moreno-Jimenez, I. et al., Tissue Eng Part C Methods 23, 938-952 (2017)
Acknowledgements:
This research was funded by Xunta de Galicia (ED481D-2021-014).
62825433604
"Introduction: Pancreatic ductal adenocarcinoma (PDAC) is one of the most fibrotic tumors, which can possess up to 90% tumor stroma of the total tumor mass (1). The tumor stroma is comprised of cancer-associated fibroblasts (CAFs), extracellular matrix (ECM) and many immune cells. The physical and biochemical characteristics of the tumor stroma control cancer cell proliferation, invasion, and metastasis. Also, the tumor stroma enhances intratumoral solid stress leading to the compression and collapse of blood vessels and also becomes a physical barrier which in turn prevents delivery of chemotherapy. Currently, there is lack of relevant in vitro models that are able to replicate these mechanical characteristics of PDAC (2).
Methodology: A multi-layered vascularized 3D PDAC model was developed consisting of primary human pancreatic stellate cells (PSC) in a collagen/fibrinogen (Col/Fib) matrix. A central endothelialized channel was introduced to study the cell-mediated contraction of vasculature in PDAC. The activation of PSCs due to mechanical and biological stimulation was studied using contraction assay and gene analysis. The clinical relevance of the model was studied for PDAC specific gene markers and compared with publicly available patient data. The effects on the blood flow inside the channel was determined using computational fluid dynamics (CFD) simulations. The effect of AV3 peptide (3), an integrin inhibitor developed by us, was tested for its effect on stroma and vasculature in this model and the effects were correlated with in vivo data in mice.
Results: PDAC samples from patients and mouse tumor models showed an abundant stroma and collapsed blood vessels. Our 3D model showed mimicking of the vasculature in the tumor tissue and adjunct healthy tissue. PSCs differentiated into myofibroblastic CAFs leading to high contraction of the matrix and upregulation of gene markers (ACTA2, Col-1a1, PDGFbR, HAS2 and CDC42). Interestingly, these genes were also positively correlated in PDAC clinical samples. The CFD simulation analysis revealed a clear pressure drop within the compressed vessels with high intravascular pressure before the compression. Furthermore, the flow velocity drastically increased in the compressed vessels. Interestingly, treatment with AV3 suppressed the compression of vessels which according to CFD simulation should result into enhanced drug delivery. The latter was proven in stroma-rich PANC-1+PSC and MiaPaCa+PSC tumor models in mice. We found that treatment with AV3 reduced desmoplasia, decompressed vasculature and enhanced delivery of chemotherapy in vivo.
Conclusion: Altogether, our 3D PDAC model provides a better understanding of mechanical characteristics of PDAC in view of stroma-induced vasculature compression as well as allows for evaluating novel anti-stromal therapeutics for the treatment of fibrotic tumors.
References:
1) Heinrich MA,….Prakash J. Translating complexity and heterogeneity of pancreatic tumor: 3D in vitro to in vivo models. Advanced Drug Delivery Reviews 2021 Jul;174:265-293.
2) Rodrigues J, Heinrich MA, Teixeira LM, Prakash J. 3D In Vitro Model (R)evolution: Unveiling Tumor-Stroma Interactions. Trends in Cancer. 2021 Mar;7(3):249-264.
3) Kuninty PR, …Prakash J. ITGA5 inhibition in pancreatic stellate cells attenuates desmoplasia and potentiates efficacy of chemotherapy in pancreatic cancer. Science Advances. 2019 Sep 4;5(9):eaax2770."
83871204329
Extrusion bio-printing is the most direct and inexpensive method for printing three-dimensional cell models. This technique provides interesting solutions to generate more complex architectures than the already existing 3D models but still presents significant drawbacks that once solved will improve its field of application in regenerative medicine and advanced 3D biological models. To print complex structures with a larger volume, it is necessary to have available an important quantity of cells. There is also significant cellular stress during the printing process when extruding the ink, due in part to shear stress, which can induce the apoptosis or inability of the deposited cells.1 One of the common answers to these problems is the use of soft hydrogels having little mechanical strength, implying a lack of structural integrity of the printed designs. For this purpose, we produced new hyper porous micro-scaffolds of PLGA reducing the shear stress experienced by printed cells.
We developed a novel method of producing porous PLGA with a simple double emulsion and enhanced the cell adhesion of the particles’ surface by adding two types of coating. With these micro-scaffolds, we put in place a 3D cell culture method using said micro-scaffolds to improve cell proliferation before printing by acting as micro-carriers. We combined these cellularized micro-scaffolds with a bottom-up method of bio-printing of complex 3D structures and integrated them into various types of bio-inks, engineered either for the maximum survival of cells or for the construction of highly complex 3D structures. Ultimately, these micro-scaffolds are capable of protecting the cells during the bio-printing process by absorbing most of the shear stress inherent to all extrusion bio-printing. We also printed more complex structures, composed of structured layers of stained mesenchymal and cancerous cells, thus creating an organoid. The observation in time of the movement of cells inside the organoid allowed us to quantify the interaction and migration of cells with and without our micro-scaffolds.
We produced Polylysine or Collagen coated PLGA micro-scaffolds with an average size of 100 µm in diameter with a porosity of 25-45 %. Our results show an augmentation up to 400% of cell proliferation when cultured with said particles. The viability after printing is augmented with the use of our micro-particles, with a survival rate between 85 and 91% with our particles and between 75 and 83% without the micro-scaffolds. When printing more complex structures with co-cultures of tumorous cells with mesenchymal cells, the presence of our micro-scaffolds increase the migration of stem cells towards the tumorous cells.
Finally, the overall results offer new insights regarding bio-printing and cellular proliferation and migration in response to the presence of micro-scaffolds. Our new process promotes high cell productivity and viability before and during bio-printing. The use of our micro-scaffolds would make it easier, faster and more efficient to produce three-dimensional cellular structures and to analyze the behaviour and interactions of different cell types in this 3D environment.
73296306486
Understanding the key players in cancer progression is essential for the development of effective therapies. Aiming to pinpoint the roles of biochemical and biophysical factors involved in malignancy, tissue engineers developed in vitro cancer models of increasing complexity [1]. Three-dimensional (3D) bioprinting techniques were extensively used in this endeavor [2,3] due to their ability to create biomimetic spatial patterning of several cell types that coexist with cancer cells in the tumor microenvironment (TME), including tumor-associated fibroblasts, immune cells, mesenchymal stem cells, adipocytes, and vascular cells [4]. Nevertheless, bioprinted cells rarely remain where the bioprinter delivers them; they remodel their extracellular matrix and take advantage of their motility to establish firm bonds with other cells and/or biomaterials [5]. Therefore, in the present work, we investigated structure formation in bioprinted models of the TME both experimentally and computationally [6]. We used extrusion-based bioprinters to build models of the TME. SK-BR-3 breast cancer cells dispersed in a hydrogel droplet (106 cells/mL) were embedded in the same hydrogel (Bioink, CELLINK, Sweeden) loaded with tumor associated fibroblasts (TAFs − 5×105 cells/mL) and peripheral blood mononuclear cells (PBMCs − 5×105 cells/mL) harvested from breast cancer patients. The bioprinted tissue constructs were cultured in vitro for two weeks and cryosectioned for histological evaluation. Hoechst staining demonstrated that cells remained viable and remodeled the hydrogel. Hematoxylin and eosin (H&E) staining of histological sections, prepared at various time points, indicated that cells proliferated and formed heterotypic aggregates of malignant and peritumoral cells. To investigate the interactions responsible for the observed phenomena, we built lattice models of the bioprinted constructs and simulated their evolution using Metropolis Monte Carlo methods [6]. The computational model was formulated on a 3D cubic lattice, representing the biological system, at single-cell resolution, in terms of 4 types of particles: tumor cells, peritumoral cells, volume elements of the hydrogel, and volume elements of the cell culture medium. Based on the differential adhesion hypothesis, computer simulations reproduced most features of the experimentally observed structure formation, but did not account for the superficial localization of the heterotypic aggregates. Depending on model parameters, peritumoral cells wrapped or infiltrated cancer cell aggregates, as expected from TAFs and immune cells, respectively. Despite their limited complexity, the tissue constructs developed in this study could be used to establish co-culture conditions for cancer cells, TAFs, and PBMCs. Future investigations should consider model tissues incorporating perfusable channels with endothelial cell lining. Also, the computational model needs to be extended to describe the self-assembly of different cell types present in the native TME.
References:
[1] Bray, L.J., Hutmacher, D.W., Bock, N., Front. Bioeng. Biotechnol. 7, 217 (2019)
[2] Li, J., Parra-Cantu, C., Wang, Z., Zhang, Y.S., Trends Cancer 6, 745–756 (2020)
[3] Datta, P., Dey, M., Ataie, Z., Unutmaz, D., Ozbolat, I.T., Precision Oncology, 4, 18 (2020)
[4] Turley, S.J., Cremasco, V., Astarita, J.L., Nat. Rev. Immunol. 15, 669–682 (2015)
[5] Robu, A., Aldea, R. et al., BioSystems 109, 430–443 (2012)
[6] Bojin, F., et al., Micromachines 12, 535 (2021)
52354508644
Introduction: Current primary liver cancer models fail to truly encompass the human tumour immune microenvironment, exacerbating a recognised discord between the preclinical and clinical successes of emerging (immuno)therapeutics. The organotypic 3D culture of human precision-cut tumour slice (PCTS) is a cancer explant model which retains tumour specific histoarchitecture, aetiological background, disease phenotype, resident immune landscape, and checkpoint expression for up to 7 days ex-vivo. Our study aims to advance culture conditions of PCTS using a proprietary Multi-well Plate (MuPL) perfusion bioreactor to extend culture lifetime and allow perfusion of immune cells through PCTS, in order to validate PCTS as a tool to assess patient-specific therapeutic responses.
Methodology: PCTS generated from primary liver cancer (hepatocellular carcinoma and cholangiocarcinoma) were treated with approved single agent or combinatory checkpoint inhibitor monoclonal antibody (CPI-mAb) or kinase inhibitor (KI) therapy for up to 7 days. PCTS/immune cells co-cultures in the MuPL bioreactor were longitudinally assessed for viability, histology, and tissue integrity. Therapeutic response was determined by evaluating histology (H&E and Sirius red), apoptosis/cell death (TUNEL, lactate-dehydrogenase release, cytokeratin 18), and proliferative capacity (PC; Ki67). Gene expression was assessed using QuantiGene RNA Assay. Resident immune cells were assessed by immunofluorescence and FACS.
Results: PCTS and immune cells co-cultured in the MuPL bioreactor maintained viability, structural integrity and histoarchitecture for >7 days. Doxorubicin was used as a positive cell death control in all treated patients, decreasing PCTS viability and PC by day 5. Compared to monotherapy, nivolumab (CPI-mAb) + regorafenib (KI) therapy decreased the tumour-to-stroma ratio and PC in all patients by day 7. Also, significantly increased apoptosis was detected in one patient, who comparatively showed higher checkpoint expression including PD-1, PD-L1 and CTLA-4. Other combinatorial immunotherapies, including atezolizumab + bevacizumab and nivolumab + ipilimumab (CPI-mAbs), reduced PC without affecting histology or viability. The overall immunotherapeutic response was patient-specific.
Conclusion: PCTS can be used as a powerful tool to study personalised responses to (immune)therapeutics. In addition, PCTS can be successfully cultured in our proprietary perfusion system, recapitulating tumour-immune cell interactions, allowing assessment of response to cell and vaccine therapy ex-vivo.
41883633129
Introduction
3D bioprinting has emerged as a promising technology for fabricating artificial tumors as it allows the fabrication of complex models recreating tumor physiology. The importance of the extracellular matrix (ECM) in tumor progression, cancer cells and stromal cells crosstalk and drug resistances, has motivated the development of more biomimetic tumor-ECM bioinks that recapitulate the high complexity of the ECM.1 In this regard, decellularized tissues-derived matrices (TDMs) can provide the native tissue biological cues, but its inadequate mechanical properties prevent their bioprinting. The aim of this work is to develop a breast TDM-like bioink suitable for bioprinting breast cancer models without the need of a sacrificial material.
Methodology
Porcine breast tissues were decellularized and delipidated, and its composition was studied. TDMs pre-gels were fabricated by digesting it with pepsin and neutralizing the pH. The addition of rheological modifiers into the bioink was also assayed. TDM bioinks were printed with a 3D bioplotter (RegenHU) and then crosslinked. The bioinks were further tuned by incorporating an ECM protein overexpressed in breast cancer, Collagen type 1 (Col1). The shape fidelity, printability and rheological properties of the bioinks were characterized and the hydrogels Young modulus was measured. For bioprinting artificial breast tumors, cell-laden bioinks were prepared by dispersing breast cancer cells (BCCs) or mesenchymal stem cells in the bioink. Cellular survival, proliferation, morphology, and the expression of adhesion molecules were studied. The bioprinted hydrogels were used to study the efficacy of anticancer drugs.
Results
Breast TDMs were successfully decellularized and rich in glycosaminoglycans and collagen. The addition of rheological modifiers allowed the TDM bioprinting without the requirement of any sacrificial material. BCCs were able to proliferate in TDM bioprinted scaffolds and form spheroids with a low expression of e-cadherin. The addition of Col1 improved the bioink printability, increases cellular proliferation and reduces doxorubicin sensitivity. TDM bioinks also allowed BCCs and stromal cells bioprinting and therefore could be used to fabricate artificial tumors.
Conclusions
Taken together, we have proved that TDM bioinks could be used for bioprinting artificial breast tumors closely recreating the tumor ECM.
Acknowledgement
European Union’s Horizon 2020 (Marie Skłodowska-Curie 712754), Spanish Ministry of Economy and Competitiveness (SEV-2014-0425, CEX2018-000789-S), FEDER and Spanish Ministry of Science, Innovation and Universities (RTI2018-096320-B-C21; MAT2015-68906-R), Spanish Ministry of Economy, Industry and Competitiveness (EUIN2017-89173), European Commission (JTC2018-103).
Reference
1. Bahcecioglu, G. et al., Acta Biomater. 106, 1-21 (2020).
94238127237
Treatment options for triple-negative breast cancer (TNBC) are limited. Current 2D cancer models fail to accurately model the tumour microenvironment of breast cancer. Alterations to extracellular matrix (ECM) composition have been shown to play a key role in the epithelial-mesenchymal transition (EMT) process involved in breast cancer progression (1). This highlights the need for the development of a representative in vitro 3D model, in which to study TNBC behaviour and identify new treatment targets. This project aims to develop a collagen-based scaffold model composed of matrix components of breast tissue, including glycosaminoglycans, hyaluronic acid (HyA) and chondroitin sulphate (CS), both elevated in tumours, to investigate their role in the EMT process, within TNBC.
Collagen-based scaffolds comprised of varying concentrations of HyA, or CS were fabricated using previously optimised protocols (2). Scaffold characterisation was performed using SEM, porosity measurements, and mechanical testing. AlamarBlue and DNA assays were performed to assess the metabolic activity and growth of TNBC cell lines, MDA-MB-231 and MDA-MB-436, in comparison to normal breast epithelial cell line, MCF10a. The migratory ability of TNBC cells in 3D was assessed with H&E staining. A cytokine array and qPCR were performed to assess the effect of varying HyA and CS concentrations on MCF10a and MDA-MB-231 behaviour and to determine expression levels of markers associated with the EMT process.
All scaffolds were highly porous and had a uniform pore distribution, with an average stiffness of 1kPa and are therefore within the stiffness range of cancerous breast tissue (1kPa-4kPa). Each scaffold type exhibited huge biocompatibility for each cell line. TNBC cells were more metabolically active on CHyA scaffolds than CCS scaffolds and TNBC cells proliferated at a faster rate than MCF10a cells. Each scaffold type supported the migration of TNBC cells. Change in HyA or CS concentration did not affect cell proliferation but altered the expression of pro-inflammatory cytokines. Interestingly, an increase in HyA increased pro-inflammatory cytokine expression and an increase in CS decreased pro-inflammatory cytokine expression in MDA-MB-231 cells. The effect of glycosaminoglycan type and concentration on cytokine expression requires further investigation. Alterations in the expression of EMT associated markers differed with an increase in HyA and an increase in CS concentration, findings which require further investigation.
Collagen-based scaffolds composed of varying ECM components have been developed. Altering the mechanical stiffness of the collagen-based scaffolds within a range that represents cancerous breast tissue is achievable. Each scaffold is highly porous and supports cell viability and proliferation. Varying concentrations of HyA and CS alters the cytokine expression profile of MCF10a and MDA-MB-231 cells, highlighting the effect of changes to the ECM composition on cancer progression. This finding will be further investigated in future studies. In summary, the collagen-based scaffolds have the potential to mimic the ECM of breast tissue and have the capacity to be used as 3D models for breast cancer research.
Acknowledgements: Funded by Health Research Board
References:
(1) Scott LE, Weinberg SH, Lemmon CA. Front Cell Dev Biol, (2019)
(2) Haugh MG et al., Tissue Eng - Part A,1201–8 (2011)
31412737539
Introduction
Lung cancer is the leading cause of cancer mortality with poor prognosis due to late stage diagnosis, drug resistance and high risk of relapse. There is a high need for tissue engineered 3D models that can recapitulate tumor heterogeneity and complexity to understand the cellular mechanisms leading to lung tumorigenesis, metastasis and drug responses. Future of precision oncology is envisioned with the ability to perform therapeutic tests on pre-clinical tumor models that can faithfully recapitulate the native tumor microenvironment combined with the use of patient-derived cells that would inform the treatment decision making. Patient-derived xenografts (PDX) and tumor-derived organoids (TDO) have emerged as methods to provide reliable pre-clinical models that account for genomic diversity and cellular heterogeneity especially for cancers with lack of established cell lines. However, PDX models are costly with low tumor formation rate that limit medium to high-throughput screening approaches. On the other hand, TDO allowed in vitro culturing of both solid tumor and liquid biopsy-derived cells in Matrigel and revealed preservation of genomic diversity. Nevertheless, Matrigel culturing hinders a systematic study of the role of tumor extracellular matrix (ECM) components and mechanics on tumor cell growth, phenotype, metastatic potential and drug responses.
Methodology
Lung tumors are marked by an increase in tissue stiffness as well as changes in the biochemical composition (i.e. increase in cell-instructive ECM ligands). We developed a biomaterial-based human in vitro human non-small cell lung adenocarcinoma model to study the effect of aberrant tumor matrix characteristics in a controlled manner on the phenotype and malignancy of pulmonary epithelium. We built models for healthy and tumorous lung matrix from hydrogels of decellularized native lung extracellular matrix (ECM) with differing ligand content and tissue stiffness. We then encapsulated non-small cell lung adenocarcinoma cells (A549) in both healthy-like (low stiffness, low ligand content) and tumor-mimetic lung matrices (high stiffness, high ligand content) and monitored cell growth and phenotype over 4 weeks. We performed analyses on gene (qRT-PCR, RNAseq) and protein expression (Western blot, immunofluorescence) to investigate the signaling mechanisms involved in the tumor matrix-mediated effects on cell growth and phenotype. We performed loss of function (small molecule, shRNA) and overexpression studies to validate proposed mechanisms triggered in the cells in the respective engineered microenvironments.
Results
Lung tumor cells in tumor-mimetic matrices demonstrated significantly higher cell growth with increased number of larger and invasive-looking (decreased circularity) colonies. Gene and protein expression analyses revealed an upregulation of the epithelial-mesenchymal transition program with significantly higher expression of known markers including N-Cadherin, Zeb-1 and Twist-1 as well as lung adenocarcinoma markers such as TTF-1 when compared to heathy-like microenvironments. Mechanistic studies revealed ECM-ligand mediated induction of aberrant growth and tumorigenic phenotype that synergizes with the increased mechanical stiffness in the microenvironment.
Conclusions
Understanding the key characteristics of lung tumor microenvironment and recapitulating the compositional and mechanical differences in tissue engineered models hold a great importance towards achieving cellular responses seen in patients to steer therapeutic approaches for better clinical outcome.
20941831204
INTRODUCTION: Most epithelial cancer cell populations undergo an epithelial to mesenchymal transition (EMT), acquiring a more aggressive phenotype1. Mesenchymal cells are more motile and have the ability to remodel the extracellular matrix. This mechanical interaction with the surrounding matrix can be measured by bulk and single cell force generation. In this study we aim to use 3D in vitro methods to assess the contractility signatures of various epithelial cancer cell-lines.
METHODS:
Contraction assays. Two colorectal cancer (HT-29, HCT 116) and two breast cancer (MDA-MB-231, MCF-7) cell-lines were incorporated into collagen type I hydrogels at 1x106 cells/mL. Contraction was observed over 96 hours. Human Dermal Fibroblasts (HDFs) serve as a positive control.
Traction force microscopy (TFM). Polyacrylamide (PA) gels containing 0.1 μm red fluorescent beads were cast on glass bottom petri dishes. The gels were functionalised using collagen type I. Cells were seeded on the gels at a density of 1.5x105 cells and incubated for 24 hours before imaging. Comparative images were taken before and 15 minutes after trypsinization of the cells.
Quantitative polymerase chain reaction (qPCR). RNA was extracted from hydrogels through the TRI- Reagent® phase-separation method2. The following genes were measured for relative expression: LOX and RAE1.
Analysis. Images were analysed using Fiji ImageJ software and statistical analysis were performed using GraphPad Prism 9.
RESULTS: HDF cells contract the collagen type I hydrogels by 60% over 24 hours. MDA-MB-231 cells cause a 22% contraction whilst HT-29, HCT 116 and MCF-7 cells did not contract the gel. TFM results validate this trend showing that HDF cells displace the matrix by 2.8±1.7 µm. MDA-MB-231 cells displace the matrix by 0.7±0.1 µm. HCT 116, HT-29 and MCF-7 cells do not displace the matrix. MDA-MB-231 cells has significant upregulation of the EMT markers LOX and RAE1 compared to HT-29 cells (p=0.0070 and p=0.0237 respectively).
CONCLUSION: This study showed a correlation between the contractility profiles of epithelial cancer cells and their EMT status: highly mesenchymal-like cancer cells such as MDA-MB-231 cells are force-generating cells. This suggests that these cells can remodel the extracellular matrix, which aids migration and hence makes them highly invasive cancer cells.
ACKNOWLEDGEMENTS: MCF-7 cells were kindly provided by Dr Nina Moderau and Mr Michael Toeller from Imperial College London. We are grateful for supports from UCL Institute of Healthcare Engineering and EPSRC DTP PhD Studentship.
REFERENCES:
1. Hanahan, D. & Weinberg, R. A. Hallmarks of cancer: The next generation. Cell vol. 144 646–674 (2011).
2. DC, R., M, A., GJ, H. & TW, N. Purification of RNA using TRIzol (TRI reagent). Cold Spring Harb. Protoc. (2010).
83767212955
The creation of bioinks that can both support cells during the fabrication process and lead to advanced tissue function post-processing remains a challenge. If the tissue engineering community wishes to unlock the potential of 3D biofabrication techniques, new materials must be designed to meet these needs. In our lab, we take a look at new strategies with dynamic hydrogels as a potential solution to the demands of both fabrication and tissue formation. Recently, we have shown the ability to engineer shear-thinning and self-healing hydrogels utilizing dynamic (dynamic covalent and supramolecular) chemistry with rationally controllable mechanical properties. These viscoelastic materials are able to be 3D bioprinted, and offer unique opportunities to control cell behavior or improve culture conditions. Combined, these strategies show a potential path forward for the design of next-generation bioinks and tissue engineering hydrogels.
31451708649
Chronic kidney diseases (CKD) affect approximately 10% of worldwide population [1]. Currently in vitro models fail to mimic the complexity of the kidney essential for relevant in vitro studies. CKD can be caused by a multitude of factors spanning from nephrotoxic events, especially when exposed to cytotoxic compounds (e.g., antibacterials, corticosteroids, anti-cancer drugs) up to viral infection. Hence, the BIRDIE project aims to develop a novel platform for studying CKD that can support nephrotoxicity and viral testing and in which both structure and function of the native kidney can be mimic. Therefore, BIRDIE combines three different technologies: bioprinting, human pluripotent stem cells (hiPSCs), and organ-on-chip.
Through bioprinting, we will build a three-dimensional organ-like structure where cells can be patterned and spatially organized as in vivo. Secondly, hiPSCs differentiation into kidney progenitors will offer a more reliable cell source compared to the canonical 2D cultures of primary cells or cell lines. Lastly, the introduction of a microfluidic system will recreate the physiological conditions of the native kidney, further sustaining progenitors development.
Overall, the in vitro model envisioned in BIRDIE will provide a physiologically relevant platform as a reliable tool to study nephrotoxicity and viral infection associated to CKD, supplanting current in vitro and in vivo models.
Reference
[1] Wilson S, et al., J Clin Hypertens (2021) 23:831–4.
41883632644
Introduction: Developing more predictive in vitro platforms for biomedical research remains a major challenge in tissue engineering. 3D bioprinting allows patterning of cell-laden biomaterials into hierarchical structures. Volumetric bioprinting (VBP) is a novel light-based approach that tackles challenges posed by conventional approaches, through the layer-less biofabrication of viable and highly complex cell-laden structures at unprecedented speeds[1]. Given the requirement of high cell densities to create functional tissue mimics, strategies to overcome the light-scattering effect of intracellular organelles are needed to resolve high-resolution prints. Herein, an optically-engineered bioresin was developed to pattern morphologically-undisrupted organoids into complex centimeter-scale assemblies. Patient-derived human hepatic organoid-laden constructs were printed to create advanced in vitro models that capture salient features of the liver involved in systemic homeostasis and detoxification.
Methods: 405nm light back-filtered projections of a 3D object are directed onto a volume of cell-laden bio-resin (gelatin methacryloyl with visible-light photoinitiator lithium phenyl-2,4,6-trimethylbenzoyl-phosphinate), to selectively crosslink the hydrogel in a spatially-controlled fashion. The resolution of VBP in the presence of hepatic cell line (HepG2) or patient-derived hepatic organoids was enhanced through the addition of refractive index-matching compound iodixanol. These optically-tuned resins were used to print high hepatic organoid densities (up to 107cells/mL). Viability and metabolic activity of bioprinted organoids was evaluated, as well as hepatic differentiation capacity of VBP-prints (hepatic markers, albumin secretion, and cytochrome activity) compared to extrusion bioprinted (EB) constructs. Finally, cell-laden, mathematically-derived architectures with different structural properties were printed at high resolution and cultured under dynamic perfusion to evaluate organoid metabolism of ammonia.
Results: VBP-printed constructs were fabricated in tens of seconds, achieving previously unattained resolutions (41.5±2.9μm positive and 104.0±5.5μm negative features). The concentration of iodixanol was optimized to match the refractive index of intracellular components of both HepG2s and organoids, resulting in a significant resolution enhancement of cell-laden constructs (50.5±6.0μm). Hepatic organoids ranging from 100μm to 1mm in diameter were successfully printed via VBP with high accuracy. Compared to EB-printed structures, where shear forces resulted in organoid fragmentation and lower viability (73.2±1.2%), VBP-printed organoids exhibited high viability (93.3±1.4%), maintained their morphology and displayed apicobasal polarity post-printing. Complex gyroid-like structures with different pore architectures printed within 16-20s were integrated in a fluidic system and exhibited differences in permeability and surface-area-to-volume ratio. This resulted in enhanced rates of ammonia metabolism (33.5±5.8-24.3±1.4nmol mgtotal protein-1) compared to static controls (12.7±0.3nmol mgtotal protein-1), as well as shape-dependent changes in metabolism.
Conclusion: This study demonstrated the contactless bioprinting of complex and labile biological structures (hepatic organoids) via VBP. Through a refractive index-matching approach, an optically-tuned gelMA resin enabled high-resolution printing of cell-laden structures. Organoids exhibited high viability and hepatic differentiation capacity post-printing. Furthermore, the dynamic culture of convoluted VBP-printed structures was demonstrated through architectures that could modulate organoid function in a shape-dependent fashion. The combination of organoid technology with the ultra-fast printing times and freedom of design offered by VBP shows promise for the development of new predictive platforms for in vitro disease modeling and drug screening.
[1] Bernal, P.N. et al., Adv. Mater. 1, 1904209 (2019).
83767225448
Introduction:
Recently, conductive hydrogels have garnered significant attention and permitted momentous improvements in neuroscience due to their tissue-like softness, chemical steadiness, and sufficient electrical conductivity.(1) They have been utilized as interfaces for neural electrode arrays to improve their biocompatibility and lower protein adsorption. In particular, these materials have the potential to circumnavigate the mechanical mismatch between the neural probes and the implanted tissue.(2) Therefore, the transition from rigid to soft interfaces can improve the performance of the recording/stimulating devices by minimizing tissue irritation and neuronal cell loss. Alas, the chronic application of such interfaces is still challenging due to the poor adhesion of soft hydrogels to metallic electrodes and their relatively low stimuli-responsive characteristics.
The utilization of porous, high surface area and stimuli-responsive hydrogels may compensate for these physiochemical shortcomings, offering multifunctional properties such as low electrical impedance, better mechanical properties, lower thickness, and on-demand controlled release of bioactive agents.
Methodology:
To this end, a conductive hydrogel with semi-interpenetrating polymer network (semi-IPN) structure comprised of temperature-responsive poly(N-isopropyl acrylamide) (PNIPAAm)-based copolymer and polythiophene (PT) was synthesized in this study and miniaturized via a nanofabrication method to be used as a neural interface.
Results:
The electrospinability of the solution was facilitated by the high molecular weight of the synthesized PNIPAAm-based block copolymer and its narrow molecular weight distribution. A cytocompatible and degradable dendrimer was used as the crosslinking agent of the semi-IPN with ample surface groups, which allowed a dual-hardening physical and chemical gelation process. Consequently, a lowered curing temperature was necessary to attain structural robustness at molecular and macroscopic levels. The copolymerization process reduced the volume phase transition temperature (VPTT) of pure PNIPAAm, and the resulted block copolymer showed lower overall transition energy. The fibrous hydrogel gave water molecules rapid access to the whole material and switched on a fast responsive characteristic. As the water impregnated the xerogel, the porosity and fiber diameter increased substantially. The developed material showed fast swelling and de-swelling responses triggered by temperature changes. Repeated hydration/dehydration cycles did not affect the physical integrity of produced electrospun fibers.
Furthermore, the conductive fibrous semi-IPN displayed a high electrical conductivity and charge storage capacitance compared to the conductive bulk hydrogel. This occurrence was attributed to the formation of a large electrochemical surface area that resulted from system miniaturization. The impedance of the developed material was in the range of physiologically relevant frequencies.
Conclusion:
The incorporation of PT chains in the stimuli-responsive hydrogel network promoted the synergetic effect between the two components leading to the fabrication of a superior fibrous interpenetrating network neural interface with remarkable electrochemical properties.
Acknowledgment:
This study was supported by the First TEAM grant number POIR.04.04.00-00-5ED7/18-00, which is conducted within the framework of the First TEAM programme of the Foundation for Polish Science (FNP) and co-financed by the European Union under the European Regional development Fund.
References:
1. Sung, C. et al., J. Mater. Chem. B, Mater. Biol. Med. 8, 6624 (2020).
2. Park, S. et al., Nat. Commun. 12, 3435 (2021).
31412730159
Chronic kidney disease (CKD) is characterised by the gradual loss of renal function. It affects approximately 10% of the population worldwide, and the only treatment is aimed to slow down the progression of kidney damage. Ultimately, CKD results in end-stage renal disease (ESRD), which can only be treated with dialysis or renal transplant. However, both options are far from ideal and cannot be considered permanent solutions.
Regenerative medicine, particularly the use of organoids, might provide a solution to this problem. Organoids are a relatively easy and scalable model that can be used to study organ development, regeneration, genetic diseases, and perform drug screening. However, the limited ability to accurately replicate adult organs' maturation level, complexity, and functions drastically restrict their application in research and clinical medicine.We implemented differentiation protocols to obtain iPSC-derived metanephric mesenchyme (MM) and ureteric bud (UB) progenitors in sufficient numbers for bioprinting. We used a microfluidic 3D bioprinter capable of extruding core-shell filaments to manufacture renal constructs containing single cell progenitors. After bioprinting, we cultured the construct with an optimised mix of growth factors for two weeks. The 3D bioprinted renal progenitors showed high viability after bioprinting. After one day in culture, the cells self-aggregated into spheroids inside the hydrogel filaments. Within one week, renal vesicles were visible. Tubular structures were observed two weeks post-bioprinting, which stained positive for lotus tetragonolobus lectin (LTL) and e-cadherin. For the first time, we were able to bioprint iPSC-derived renal progenitors that generated renal organoids inside the bioprinted hydrogel constructs.
83767228888
Native tissues are characterized by its 3D organization and distribution of cells, with specific cell-cell and cell-extracellular matrix (ECM) interactions dictating tissue function. The spatial distribution of cells and ECM in tissues is not arbitrary. There are specifically located cell populations for generating interconnected lumen structures, creating a fundamental structure-function relationship that determines the role of numerous tissues. Therefore, the capability to mimic this 3D environment is key for a correct in vitro modeling of tissues and for future tissue engineering applications.
Here we present a templating strategy using a thermally responsive polymer and we show the fabrication, in one step, of a network of interconnected channels within a hydrogel. While other polymers have been previously described for similar applications, we uniquely show that our template can create a defined 3D polymer scaffold to which cells can adhere, leading to the subsequent formation of a channel network that directly incorporates cells. This approach is based on a family of oxazoline polymers (POXA) that have been specifically designed to have a uniquely tunable range of lower critical solubility temperature (LCST), above which it remains insoluble and below which it is triggered to dissolve.
We were able to produce fibers with our POXA polymer with diameters as small as 5µm, up to the millimeter scale using melt electrowriting (MEW). We recorded stereomicroscope time lapses of their dissolutions while decreasing the temperature in a controlled way from 37°C to 4°C. Additionally, we monitored its water contact angle at different temperatures, confirming their change in stability and solubility. By using a customized fabrication method, we could include microchannels in a set of hydrogels with different crosslinking mechanisms: collagen, PEGDA, polyacrylamide and fibrin. Additionally, we were able to culture Schwann cells (SC) and HUVECs on top of these micro-scale fibers and transfer them to a hydrogel where the polymer would be dissolved, leaving cells growing in specific patterns inside of microchannels. With our method, we could fabricate in vitro models for vasculature by culturing HUVECs on the lumen of the channels and other cell types such as fibroblasts embedded in the surroundings. Allowing us to control the arrangement of the vascular channel as well as their interactions with the environment. We also showed how SC could migrate along these microchannels, as they would do in vivo, this is a key mechanism during tissue innervation, guiding axonal growth towards other tissues. Similarly, we cultured sensory neurons (nociceptors) derived from human iPSCs and showed how their axons could grow along the microchannels. Potentially becoming a new tool for nerve repair and tissue innervation studies or drug testing in clinical applications.
Here, we demonstrate the value and versatility of this novel templating technology to generate complex 3D cell culture models. We believe that this system will allow the tissue engineering field to fabricate more realistic in vitro models considering cellular arrangement and EMC interactions.
20941849987
INTRODUCTION: Major challenges in bioprinting tissues with functional, native-like behavior revolve around enabling the use of hydrogels with low elastic modulus, while also ensuring high shape fidelity and printing resolution. Such materials are necessary to allow cells to migrate, and to facilitate intercellular communication and reorganization of the neo-synthesized extracellular matrix. In this perspective, suspended bath bioprinting was previously developed as a printing technique that solves this problem by extruding bioinks within a yield-stress support bath that keeps bioinks with low viscosity in place until cured. Moreover, in order to increase the printing speed and overcome the geometric constraints of conventional layer-by-layer AM approaches, volumetric bioprinting was recently developed as a new light-based approach. However, the possibility to create high resolution features comprising multiple, independent structural elements intertwined into a single construct remains a major challenge, especially when using multiple materials and cell types in a single printing process. The current study describes a new biofabrication strategy that synergizes the multimaterial printing ability of extrusion in suspension media, and the layerless 3D patterning provided by visible-light tomographic printing, in order to rapidly fabricate complex tissue models with tunable mechanical properties, while embedding different cells types.
METHODOLOGY: A novel photo-crosslinkable bioresin was designed and characterized, based on Gelatin-Methacryloyl (GelMA) hydrogel microparticles that act like a Bingham plastic. This bioresin was used both as a support bath to enable deposition of soft hydrogels, and subsequently sculpted into a desired architecture via volumetric bioprinting, to leverage the microporosity provided by the packing of the microgels for cell infiltration and nutrient diffusion. In addition, two (bio)inks for the extrusion process were designed and mechanically characterized: i) a gellan gum (GG) and Poly(ethylene glycol) diacrylate (PEGDA) blend, used to create different mechanically-competent, reinforcing scaffolds within the volumetrically crosslinked GelMA microgels, ii) a blend of methylcellulose and fibrinogen, used as medium for cell printing.
RESULTS: Features smaller than 500 µm can be volumetrically printed in less than a minute with the microgel-bioresin, and ⁓300 µm width filaments can be extruded within it with both bioinks. With a compression modulus ranging between 3 and 4 kPa, microgel-based samples have shown lower mechanical properties than bulk GelMA gels, but these could be enhanced and tuned using the GG/PEGDA ink. Printed reinforcing GGPEGDA/GelMA meshes taking up a 2.5% volume fraction of the whole slurry-based construct lead to increasing the compression modulus of the composite by 40%. Printing of multiple cell types including vasculature forming endothelial cells and pancreatic cells was finally investigated to build complex biofabricated constructs for vascularized tissue engineering.
CONCLUSIONS: Combining extrusion-based bioprinting in a suspension media and volumetric bioprinting is an advantageous approach that allows to create complex cm3-scale and vascularized structures in a fast and accurate process, combining different biomaterials to tune both mechanical and biological characteristics. These features are crucial to better mimic the heterogeneous characteristics of living tissues (e.g., the complex architecture of the trabecular bone and the bone marrow, or the endocrine pancreatic tissue within the exocrine one).
41883640155
Introduction
The kidney plays a crucial role in drug development, as it dictates drug clearance and is a target for drug-induced toxicity. Nephrotoxicity of candidate drugs is one of the major reasons for drug attrition during preclinical, clinical and post-approval stages of drug development. These failures during the final stages of the drug development process are partially caused by the use of inaccurate preclinical nephrotoxicity models. Therefore, an accurate kidney model for Multi-Organ-Chip applications could revolutionize drug trials by providing a relevant in vitro platform.
Methodology
In this study, we generate an autologous kidney-on-a-chip that encompasses a glomerular and a tubular model. Induced pluripotent stem (iPS) cell-derived podocytes and tubular cells are seeded into the HUMIMIC Chip4, which enables the long-term co-cultivation of the renal model with up to three additional organ equivalents with a defined fluid flow and shear stress. The final maturation of the iPS cell-derived podocytes and tubular cells occurs within the Multi-Organ-Chip. After the renal cells’ final maturation, the co-culture can be maintained for at least 14 days.
Results
The kidney-on-a-chip exhibits a stable metabolism, a cellular barrier that prevents albumin from entering the excretory circuit, and the cells demonstrate a steady expression of key podocyte and tubular markers.
Conclusion
The kidney-on-a-chip can be employed for elaborate safety, efficacy and nephrotoxicity studies, as wells as for mechanistic studies of renal development or disease. The combination of the renal model with other organ equivalents further enables systemic studies, including ADME experiments. Therefore, the kidney-on-a-chip presents a human and systemic alternative to current in vivo and in vitro models.
73296308379
"Organs are complex systems, comprised of different tissues, proteins, and cells, which communicate to orchestrate a myriad of functions in our bodies. Technologies are needed to replicate these structures towards the development of new therapies for tissue and organ repair, as well as for in vitro 3D models to better understand the morphogenetic biological processes that drive organogenesis. To construct tissues and organs, biofabrication strategies are being developed to impart spatiotemporal control over cell-cell and cell-extracellular matrix communication, often through control over cell and material deposition and placement. These technologies could also play a role in protecting seeded or encapsulated cells in case of off-the-shelf products are desired for biobanking purposes.
Here, we present some of our most recent advancements in biofabrication that enabled the control of cell activity, moving towards enhanced tissue regeneration as well as the possibility to create more complex 3D in vitro models to study biological processes, with a particular attention to biobanking requirements to preserve tissue construct functionality before and after storage."
31451702248
"Almost 160 clinical trials of tissue engineered products (TEPs) currently registered in ClinicalTrials.gov databases as well as the continuously growing record for TEPs under clinical investigations starting from the year 2006, can be taken as one of the many measures of success of tissue engineering as an applied science discipline. On the other hand, the number of TEPs on the medical market remains at a level that can be considered below expectations. Obviously, the road of the medicinal products to the market is costly and time consuming. Extremely complicated, unstable and demanding regulatory issues concerning advanced therapy medicinal products make it even more difficult. Nevertheless, it is hard to disagree that - so far - TEP-based therapies have failed to bring medical breakthroughs. Among the substantive reasons, one of the most important seems to be the multiplicity of variables at all stages of design and validation of TEPs. On one hand it opens more possibilities, but on the other one, it often limits repeatability of results. The reproducibility of the active substance of TEPs, i.e. the cells, is already extremely difficult to achieve. Even when the requirements of pharmaceutical law are met, the donor-dependent biological diversity of the cells makes it difficult to obtain reproducible results. Advanced preclinical studies of TEPs in vitro, repeated on material from many donors, may bring significant progress. At this point, tissue engineering come close to biobanking, which meets this need. By the most general terms, biobanking means collecting all types of biological material, such as blood, tissue, cells, RNA or DNA, preferably in connection with the data related to the samples. For health research, biobanking material of human origin is now considered an extremely important tool. In particular, acquisition, preparation and storage of well-characterized biological material from multiple donors - under the control of quality standards - is the response of the scientific community to reports stigmatizing the lack of repeatability of preclinical studies. Strong collaboration between tissue engineers – model beneficiaries and at the same time potential suppliers of biological material for biobanks and biobankers - who provide biological material of confirmed quality necessary for research, opens new possibilities for the development of well-defined TEPs of repeatable properties.
Some examples illustrating the problems mentioned here, based on our own experience from clinical trial on TEPs carried out with the participation of 100 volunteers will be discussed. On the other hand, the activities of BBMRI-ERIC - a European research infrastructure for biobanking will also be presented. The combination of both of these issues will be shown in the context of a possible common added value of significant importance for medical research.
Supported by the National Center for Research and Development (grant number STRATEGMED2/267976/13/NCBR/2015) and by the Ministry of Science and Higher Education in Poland (Grant DIR/WK/2017/2018/01-1)."
94355107448
Biobanking refers to the process by which samples of human body fluids, tissues or cells are collected to provide samples for different research purposes. Initially, the term biobank appeared in the scientific literature to describe human population-based biobanks.
Biobank is nowadays recognised as an organized collection of samples and associated data describing donor, stored for future research in biomedicine.
A tissue and cel bank is an establishment that and procure human tissues and cells from deceased and living donors which are than processed, stored and distributed for allo- and autograft transplantations.
A problem is raised when human tissues and cells are used in clinical studies. How the procedurę of Transplantation should be clasified, what are legal requirements to be followed by bank? Does bank of tissues and cells became a biobank. What are the conditions, if biobank distribute tissues or cells for clinical use? Is it possible that biobank distribute tissues or cells as starting materils for manufacturing of advanced therapy medicinal products (ATMP) including tissue engeenired productcs or for gene therapy?
62825471109
Introduction: The surge in clinical need for bone tissue restoration together with limitations of existing treatments calls for the development of alternative strategies. Tissue engineering has been proposed towards the formation of bone graft substitutes capable of driving repair. Previously, we demonstrated the possibility of generating engineered cartilage grafts using a human mesenchymal stromal cell (hMSC). Here, we aim to develop an efficient decellularization protocol and assess the osteoinductivity, immunogenicity and regenerative potential of decellularized cartilage in both immunodeficient (ID) and immunocompetent (IC) settings.
Method: Our graft consists of in vitro engineered cartilage tissue produced by human mesenchymal stromal cell (hMSCs) lines. After cartilage formation, the tissue was subsequently decellularized using a combination of hypertonic and hypotonic baths, Sodium dodecyl sulfate (SDS), and DNase to effectively remove cells, thus resulting in a cell-free graft aiming at instructing bone formation by endochondral ossification. The decellularized cartilage is implanted subcutaneously in the back of IC and ID animals for a maximum of 12 weeks. The early recruitment of immune cells (dendritic cells, monocytes, macrophages, natural killer, T and B cells) was assessed quantitively by flow cytometry at 3-, 7- and 10-days post-implantation (Immune prints).
Results: We demonstrated the reproducible engineering of decellularized human cartilage, as cell-free grafts capable of bone formation by exploiting a dedicated human Mesenchymal Stromal Cell line (MSOD-B). Following subcutaneous in vivo implantation, a complete remodeling into bone was achieved in immunodeficient mice (ID) through recapitulation of the endochondral ossification pathway. In contrast, only minor calcification was observed upon implantation into immunocompetent mice (IC). Initial immune responses during successful bone formation in ID seemed to correlate with an early M2 macrophages polarization and recruitment. Moreover, we showed that despite the absence of cellular material, human-derived grafts were able to induce a pro-inflammatory response in IC, detrimental to effective bone formation.
Conclusion and discussion: Using our decellularization method, we efficiently removed significant amount of cells without affecting the overall structure and composition of cartilage graft. Ectopic evaluation of decellularized tissues displayed excellent osteoinductive properties, correlating with early M2 polarization in ID mice but not in IC mice. This indicates ECM interspecies variations could still result in the immune rejection of cell-free tissues. Compiling immune prints may offer understanding the immunogenicity of engineered grafts, and help designing biomaterials with tailored immune profile for effective repair.
52354574349
"Introduction: Musculoskeletal ailments caused by cartilage damage are common, and thanks to more modern diagnostic methods, they are more often recognized. Moreover, cartilage diseases progress with age and result from injuries, becoming a dominant problem in orthopedic surgery. The conducted research aims to produce conjugates for cartilage regeneration based on chondrogenic differentiation of mesenchymal stem cells isolated from Wharton's jelly (hUC-MSC). Composed conjugates are based on lipid carriers of kartogenin and glycosaminoglycans derivatives stabilize the whole system.
Methodology: Kartogenin (KGN) – an active substance – was encapsulated in the liposomes. The lipid carriers were covered with hydrophobically modified chondroitin sulfate (CS) or hyaluronic acid (HA). The physicochemical analysis of the obtained systems was carried out by dynamic light scattering, zeta potential, and fluorescence measurements. The thermotropic behavior of lipid membranes was studied using a Nano DSC calorimeter (TA Instruments). In addition, the interactions of polymers with liposomes (loaded or unloaded by KGN) were analyzed by microscale thermophoresis (Monolith, Nanotemper). hUC-MSC morphology imaging after incubation with the prepared formulations and metabolic toxicity test were performed. Additionally, these systems have been tested to differentiate stem cells into chondrocytes using real-time PCR (rt-PCR).
Results: CS and HA substitution by alkyl domains were confirmed by XPS spectra. KGN was successfully incorporated into the lipid bilayer. Composed formulations were stabilized by covering their surfaces with CS or HA derivatives. The changes in thermograms for an aqueous dispersion of DPPC confirmed the incorporation of polymers' hydrophobic domains into the lipid bilayer. Moreover, an increase in the liposome size and decrease in the zeta-potential values confirmed the presence of polymers on liposomes surfaces. Despite both systems: modified CS and liposomes with KGN have high negative charge, they interact because of the hydrophobic effect. According to cytotoxicity results (MTT assay), all polymers used in lipid formulations were significantly non-toxic, than pure polymers. This is explained by the fact that hydrophobic anchors are hidden in the lipid bilayer and their exposure to the cell surface is minimized. The selected genes expression was analyzed by real-time PCR. All systems have induced ACAN and SOX9 gene expression compared to untreated cells.
Conclusion: Composed hybrid lipid-polymer formulations are stable vesicles of KGN. Moreover, they can be embedded into hydrogels and provide control released of the cargo. The resulting systems are promising conjugates for the regeneration of cartilage tissue."
20941857789
"Introduction: Vascularization is a critical aspect of every tissue engineering (TE) approach, especially in 3D constructs. The formation of a network of capillaries is necessary to ensure adequate delivery of nutrients and oxygen to cells within the constructs, as well as fast anastomosis with the host’s vasculature after implantation. Pre-vascularization of these constructs before implantation can be a solution. However, cell sourcing is a limiting issue. Adipose tissue is regarded as a privileged source of mesenchymal progenitor cells due to its easy accessibility and abundancy. This tissue hosts adipocytes, as well as a stromal vascular fraction (SVF) comprising several other cell types including fibroblasts, endothelial progenitors, endothelial cells and hematopoietic cells. Due to this composition, the SVF of adipose tissue is highly angiogenic and has been proposed for the growth factor-free vascularization of TE constructs[1]. To produce such constructs, collagen from mammalian sources is widely used. However, regulatory issues associated with the risk of disease transmission have boosted the search for new collagen sources such as from marine organisms. Collagen from otherwise wasted blue shark skin was herein used to produce sponges that were then seeded with cryopreserved SVF for growth factor-free vascularization.
Methodology: A blue shark skin collagen hydrogel was created by acid solubilization of blue shark skin collagen, followed by cryogelation with crosslinking reaction carried out at low temperatures using 1-[3-(dimethylamino) propyl]-3-ethylcarbodiimide hydrochloride. Finally, cryogels were freeze dried to form the collagen sponge. SVF was isolated from human adipose tissue subcutaneous tissue and cryopreserved in 10% DMSO in FBS with a controlled freeze rate of 1ºC/min, for at least 7 days. SVF was then seeded on the collagen sponges and cultured for 7 days to create a pre-vascularized construct. Sponges’ pre-vascularization was assessed in vitro by immunohistochemistry and secretome profiling and their functionality was tested in ovo using a chick chorioallantoic membrane (CAM) assay.
Results: After 7 days of in vitro culture, CD31 expression pattern demonstrated the formation of a vessel like network. The secretome profile of angiogenic-related factors changed with culture time. From 5 to 7 days of culture, there was an increase in the secretion of both pro angiogenic proteins (VEGF, MMP-9, IL-8) and angiogenesis inhibitors (TIMP-1, SERPIN E1, Thrombospondin-1). Upon in ovo implantation, vessel number quantification demonstrated an increase in vessel recruitment in pre-vascularized sponges when comparing with sponges without SVF cells. CD31 expression pattern demonstrated the integration of the pre-vascular network within the CAM, while in situ hybridization confirmed the presence of the seeded human cells.
Conclusions: These results demonstrate the potential of cryopreserved SVF to assist in the vascularization of TE constructs in an extrinsic growth factor-free manner, allowing a simplified and cost-efficient methodology to ensure construct integration after implantation.
Acknowledgements: EU Horizon2020 ERC grant CapBed (805411); FCT fellowships PD/BD/135252/2017, IF/00347/2015; INTERREG España-Portugal 2014-2020 project 0474_BLUEBIOLAB_1_E; Atlantic Area Programme project BLUEHUMAN (EAPA_151/2016) and NORTE2020/PT2020 project ATLANTIDA (Norte-01-0145-FEDER-000040). Dr. Cármen G. Sotelo (IIM-CSI, Vigo, Spain), for the kind offer of blue shark skin collagen."
20941850409
Articular cartilage and osteochondral defect repair remain major clinical challenges. Biomaterial scaffolds currently in clinical use in orthopaedic medicine do not accurately mimic native tissues, and therefore do not preferentially promote tissue-specific regeneration when they are colonised by endogenous stem/progenitor cells post implantation. Tissue-specific extracellular matrix (ECM) derived scaffolds have been shown to promote tissue repair by providing both structural and functional cues to cells, suggesting that such natural, biomimetic materials may provide a cell-inductive platform for the regeneration of musculoskeletal tissues. Indeed, decellularized articular cartilage ECM derived scaffolds have been shown to promote the chondrogenic differentiation of mesenchymal stem/stromal cells (MSCs), while bone and growth plate ECM derived scaffolds have been shown to support osteogenesis. However, directing the phenotype of stem/progenitor cells is only the first step in ensuring successful cartilage or osteochondral defect repair following scaffold implantation; such biomaterials are also required to direct the structural organization of the repair tissue to promote functional regeneration. This talk will summarise our efforts towards developing such ECM derived scaffolds for cartilage, bone and osteochondral repair, and will provide preclinical data in relevant in vivo models to support their continued development in the field of orthopaedics.
62903403044
"Introduction: Decellularization creates cell-free collagen-based extracellular matrices from native organs, which can be used as scaffolds for regenerative medicine applications1-8. This technique has gained much attention in recent times. However, there is still a limited understanding of scaffold responses in vivo post-transplantation and ways we can improve scaffold durability to withstand the in vivo environment9. This study uses intravital microscopy (IVM) to gain instant feedback on their structure, function, and deformation dynamics.
Methodology: In vivo assays were developed to evaluate the effectiveness of decellularization and structural and functional integrity of the acellular nephron in the post-transplantation environment. Cohorts of 2-3-month-old male Sprague Dawley rats were used: non-transplanted (n = 4), transplanted day 0 (n = 4), transplanted day 1 (n = 4), transplanted day 2 (n = 4), and transplanted day 7 (n = 4). Qualitative and quantitative assessments of scaffold DNA concentrations, tissue fluorescence signals, structural and functional integrities of various decellularized nephron segments, and velocity within the microcirculation were acquired and compared to the native (non-transplanted) organ.
Results: Large molecular weight dextrans, which lined the vasculature, provided real-time evidence of ischemia onset and microvascular permeability increases. We observed substantial translocation of macromolecules from glomerular/peritubular capillary tracks as early as 12 hours post-transplantation. Blood extravasation continued across a week. During that time, the decellularized microarchitecture was significantly compromised and thrombosed.
Conclusions: Models examining the microvasculature primarily utilize in vitro/in vivo techniques that cannot provide adequate spatial/temporal resolution. These results identifies IVM as a powerful approach for studying scaffold viability and identifying ways to promote scaffold longevity, and angiogenesis in bioartificial organs. We also created the basis to develop a fractal model that can be used to explore ways to improve scaffold integrity to support recellularization and withstand deformation in transplantation environments.
References
94238104239
"Introduction:
Articular cartilage facilitates the frictionless movement of synovial joints, however, due to its avascular and aneural nature, it has limited ability to self-repair. Current treatments for cartilage defects elicit variable results – an issue that the field of tissue engineering has aimed to address; however, the inability to mirror the complexity of native tissue with current biomaterials has hindered progress 1, 2. The advent of 3D-printing has provided a potential solution. 3D-printed (3DP) scaffolds, fabricated using biomaterials native to articular cartilage, can be designed to mimic native articular cartilage. These biomaterial-based printable inks can also be functionalised with cells, bioactive factors and/or gene therapeutics to form biomimetic ‘bioinks’, capable of repairing cartilage 3.
The aim of this study is to develop a novel 3DP scaffold composed of biomaterials native to human articular cartilage, such as collagen and hyaluronic acid, which can also be incorporated with mesenchymal stem cells (MSCs) and/or therapeutic biomolecules to promote regeneration of the native tissue.
Materials and methods:
To this end, 3.5% neutralised collagen type I was mixed in a 1:1 ratio with methacrylated hyaluronic acid (MeHA) at concentrations of 0.5-3% to formulate four distinct bioinks. The printability of each bioink was first assessed, and three formulations were carried forward to 3D print 10mm x 2mm circular mesh scaffolds. The mechanical and physiochemical properties of the scaffolds were then determined. Two suitable formulations were selected and incorporated with rat MSCs, and the cell viability of the MSCs within 3DP cell-laden scaffolds was determined over 7 days. An optimal bioink formulation was then selected and incorporated with rat MSCs at three respective cell densities. The production of articular cartilage matrix components within these cell-laden 3DP scaffolds was assessed following 21 days culture.
Results and discussion:
Three bioink formulations were found to have desirable 3DP properties and were carried forward for further analysis. Subsequent studies showed no significant difference in the mechanical properties or macro-pore size of scaffolds 3DP with each bioink. However, 3DP scaffolds with a higher concentration of MeHA did have a higher mass swelling ratio. 3DP scaffolds containing the lowest MeHA concentration were excluded from subsequent studies as they could not withstand physical manipulation.
Cell viability studies with the remaining two bioink formulations showed that 3DP scaffolds containing the highest MeHA concentration facilitated greater levels of cell proliferation. Subsequently, this optimal bioink formulation was shown to facilitate deposition of articular cartilage-specific matrix components in 3DP scaffolds in a cell density-dependent manner. Ongoing work includes improving the mechanical properties of the 3DP scaffold by co-printing with a polymer, and incorporation of therapeutic chondrogenic nanoparticles to enhance the chondrogenic potential of the bioink.
Conclusion:
A biomimetic collagen and hyaluronic acid-based bioink with favourable 3D-printing properties was successfully developed. Scaffolds printed using this bioink facilitated proliferation of MSCs and deposition of new articular cartilage matrix.
References:
1Moroni (et al.), Nat. Rev. Mater. 3:21-37, 2018.
2Raftery (et al), Biomaterials 216: 119277, 2019.
3Gonzalez-Fernandez (et al.), J. Control. Release 10:301, 2019.
Acknowledgements:
Funding: ReCAP: ERC Advanced Grant number 788753"
31412705928
"Introduction
Prolonged alveolar air leaks are post-surgical complications to routine lung resections and biopsies that are a significant cause of patient morbidity. Extended duration of chest tube drainage and emergency revision surgeries are the standard approaches for its clinical management. Transplantable decellularised pleural membrane patches as adjuncts to traditional intraoperative closure techniques could reinforce the mechanical barrier, reducing incidence and severity of sustained air leaks. As a treatment modality, it can provide the physiological cues that stimulate endogenous tissue regeneration. We aimed to optimise a decellularisation and characterisation protocol for porcine pleural membranes (PPM), with minimal disruption to the microarchitecture, biochemical composition, and mechanical integrity of the native tissue.
Method
PPM decellularisation was performed with physical (freeze-thaw cycles) and chemical (0.5% sodium deoxycholate and 1% Triton-X100 in 10mM Tris buffer) treatments. Protocol efficiency was determined via histological analysis (Hematoxylin and Eosin, Alcian blue and Picrosirius red), nuclear membrane integrity (DAPI staining), and quantitative bioassays (Picogreen dsDNA quantification and dimethyl methylene blue (DMMB) glycosaminoglycan assay). Decellularised PPM were assessed for their cytotoxicity (Live-Dead cytotoxicity kit, Invitrogen™, and Trypan blue exclusion assay) and biocompatibility (MeT-5A cell-line seeding and culture). Proteomics was carried out using antibody microarray technology (scioDiscover™, Sciomics GmbH)
Results
H&E staining of decellularised PPM showed absence of stained nuclei, consistent with significant reduction (p < 0.0001) in DAPI stained nuclei counts against native controls. Residual DNA quantification in the decellularized PPM reflected over 90% reduction in native nuclear dsDNA (p < 0.001). Staining for sulphated glycosaminoglycans (sGAG) and collagen exhibited minimal disruption to the structural alignment of the native ECM. sGAG content in the decellularised PPM showed a significant reduction in comparison with native controls (p < 0.01). Mechanical characterisation studies showed that increased decellularised membrane thickness (p < 0.05) did not affect the inherent membrane stiffness, as the estimated Youngs modulus in the decellularised PPM (12782.7 kPa ± 3874) was comparable with the native controls (9259.5 kPa ± 2079). In vitro cytotoxicity and scaffold biocompatibility studies exhibited minimal inhibitory effect on MeT-5A cell line attachment, proliferation, and viability. Proteomics provided molecular readouts of the native and decellularised PPM proteome, reflecting differential protein expressions and enabling decoding of our decellularised PPM matrisome.
Conclusion
Our pilot study represents a step forward in deriving bioactive ECM scaffolds in the form of decellularised PPM. Studying the recellularisation dynamics of the cell-seeded scaffolds using primary mesothelial cultures will underpin our research towards developing proof of concept for the application of the relatively unexplored decellularised pleural membranes in biological ECM scaffold-based therapeutic approaches."
83767207686
"INTRODUCTION
Cancer early detection is pivotal to patient survival. The small non-coding nucleic acid sequences, microRNA (miRNA) are a captivating molecular target for cancer early detection. miRNA are dysregulated during the early stages of cancer1, it is found in stable amounts in blood plasma and serum. Therefore, a minimally invasive liquid biopsy screening device would allow for point of care diagnostics. Current miRNAs detection methods are cumbersome and lack reproducibility along with poor sensitivity and low accuracy. To overcome these challenges we aim to develop a new diagnostic platform using a functional 3D peptide hydrogels for sequence specific2, 3, PCR-free, fluorescent detection of miRNAs in a “one-pot” assay. This work will assess the suitability of the novel hydrogel-based technology for rapid, robust and reliable screening of the unique miRNA fingerprint of difficult to detect cancers.
EXPERIMENTAL METHODS
Using a split probe system with a FRET pair conjugated to an anti-parallel β-sheet peptide hydrogel, can be formulated that allow complementary strands of cancer miRNA biomarkers to be identified via fluorescence.
Diffusion characteristics were evaluated via plate reader and Fluoroblok well insert. Allowing to measure the rate at which fluorescently label DNA analogue of miR-21 diffuse into peptide hydrogels.
Cell culture of Panc-1, MIA PaCa-2, LNCaP and PC-3 cells in 2D and 3D (multicellular tumour spheroids) forms was undertaken to validate the sensor. The hydrogels under investigation were also evaluated in their ability to support the four cancer cell lines spheroid structure.
RESULTS & DISCUSSION
Four de novo designed self-assemble peptide hydrogels (SAPH) were tested to understand the diffusion characteristics of miRNA and select system that allow fast trapping and detection of miRNA. MiRNA being negatively charge it was found that positively charted hydrogels promoted miRNA trapping. The mesh size of the hydrogel used (<40nm) also allowed to filter and avoid interference from larger cell debris usually present in biological samples.
The bio-compatibility of the four peptide hydrogels provides a 3D platform for cancer cell culture, and in situ bio-sensing.
CONCLUSIONS
The self-assemble peptide hydrogel is an extremely versatile material. Has the potential to harbour fluorescent properties, to allow for biosensor application in the early detection of cancer.
Future work on 3D culturing of other pancreatic and prostate cancer cells for quantification of the key secreted biomarkers linked to the two cancers that require early detection.
ACKNOWLEDGEMENTS
The work was funded in part by DTA EPSRC Funding from the Department of Materials at University of Manchester, MERCARDO associated with Cancer Research UK Manchester Centre, and ACED.
REFERENCES
[1] Kosaka, Nobuyoshi, Haruhisa Iguchi, and Takahiro Ochiya. ""Circulating microRNA in body fluid: a new potential biomarker for cancer diagnosis and prognosis."" Cancer science 101.10 (2010): 2087-2092.
[2] Yousaf, Sameen, et al. ""Sequence-Specific Detection of Unlabeled Nucleic Acid Biomarkers Using a “One-Pot” 3D Molecular Sensor."" Analytical chemistry 91.15 (2019): 10016-10025.
[3] King, Patrick JS, et al. ""A de novo self-assembling peptide hydrogel biosensor with covalently immobilised DNA-recognising motifs."" Chemical Communications 52.40 (2016): 6697-6700."
62825410577
"Introduction
Growing clinical demands for electrical stimulation-based therapies for central nervous system applications requires the development of conductive biomaterials balancing conductivity, biocompatibility, and mechanical performance. Traditional conductive materials often induce scarring, due to their stiffness and poor biocompatibility, hindering their clinical translation and efficacy. To address these issues, we report the development of a pristine graphene-based (pG) composite material consisting of type I collagen and 60 wt% pG, yielding conductivities (~1 S/m) necessary for efficient electrical stimulation, and with versatile processability.
Materials and Methods
Pristine graphene and collagen films (60 wt%, CpG) (CpG60%) were synthesised[2]. Different neurons (SHSY-5Y, NSC-34, iPSC-derived) and glial cells were seeded on the composites, and the metabolic activity, DNA content, cell morphology and release of inflammatory cytokines were assessed. Electrical stimulation was applied to mouse primary cortical neurons to enhance neurite outgrowth and viability. Finally, to demonstrate the versatility of CpG composites for a number of applications, the CpG was fabricated into porous 3D scaffolds, microneedle arrays, and bioelectronics circuits, using freeze drying, dry casting, and 3D printing approaches respectively.
Results
Of all composites tested (N=4), CpG60% exhibited physiologically relevant conductivities (~1 S/m), and robust mechanical properties (~17.8 MPa). Four neuronal and glial cell types exhibited robust growth when grown on composite films with no change in inflammatory markers IL-6, IL-10, or IL-1β, and good biocompatibility. Induced pluripotent stem cell-derived neurons exhibited typical cellular morphology after 15 days growth on the films. The achieved conductivity enabled the efficient delivery of electrical stimulation to mouse primary cortical neurons on the composite (200mV/mm, 12Hz, 4h/day, 5 days), and enhanced neurite outgrowth, cellular viability and morphology compared to collagen controls. Finally, the diverse potential applications of the composite were demonstrated using a range of neural-interfacing structures, including porous scaffolds with aligned pores visible under SEM, microneedle arrays, and 3D-printed working LED circuits for bioelectronics.
Discussion
These results show that (CpG60%) composites form a versatile neurotrophic platform that balances the requirements for physiologically relevant conductivity, robust mechanical properties, and excellent biocompatibility. The mechanical properties of the composite give it an advantage over stiffer traditional electrode materials, which can cause scarring due to extreme mechanical mismatch. The CpG60% composite supported robust neuronal and glial cell growth, with an absence of neuro-inflammatory responses. In addition, CpG60% efficiently delivered electrical stimulation to neurons, which when coupled with these conductive materials enhanced neurite outgrowth, viability, and cellular morphology. Finally, the versatile processing capabilities of the CpG composites using various fabrication techniques demonstrate its potential as platform for fabrication of next-generation neuronal medical devices.
References
[1] Gouveia, P.J., Maughan, J., Gutierrez Gonzalez, J., Leahy, L., Woods, I., O’Connor, C., McGuire, T., Garcia, J., O’ Shea, D., McComish, S.F., Kennedy, O.D., Caldwell, M.A., Dervan, A., Coleman, J.N., O’Brien, F.J., Collagen/Pristine Graphene as an Electroconductive Interface Material for Neuronal Medical Device Applications, Materials Today, 2022, Under Review.
[2] Ryan et al. Electroconductive Biohybrid Collagen/Pristine Graphene Composite Biomaterials with Enhanced Biological Activity, Advanced Materials 30(15)(2018)(1706442).
Funding
Science Foundation Ireland AMBER Centre, IRFU Charitable Trust, and the Anatomical Society"
62825408605
By its avascular nature and limited healing potential, articular cartilage (AC) defects are still challenging to cure, resulting in degenerative diseases such as osteoarthritis. Several clinical techniques aim to repair the AC; however, load-bearing and fully functional tissue recapitulation remain a significant hurdle. In the last few decades, tissue engineering has given hope for resolving the issues associated with the existing therapy methods. Essential innovations in 3D bioprinting technology have led to a greater focus on successfully implementing engineered tissue constructs. For cartilage regeneration, Mesenchymal stem cells (MSCs) are a potential cell source in a unique milieu known as the stem-cell niche, characterized by low oxygen levels. Cobalt is well known for its hypoxic effects in vitro by stabilizing hypoxia-inducible factor (HIF-1α), a central regulator of stem cell fate. The main aim of this study was to evaluate the impact of Cobalt nanowires (Co NWS) on the chondrogenic potential of human umbilical cord MSCs (UMSCs) encapsulated in the PEGDA/Alginate hydrogel. In the current study, cell proliferation, mechanical properties, and the expression of chondrogenic markers were analyzed. Co NWS supplementation into the PEGDA/Alginate hydrogel enhanced the cell proliferation and mechanical properties and showed the upregulation of chondrogenic markers such as SOX 9, COL2A1, and ACAN through the HIF-1α pathway. Together these findings are taken into consideration the potential of hypoxia mimicking hydrogels in the treatment of osteoarthritis.
83767225884
"Introduction. Large critical size bone defect is one of the most challenging pathologies in orthopaedic surgery. NVD‑003 is an autologous scaffold-free cell-based osteogenic implant intended to improve bone healing in severe pathophysiological conditions. This study aims to investigate the therapeutic potential of NVD-003, an osteogenic graft derived from human adipose stem cells.
Methods. NVD-003 consists of autologous osteogenic cells cultured from adipose tissue derived stem cells embedded in their extracellular matrix with hydroxyapatite/beta-tricalcium phosphate (HA/TCP) particles. The bioactivity of NVD-003 was studied in nude rat models: (i) to compare the impact of fresh or decellularized grafts in term of angiogenesis (up to 1 month) in a fibrotic tissue (in a cauterized muscular pocket); (ii) to assess its in vivo osteogenicity (in comparison to HA/TCP particles alone), at 1/2/3 months post-implantation, in an irreversible femoral critical size bone defect. The angiogenesis was quantified by histomorphometry while the osteogenesis was studied by micro-CTscan, histomorphometry and Q-RT-PCR on graft explants. Four paediatric patients (5 to 15 years old) suffering from a congenital pseudarthrosis of the tibia were treated in a compassionate use program. Three months after the adipose tissue procurement, the 3D-grafts were placed into the defect and followed clinically and radiologically.
Results. (i)- Preclinical: After 1-mo of intra-muscular implantation, cellular survival of human cells and the promotion of angiogenesis were observed. The number of blood vessels number per tissue area at 12 weeks post-implantation and the blood vessels area per tissue area at 8 weeks post-implantation were noticeably increased in NVD-003 implants as compared to HA/TCP particles. Quantitative analysis of µCT images at successive imaging time points (4, 8 and 12 weeks) showed that a similar level of mineralization was observed at each time point, indicating the absence of resorption of the test item up to 3 months post-implantation. A complete integration and bone fusion were found for the 3D graft in comparison to HA/TCP alone which revealed a lack of tissue remodelling and osteogenesis. Specific genes of the skeletal development were overexpressed in the bone defect treated with the NVD-003 (at 4/8 weeks post-implantation) while no osteoinduction was found for the HA/TCP particles alone. Changes in bone formation were assessed using histomorphometry measurements of the entire implant site. Bone area showed a trend towards an increase with NVD-003 already at 8 weeks post-implantation, while a noticeable increase in bone area was observed with NVD-003 as compared to HA/βTCP samples at 12 weeks post-implantation.
(ii) Clinical: A large volume (>15cm3) of the autologous 3D graft was manufactured in aseptic areas under GMP requirements for each patient and then implanted without any modification of the surgical procedure. The graft was easily handled, shaped, and implanted. NVD-003 implant demonstrated a continuous remodelling (with bone formation) up to 14 months post-implantation to obtain a sufficient bone fusion (allowing walk without pain) and no recurrence of the disease.
Conclusion. The NVD-003 graft plays a major role to induce angio- and osteogenesis in a complex environment and to recover a bone fusion in a critical-sized bone defect."
41883642808
Supplement-free induction of macrophage polarization solely through the topography of materials is an auspicious strategy but has so far significantly lacked behind the efficiency and intensity of media-supplementation based proto-cols. We investigated Melt-Electrowriting (MEW) for the fabrication of fibrous 3D scaffolds made from poly(ε-caprolactone) (PCL) and advanced the precisely defined inter-fiber spacing from 100 µm down to 40 µm for a variety of pore geometries (rectangular, triangular and round) with the aim to identify structural design criteria for the fabrication of scaffolds with strong topographic immunomodulation for human monocyte-derived macrophages. These scaffolds did facilitate primary human macrophage differentiation towards the M2 type, which was most pronounced for box-shaped pores with 40 µm inter-fiber spacing, but not with the desired efficiency [1].
We then found that human monocyte-derived macrophages show a strong M2a like pro-healing polarization when cultured on type I rat-tail collagen fibers but not on collagen I films. Therefore, we hypothesized that highly aligned nanofibrils also of synthetic polymers, if packed into larger bundles in 3D topographical biomimetic similarity to native collagen I fibers, would induce a localized macrophage polarization. Through integration of flow directed polymer phase separation into MEW we developed Melt-Electrofibrillation, a process that yields nano-fiber bundles with a remarkable structural similarity to native collagen I fibers, particularly for medical grade PCL. These biomimetic fibrillar structures indeed induce a pronounced elongation of human monocyte-derived macrophages and unprecedentedly triggered their M2-like polarization similar in efficacy as IL-4 cytokine treatment [2].
INTRODUCTION: Mesoporous bioactive glass nanoparticles (MBG NPs) have received a significant amount of interest for their potential to treat cancer cells with high drug loading capacity and excellent stability, providing a controlled drug release platform into the malignant tumor [1]. Although enhanced permeability and retention (EPR) effects allow MBG NPs to accumulate at tumor sites, the inferior tumor targeting and ineffective immune escape performance of MBG NPs remain significant challenges. Herein, the tumor targeting and immune escape properties of MBG NPs that were camouflaged with tumor-derived cell membrane and macrophage-derived cell membrane were initially evaluated in vitro and in vivo. And the macrophage cell membrane bionic modified MBG NPs embedding glucose oxidase (QO) drug delivery system (RAW@MBG@QO DDS) was constructed based on its superior performance in immune escape. Finally, we systematically investigated the tumor treatment effect and biosafety of RAW@MBG@QO DDS in order to expand the clinical application of MBG NPs as a drug delivery.
METHODS: MBG NPs were synthesized by a sol-gel method combined with a hydrothermal reaction. For cell membrane camouflage, 4T1 cells and RAW 264.7 cells were separately suspended in a hypotonic lysing buffer and then disrupted using a Dounce homogenizer. The homogenized solution was secondary centrifuged. The two kinds of membranes were collected and mixed with MBG NPs under the sonication induced assembly, respectively. The tumor target and immune escape performance of both cytomembrane coated MBG NPs were analyzed using the in-vitro cell model and the in-situ tumor model, and the RAW 264.7 cells were filtered for developing RAW@MBG@QO DDS. The cytotoxicity, oxidative stress, and tumor suppressive effects mediated by RAW@MBG@QO DDS were further investigated.
RESULTS & DISCUSSION: In the present study, we have successfully constructed two kinds of cytomembrane coated MBG NPs, both of which showed significantly higher tumor targeting compared to MBG. MBG NPs coated with macrophage membrane were more effective at avoiding being phagocytosed and eliminated by the immune cells. Following that, our results indicated that RAW@MBG@QO DDS could release QOs in a controlled fashion to induce the oxidative stress and apoptosis on tumor cells using the catalytic reaction of glucose oxidase, and showed excellent tumor suppressive effect in vivo. Meanwhile, RAW@MBG@QO DDS had the advantage of QO-controlled release, which effectively mitigated blood homeostasis interference and avoided potential toxicological risks.
CONCLUSIONS: Our findings suggest that the cell membrane camouflage MBG NPs embedding glucose oxidase drug delivery system exhibits high efficiency for tumor suppression, which can promote the development of MBG NPs serving as as excellent drug delivery vehicles for cancer therapy.
ACKNOWLEDGEMENTS: This work was supported by grants from National Natural Science Foundation of China (31900956, 81971751) and Shanghai Sailing Program (19YF1427400).
REFERENCES
[1] E. Sharifi, A. Bigham, S. Yousefiasl, M. Trovato, M. Ghomi, Y. Esmaeili, P. Samadi, A. Zarrabi, M. Ashrafizadeh, S.J.A.S. Sharifi, Mesoporous Bioactive Glasses in Cancer Diagnosis and Therapy: Stimuli‐Responsive, Toxicity, Immunogenicity, and Clinical Translation. Advanced Science, 2021: 2102678.
94238155587
The reconstruction and replacement of musculoskeletal tissues have been extensively investigated in the last decades. Trauma injuries and degenerative diseases are the most common causes reported worldwide. Stem cells play an important role in tissue regeneration and have been successfully applied in musculoskeletal research, especially due to the low self-repair capability of tendons, ligaments, and joints. Due to the specialized architecture of native musculoskeletal components, mostly related to the complex interplay between multiple tissues, interface regions between hard (bone) and connective tissues (cartilage) are challenging to be engineered. When it occurs, they often fail during implantation due to the lack of appropriate mechanical properties. This challenge is even more exacerbated in the temporomandibular joint (TMJ), which is composed of several anatomical structures such as the articular disc, jaw, mandible, muscles, and tendons that connect the scapula, sternum, and neck. The TMJ performs complex movements under compression and tension during common activities such as talking, chewing, and biting. In this context, multi-material approaches that combine different manufacturing techniques can be very promising for interfacial tissue engineering of the TMJ. Hence, the objective of this work was to evaluate the integration of polycaprolactone-polylactic acid (PCL-LA copolymer) fibrous scaffolds produced by melt-electrowriting (MEW) with bioprinted constructs made of xanthan gum (XG) hydrogel and mesenchymal stem cells. MEW meshes were manufactured at 10 mm/s, 170 °C, 0.8 bar of pressure, 6 kV and 4 mm of height. Four-layered constructs were bioprinted varying speed from 40-60 mm/s and pressure from 50-70 KPa using smooth flow tapered tip. Morphological aspects regarding filaments size and porosity of both manufacture techniques were quantified through optical and scanning electron microscopes. Stability in culture media for 28 days was also analyzed. Regular and well-defined PCL-LA meshes were obtained using MEW. Constructs with satisfactory shape fidelity were also obtained through bioprinting. To analyze the most appropriate strategy to improve integration, stability, and mechanical properties between PCL-LA meshes and XG bioprinted constructs, a double crosslinking network has been investigated. First, ionic crosslinking of XG using trivalent iron ions, followed by a photocrosslinking step using acrylate groups in MEW meshes. Overall, based on the hybridization between both processing techniques, employing a multi-material approach, as well as including a double crosslinking strategy we hypothesize that promising interfacial tissues with improved mechanical properties can be obtained. The potential application of the multi-material herein explored are analyzed as a replacement for the multi-tissue temporomandibular joint.
83767238355
"Introduction
Biodegradable polymeric scaffolds face a growing use in tissue engineering. However, changes in material properties during degradation can impact drastically the scaffold durability and therefore the efficiency of tissue reconstruction. Few studies focus on approaches allowing the prediction of the scaffold lifetime, while there is a need for strategies using accelerated testing protocols and versatile tools to easily investigate on the material degradation rate. In the present study, we investigated the thermally-accelerated ageing and lifetime prediction in culture medium of cross-linked poly(ester-urethane-urea) (PEUU) scaffolds [1].
Methodology
Elastomeric cross-linked poly(ester-urethane-urea) (PEUU) scaffolds have been developed through an emulsion technique allowing to produce highly interconnected porous structure [2]. Thermally-accelerated ageing was performed in cell culture medium at different temperatures: 37°C, 55°C, 75°C and 90°C. The degradation process was followed by gravimetry, swelling measurements, compression tests and Fourier-Transform infrared spectroscopy (FTIR). Compressive set measurements were also used as an indicator of the scaffold lifetime at 90°C.
Results
The study revealed that the PEUU scaffold degradation was associated with the hydrolytic instability of ester groups. As expected, the scaffold chemical composition variation over degradation was temperature dependant since the absorbance intensity associated to the ester stretching vibrations decreased with rising incubation time and temperature. Therefore, FTIR spectroscopy was used as a quantitative indicator of the hydrolysis content. The dependence of ester group cleavage on time of incubation was determined for each degradation temperature by regression analysis and Arrhenius type extrapolation was used to estimate the activation energy of the hydrolytic degradation reaction (80.84 kJ mol−1).
In the present study, the compressive set was selected as the failure criterion from the point of view of the scaffold functionality. For elastomeric material, the compressive set should not equal or exceed a value of 25%. Since the compressive set measurements set the scaffold lifetime at 90°C around 11.6 days of incubation in the degradation medium, the scaffold lifetime at 37°C was estimated to 1131 days (3.1 years) using an acceleration factor f equal to 97.5 as derived from the activation energy value.
Conclusion
It is well known that it is difficult to correlate in vitro degradation with in vivo expectation since in vivo conditions are more complex and lead to variation of the scaffold lifetime. However, the approach developed in this study could be a convenient way to simply and straightforwardly screen the durability of scaffolds when performing experimental design aiming to tailor scaffold lifetime.
References
1. Langueh, C. et al., Polym. Deg. Stab. 183, 109454 (2021).
2. Changotade, S. et al., Stem Cells Int. Article ID 283796 (2015).
Acknowledgements
The authors thank the Ministère de l’Enseignement Supérieur, de la Recherche et de l’Innovation for the MENRT scholarship granted to Credson Langueh."
41883607404
"Introduction:
Highly porous biodegradable scaffolds made of polycaprolactone (PCL) and ceramic designed as three-dimensional (3D) guiding structures with rectilinear filling to facilitate bone regeneration have successfully been translated from preclinical studies into clinics as part of a scaffold-guided bone tissue engineering (SGBTE) concept. However, advances in 3D printer technology now allow the fabrication of novel scaffold structures, such as those with Voronoi design, which are potentially better able to mimic natural bone properties than those printed with rectilinear infill. The 3D Voronoi tessellation is based on random discrete seed points used to create cells that form a highly porous network structure with high mechanical strength. To pave the way for successful implementation, further developments of the SGBTE concept, such as the use of novel Voronoi scaffold design, are based on the principles of rigorous in vitro and preclinical in vivo testing to evaluate biocompatibility, biomechanical stability and tissue integration capacity.
Methodology:
Tubular composite medical-grade PCL hydroxyapatite (HA; wt% 96:4) scaffolds (outer diameter 10 mm, inner diameter 4 mm, height 15 mm) with 3D Voronoi tessellation were 3D-printed using additive manufacturing (BellaSeno GmbH, Germany). Scaffold porosity was assessed using micro-CT scanning (μCT 50, Scanco Medical AG, Switzerland). To determine the Young’s modulus, unconfined, uniaxial compression tests (2 kN load cell) were performed under simulated physiological conditions of 1% phosphate-buffered saline (PBS) solution (37°C) with strain rate of 0.1 mm/s (30 kN Instron 5567, Melbourne, Australia). In vitro hydrolytic degradation was assessed over time by performing scanning electron microscopy (SEM), mass loss, gel permeation chromatography (GPC) and differential scanning calorimetry (DSC) at time points of 0 (baseline), 30, 60, 90, 120, 150, and 180 days. During the 180 days PCL-HA scaffolds were immersed in sterile 1% PBS (10 ml) in closed 15 ml tubes, to avoid evaporation, and maintained in incubator at 37°C. At each time point, samples were washed three times with deionized water and incubated in vacuum overnight at 37°C before assessment. Biocompatibility of subcutaneously implanted scaffolds loaded with freshly harvested sheep bone graft materials was assessed using an an ectopic bone formation model of athymic nude rats. Assessment methods for in vitro and in vivo characterization included histology, immunohistochemistry, SEM and histomorphometry.
Results:
Scaffold µCT assessment revealed high mean porosity of 72.8% (± 0.94) (n=8). Further, the Youngs modulus of scaffolds (n=5) was 11.4 MPa (± 1.1). The PCL-HA scaffolds (n=7) exhibited slow degradation behaviour over the 180-day assessment period as observed with SEM, mass loss calculations, and molecular weight changes as determined by GPC and crystallinity with DSC. Immunohistochemistry, Goldner's trichrome staining and SEM analysis of specimens (n=8) collected from rats (n=2) eight weeks after implantation show integrative physiological response at the interface between scaffold and different types of bone graft without signs of inflammatory reaction.
Conclusion:
High porosity and favourable biomechanical properties, along with slow and predictable degradation was observed. Histological examination showed good biocompatibility with no adverse host tissue reactions, making PCL-HA scaffolds with Voronoi design a suitable candidate for use in SGBTE."
52354525067
Inflammation is a protective response to damaged tissue and foreign bodies, such as biomaterials, and is usually considered to be negative. More recently, however, the active anti-inflammatory, pro-regenerative role of various mediators and inflammatory cytokines from immune cells have become widely recognised. In particular, macrophages play an essential mediating role in modulating inflammation and thus macrophage phenotype and function has received considerable attention.
Nanoclay has attracted attention in the field of regenerative medicine due to the inherent osteogenic bioactivity and ability of nanoclay to interact with proteins [1,2]. However, to date, no studies have explored how macrophages respond to nanoclay in terms of immunomodulation potential.
In this study, we have evaluated macrophage responses to nanoclay particles. Mouse bone marrow-derived macrophages were isolated from balb/c male mice (4-8 weeks old) and were cultured with various concentrations of nanoclay particles (50, 100, 500, and 1000 µg/ml) for 1 and 3 days. Intracellular and extracellular macrophages were observed by transmission electron microscopy, and the localisation of nanoclay particles in the cells was confirmed by energy-dispersive X-ray spectroscopy (EDX). Macrophage phenotype was evaluated by flow cytometry, and the concentrations of pro-and anti-inflammatory cytokines in culture media were measured by enzyme-linked immunosorbent assay (ELISA). Furthermore, the expression levels of pro-and anti-inflammatory cytokine related genes were assessed by quantitative polymerase chain reaction.
Macrophages actively phagocytosed nanoclay particles, regardless of nanoclay concentration. The nanoclay particles were found in extracellular macrophages, near actin filaments and intracellularly, e.g. within phagosomes and lysosomes. In the presence of 100 µg/ml nanoclay particles, in cell culture media, the population of M1-like macrophages dramatically increased for 24 hours (p<0.034). However, after 3 days, the population of M1-like macrophages decreased, but the number of M2-like macrophages increased. Similarly, significantly higher levels of anti-inflammatory genes, interleukin (IL)-10 and transforming growth factor (TGF)-β1, were observed in macrophages cultured in 100 µg/ml nanoclay particles for 3 days (p<0.017, p<0.032). For Tumour Necrosis Factor (TNF)-α, a pro-inflammatory cytokine, macrophages cultured with nanoclay particles, regardless of their concentrations, showed a significantly lower gene expression level compared to the macrophage without nanoclay (p<0.039). The current studies demonstrate the potential of nanoclay to modulate the phenotype of macrophages in vitro. Nanoclays can promote the development of M2-like macrophages expressing enhanced levels of the inflammatory genes, IL-10 and TGF-β1 with important implications therein for reparative processes in tissue engineering.
Reference
1. Shi P. et al. Adv. Healthcare Mater. 7, 1800331 (2018).
2. Dawson J.I. & Oreffo. R.O.C. Adv. Mater 25, 4069-4086 (2013).
Acknowledgement
Supported by an MRC-AMED Regenerative Medicine and Stem Cell Research Initiative (ref.MR/V00543X/1).
94238143146
The main risk factors for aseptic loosening and implant failure are wear and corrosion of metal implants. Despite a passive oxygen layer forming on many implant materials, electrochemical reactions can occur [1]. Oxidation and reduction reactions take place, resulting in a constant exchange of electrons and ions between the metal and the surrounding fluid [2]. The release of metal ions in different oxidation states can be promoted by the acidic pH caused by specific cells [3]. This in turn may have direct effects on the surrounding tissue and its cells [4]. The cytotoxic effects of nickel, cobalt, and chromium are mainly manifested by apoptosis, necrosis, and inhibition of DNA repair mechanisms [2,5]. However, the various metal ions differ in their local and systemic effects [2,3]. It is known that some metallic corrosion products can induce an inflammatory response in the implant periphery [4]. In this context, signal transduction processes influenced by metallic corrosion products may have an impact on cellular differentiation and immune response. The aim of this study is to better evaluate the low-threshold, "adapted" cellular responses.
In the present study, we treated mesenchymal stem/stromal cells (MSC) in vitro with different metal ion concentrations (starting with 10 µM nickel(II) chloride, NiCl2; cobalt(II) chloride, CoCl2, and chromium(III) chloride, CrCL3) and subsequently analyzed cell number and metabolic activity. In addition, the influence of the non-toxic metal ion concentrations (10 and 100 µM) on energy metabolism (more specifically, mitochondrial activity and extracellular acidification) was investigated by the Seahorse Analyzer (Agilent).
When exposed to higher ion concentrations (100 µM and above), the number of cells was reduced over the 3-day period with no evidence of cell death. This decrease correlated with relative metabolic activity (determined by MTS conversion assay). Specific examination of energy metabolism showed a reduction in basal respiration upon treatment with the metal salts CoCl2 and NiCl2. The trivalent chromium salt had no effect on basal respiration. In contrast, extracellular acidification, indicating glycolytic energy metabolism, was shown to be increased by NiCl2 and CoCl2 in a concentration-dependent manner over the course of the 3-day treatment. Again, CrCl3 exposure had no marked effect.
These in vitro results demonstrate that metal ions, as potential corrosion products of metal implants, can have a significant effect on cells even at non-toxic concentrations. Therefore, in order to prevent or treat aseptic loosening, the complex mechanisms of corrosion-induced biological reactions should be fully elucidated.
This work was financially supported by funds from the Rostock University Medical Center (KOBE project "Entzündungsmodulierende Eigenschaften metallischer Korrosionsprodukte") and the European Union as well as the Federal State Mecklenburg-Vorpommern (EFRE project-No. TBI-V-1-141-VBW-116).
References:
1. Matusiewicz, H. et al., Acta Biomater. 10, 2379–2403 (2014)
2. Sansone, V. et al., Clin. Cases Miner Bone Metab. 10, 34–40 (2013)
3. Scharf, B. et al. Sci. Rep. 4, 5729 (2014)
4. Jonitz-Heincke, A. et al. Materials 12, 2771 (2019)
5. Gibon, E. et al. J. Biomed. Mater. Res. B Appl. Biomater. 105, 2162–2173 (2016)
73296356408
Although the secretome of mesenchymal stem cells (MSCs) has a great potential to be used in CNS regenerative therapies, tissue derived MSCs are a limited source which often need to be surgically harvested and present intrinsic donor variability. Induced pluripotent stem cells (iPSCs) conversely, can be easily generated and highly expanded from accessible somatic cell sources and differentiated into mesenchymal stem cells (iMSCs), which have been described to present a rejuvenated phenotype. Here, we aim to compare the replicative senescence of bone marrow derived MSCs (BM-MSCs) and iMSCs under human platelet lysate (hPL) supplementation and address its impact on secretome composition. For that, we have compared the proliferation and replicative senescence of iMSCs and BM-MSCs on a long-term culture, by cumulative population doublings, expression of senescence associated β-Gal (SA-β-Gal), P16-INK4A, P53 and P21 gene expression and the increase in cell area (hallmarks of senescence). Finally, we compared BM-MSC and iMSC secretory profiles by a non-targeted mass spectrometry approach, evaluated the concentration of important neuroregulatory factors on the secretomes of early and late passage cells from both populations with a membrane-based antibody array, and assessed how the differences seen in these factors could affect their immunomodulatory capability with a mouse mixed glial culture. We show that iMSCs and BM-MSCs under hPL supplementation maintain their MSC properties and that iMSCs displayed higher proliferation, were capable of a higher total number of duplications and presented a decreased percentage of SA-β-Gal positive cells, decreased P16-INK4A and P21 gene expression and decreased increase in cell area compared with BM-MSCs. Furthermore, iMSCs and BM-MSCs presented a very similar secretory profile, with only 2 out of 136 proteins being significantly different in concentration in the proteomic analysis. Finally, the secretomes of both cells presented important neuroregulatory factors, with both having an upregulation of IL-6 and IL-8 at late passages which corroborated with a decreased immunomodulatory capability. In conclusion, we show that iMSCs can be expanded in hPL and that they have a similar secretory profile to BM-MSCs, but present decreased replicative senescence, therefore being a promising and more standardizable alternative to produce large quantities of secretome as needed for clinical purposes.
62825430249
"Bioinspired polymer processing, with focus on improved control over biomaterial structure-function, is a research strategy that can play a critical role in facilitating the translation of a biomedical device. In this work, we utilize the specific example of tissue engineered heart valves to demonstrate this notion.
Valvular heart disease is currently treated with mechanical valves, which benefit from longevity, but are burdened by chronic anticoagulation therapy, or with bioprosthetic valves, which have reduced thromboembolic risk, but limited durability. Tissue engineered heart valves (TEHV) have been proposed to resolve these issues by implanting scaffolds designed to be replaced by endogenous tissue growth, leaving autologous, functional leaflets. This approach would putatively eliminate the need for anticoagulation and avoid calcification. Human heart valve tissue structure-function is still inadequately characterized and, despite the progress in scaffold fabrication strategies and encouraging results in large animal models, control over engineered valve structure-function remains at best partial. Moreover, while the notion of bioinspired control of structure and function is recognized as a promising strategy to enhance TEHV performance, the approach and its potential impact remain relatively unexplored in vivo.
We face these challenges by introducing double component deposition (DCD), a polymer electrodeposition technique that employs multi-phase electrodes to dictate valve macro and microstructure and resultant function. Engineered valve in vitro characterization included: leaflet thickness, biaxial and bending properties, and quantitative structural analysis of scanning electron micrographs. Results demonstrated the capacity of the DCD method to simultaneously control scaffold macro-scale morphology, mechanics, and microstructure while producing fully assembled multi-leaflet valves composed of microscopic fibers. The efficacy of this technology was further assessed in vivo in an acute (24 hrs) porcine model with the evaluation of three different devices: stented pulmonary valve (n=5), stentless tricuspid valve (n=5), and stentless mitral valve (n=2). Processing variables for these scaffolds were set to duplicate native heart valve tissue structural properties.
More recently, bioinspired DCD processed scaffolds have been implanted in an ovine model of pulmonary valve replacement with time point 1 (n=4) and 3 months (n=4,). Two groups were compared: scaffold with physiological leaflet thickness (120 µm) and scaffolds with over physiological leaflet thickness (240 µm). Explants at 1 month have shown a substantially higher extracellular matrix (ECM) production for the physiological thickness group. While these results suggest a more favorable tissue remodeling outcome for the physiological group and support the biomimetic approach, the mechanism for these preliminary observations remains unknown and re-iterate the urgent need for in-vitro platform able to elucidate the complex ECM process of ECM formation in vivo."
41935604205
Introduction
There is a large number of synthetic polymers, which are generally suitable as implant materials due to their chemical, biological or mechanical properties. What many of them have in common, however, is the challenge of growing properly into the body, which poses design demands specifically to the implant’s surface. Requirements that many conventional plastic processing methods cannot meet, especially in the small size range. Here, we leverage the fiber forming technology Melt Electrowriting to additively structure polymer surfaces.
Methodology
We developed a method to apply a structured fiber layer onto solid polymer surfaces via Melt Electrowriting and showed this for various polymers used in biomedical applications. The resulting surfaces were assessed via contact angle measurements and cell adhesion tests. SEM confirmed the accuracy of the surface structuring patterns.
Results
Fiber pattern and sizes characteristic of Melt Electrowriting were successfully obtained on top of solid polymer surfaces. The effect of the deposited fibers on the surface properties were shown for different biocompatible polymers. Specifically, contact angle measurements and cell adhesion experiments showed promising results for the application as tissue engineering scaffolds.
Conclusion(s)
This study shows a valuable approach to optimize implant surfaces via Melt Electrowriting.
83767274888
Introduction
Melt Electrowriting (MEW) is an versatile electric-field assisted fiber forming technique that has convincingly shown its potential for tissue engineering scaffolds both in vitro and in vivo. The additive manufacturing principle of MEW offers unparalleled possibilities to create precisley defined fibrous 3D architectures. The potential of design freedom with MEW is still largely unexplored. Here we present strategies for the automated design and digital fabrication of highly tunable ansisotropic scaffolds for in situ tissue engineering.
Methodology
We developed a MATLAB G-code design suite to automatically generate toolpath commands for the MEW setup. Scaffolds were fabricated from medical grade polycaprolactone (PCL). Their architecture and fiber diameter were assessed by scanning electron microscopy (SEM). Mechanical properties were determined by tensile testing, ingrowth of human umbilical artery smooth muscle cells was verified after 1 and 7 days of culture via SEM and (immuno)histology. Finally, the design strategies were validated for tubular constructs.
Results
All MEW scaffolds closely matched the coded designs. Highly tunable architectures were obtained, with fiber orientation and pattern strongly affecting the mechanical properties and anisotropy. Progressing cell ingrowth was verified in vitro after 1 and 7 days. Tubular scaffolds were exploited to show their potential for cardiovascular tissue engineering applications.
Conclusion(s)
This work further expands the capabilities of MEW towards the rational design and digital fabrication of fibrous scaffolds with controlled architectures and corresponding mechanical properties.
73296336444
Cardiovascular diseases are the major cause of death worldwide. The lack of autologous vessels that can be used in cardiovascular surgeries compel engineers to look for nowel solutions. The main assumption of vascular tissue engineering is to design and produce functional materials that replace damaged blood vessels and restore their proper functions. Tissue-engineered vascular grafts with diameters >6mm are available on the market. However, the design and manufacture of prostheses with diameters ≤6mm is still a challenge for scientists due to their low hemocompatibility and thrombogenicity.
In this study two types of cylindrical layered structures with internal diameters ≤6mm were produced by the solution blow spinning method. The prostheses differed in the morphology of the internal surface. The first type of prostheses was characterized by a nanofiber inner surface, and the second was characterized by a solid one with small fibrous areas. The mechanical properties of the manufactured dentures were tested and compared. Then the surfaces of the prostheses were coated with polydopamine and biomolecules such as amino acids, short peptide sequences or polysaccharides were attached. The influence of the morphology of the internal surface and the presence of biomolecules on the hemocompatibility of the structures was investigated.
SEM analysis of grafts cross-sections has shown that manipulation of the solution blow spinning process parameters allows for the production of layered structures with differentiated morphologies of layers. Designed prostheses show high flexibility (Young’s modulus value of about 2.5MPa) and tensile strength (maximum load value of about 60N). Grafts produced of medical-grade polyurethanes do not cause hemolysis. Activation and adhesion of blood elements to the inner surface of the prosthesis depend on its morphology. Fewer platelets were observed on nano-fibrous surfaces than on microfibrous/compact surfaces. Modification of the surface of prostheses with biomolecules also reduced the number of attached platelets.
In conclusion, the solution blow spinning method allows the production of layered cylindrical structures with internal diameters ≤6mm and desired mechanical properties, while the surface morphology and attached biomolecules affect the number of attached and activated platelets.
This project was funded in part by National Science Centre, Poland, grant number: 2020/39/I/ST5/01131.
62825427755
Collagen fiber network architecture in the native heart valve leaflets is characterized by preferential orientation and curvilinear arrangement that allow adequate stress distribution and effective leaflet coaptation. Specifically for the mitral valve, collagen fibers are preferentially aligned towards the circumferential direction with a curvilinear arrangement that runs from the posteromedial to the anterolateral commissure1. Tuning the collecting target tangential velocity is a common strategy shared by several techniques such as melt-electrowriting2, jet-spinning3, and Double-Component-Deposition (DCD)4 to achieve physiologically relevant structural anisotropy and circumferential alignment in the belly region of the valve scaffold. Similarly, conical shape collecting targets have been previously presented to obtain curvilinear arrangement in valve scaffolds5. However, the two methodologies cannot be combined. Tangential velocities can only induce circumferentially aligned straight fibers along the 3D geometry of a scaffold, while using conical mandrels produces curvilinear arrangement, which is strictly limited to 2D, planar scaffolds. In addition, the high tangential velocities requested to achieve physiologically relevant anisotropy are generally associated with deposition artifacts in complex 3D scaffold geometries. While the DCD processing method we previously introduced4, utilizes a collecting target made of an electrically conductive and a non-conductive component, this target design enables to manipulate the electrical field at the macro-scale and allows to recapitulate valve anatomy and to dictate various microarchitectural parameters, including fiber diameter and pore size. Yet, the curvilinear arrangement of the fiber network could not be achieved. In this study, we further advance the notion of DCD by manipulating the electrical field of the collecting target with mesoscopic grooves designed to induce local anisotropy and fiber undulation. A micro-grooved cylindrical copper mandrel was used as collector. To evaluate the effects of the groove geometry on the fiber deposition, three variables were considered: width, depth, and frequency which were set as equal to 50, 100, and 150µm. A cylindrical smooth mandrel was utilized as control. A tangential velocity of 0.26m/s, which normally generate isotropic scaffold on flat surfaces, was used for all the fabrications. The spatial electric field distribution was simulated in COMSOL-Multiphysics®. Morphological and mechanical properties of fabricated PEUU scaffolds were characterized by scanning electron microscopy, and biaxial tensile test. The width resulted in being the most effective parameter in terms of its capacity to induce statistically significant levels of circumferential fiber alignment and mechanical anisotropy. This notion was transferred to a collecting target specifically designed to reproduce the three-dimensional anatomy of the mitral valve, demonstrating control over fiber alignment and posteromedial-anterolateral commissure curvilinear arrangement. The in-silico model simulations allowed to visualize the electrical field distribution produced by the groove pattern and elucidate the likely mechanism of fiber deposition associated with local anisotropy at the tissue scale and the curvilinear fiber network at the organ level scale. This seminal study introduces a novel approach to design collecting targets for electro-deposition to advance biomimetics in HV engineering.
1.Rausch, et al., Biomech&mod. in MB, 12.5:1053-1071(2013).
2.Saidy, et al., Front.BEBT, 8:793(2020).
3.Capulli, et al., Biomaterials, 133:229-241(2017).
4.D'Amore, et al., Biomaterials, 150:25-37(2018).
5.Hobson, et al., JBMMR-Part A, 103.9:3101-3106(2015).
83767237387
INTRODUCTION
Cardiovascular disease is one of the major causes of death worldwide [1]. Synthetic vascular grafts (SVG) and autograft vessels are the current treatment modalities but, are ineffective for vessels with a diameter lower than 6 mm due to compliance mismatch [2] and limited in both supply and anatomical variability, respectively. An alternative solution is via tissue engineered vascular grafts (TEVG) [2-3] which aim to match the mechanical and biological properties of native vessels [3].
Melt electrospinning writing (MEW) is a recently developed technique that allows for the layer by layer assembly of micron diameter fibres in highly organised architecture that can be tuned to mimic the collagen fibre orientations found in the native vessel wall [3]. Therefore, the aim of this project is the development of a VG able to overcome current limitations of compliance mismatch and poor endothelialisation causing clot formation.
METHODOLOGY
A custom-made MEW printer was used to direct the deposition of polymeric micron-scale fibres in a bioinspired direction. Different aspect ratios were investigated in planar conformation to better tune mechanical behaviour, cell alignment and matrix deposition, which was then translated into a tubular conformation, through the use of a rotating mandrel.
MEW bioinspired scaffolds were infiltrated with a lyophilised fibrinogen sponge functionalised with heparin to prevent clotting. This hybrid construct was further wrapped in an electrospun elastic PLCL sheath to seal the graft.
Scanning electron microscopy (SEM) was used to investigate morphological characteristics. Pore size, porosity and degradation rate of the fibrinogen was also assessed for different crosslinking agents. Ring tensile test was used to investigate the mechanical properties of the grafts and compare them to those of a native porcine tissue. Biological evaluation of cell behaviour and extra-cellular matrix (ECM) production were performed to identify the best aspect ratio. Hemocompatibility and endothelialisation assay were also performed to validate the use of this off-the-shelf VG.
RESULTS
Our data demonstrates a preferential alignment of cell as well as ECM deposition along the major diagonal. The presence of fibrinogen enhanced cell seeding efficiency and ECM production while not effecting alignment and orientation. Mechanical data reported a response that resembles the typical J-shape of native tissue. The addition of a highly elastic layer of electropsun PLCL allowed for a higher resistance in deformation and recovery, additionally, the permeability was improved. The successful implementation of heparin allowed for a reduction of platelet adhesion that combined with a non-haemolytic behaviour demonstrate the suitability for vascular system application.
Thus, this bio-hybrid multi-layered graft represents a novel off-the-shelf solution to overcome current limitations of TEVG.
CONCLUSIONS
We successfully tuned tubular scaffold architecture, demonstrating high control and versatility. The proposed mimetic bio-hybrid scaffold was identified as the ideal candidate to recapitulate mechanical properties, anatomical fibre orientation and ECM deposition of native vessel. Moreover, implementation of heparin demonstrates it suitability in an environment in contact with blood.
References
[1] H.H.G.Song et al. CellStem 22, 2018. [2] A.Hasan et al. Acta Biomater.10, 2014. [3] M.J.McClure et al. J.Drug Deliv.Sci.Technol. 21, 2011.
20941801806
"Introduction: Ischemic heart disease is a major cause of human death worldwide owing to the heart’s minimal ability to repair following injury. Despite medical advances, current treatments are not able to regenerate the damaged heart tissue. Therefore, alternative strategies are being assayed to identify the proper strategy to induce heart regeneration. In this sense, cardiac tissue engineering aims at obtaining cardiac constructs that mimics the native myocardium. Although major advances have been achieved in this respect, the generation of functional human mature tissue with physiological myocardial architecture and function to native adult myocardium remains a major obstacle. To address this, we have generated a 3D printable design that recapitulates not only the physical myocardial milieu, but importantly, 3D myocyte alignment, which is key to generation of maximum contractile force generation and therefore, maximum therapeutic efficacy.
Methodology: The designs have been 3D printed using Melt Electro Writing, an advanced printing technology uniquely capable of reproducing the properties of the cardiac extracellular matrix. Human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (hiPSC-CMs) and cardiac fibroblasts (CFs) were embedded in a hydrogel (Fibrin or GelMA) and combined with MEW-designs in a composite system. Different ratios of hiPSC-CMs and -CFs (100:0, 95:5, 90:10 and 80:20 for CM:CF), as well as scaffolds fiber thickness (0.20, 0.35 and 0.70 mm) were evaluated for optimal tissue formation. Cardiac engineered tissues were maintained in culture for up to 4 weeks and characterized by histology, staining, ultrastructure, RNAseq, metabolic and electrophysiological analysis.
Results: Overall, contractile human cardiac minitissues can be generated using both fibrin or GelMA hydrogels reinforced with 3D MEW printed structures displaying cardiomyocytes well distributed throughout the scaffold. No statistical differences were found between fibrin-based tissues containing only cardiomyocytes compared with those containing fibroblasts, in terms of beat rate, metabolic activity, gene expression, conduction velocity and activation frequency. However, contractile capacity for fibrin-based engineered tissues was markedly superior. Additionally, a longer remodeling process was required for GelMA-based tissues, whereas fibrin ones displayed an earlier coordinated beating. Also, cell and gel detachment were observed in GelMA-based scaffolds, making necessary the addition of high number of fibroblasts (20%) for optimal tissue formation. Metabolic maturation and transcriptomics analysis also highlighted the differences elicited by the choice of hydrogel. In summary, fibrin-derived tissues exhibited improved biological, structural, and mechanical properties compared with GelMA-based constructs. In vitro findings suggested that the composite fibre-hydrogel system may be a more suitable option for tissue-engineered heart repair.
Conclusion: We are progressing towards the rational development of engineered human cardiac tissues by a precise assessment of the main components, mimicking the unique 3D organization of the native heart architecture. Our results highlight the relevance of the choice of ECM-mimic (hydrogel), and provide and in-depth characterization of their differential effects upon the biology of the resulting tissues."
52354521444
Introduction
Currently used prosthetic heart valves show multiple limitations, including a reduced ability to regenerate. In this study we developed a three-layered electrospun heart valve using a dual electrospinning setup with a special 3D printed collector. In this manner, not only the microscopic but also the macroscopic structure of native heart valves was imitated. Biocompatibility of the used material constitutes an important property for clinical application. Re-seeding with human cells could allow for a regenerative approach.
Methodology
A heart valve shaped template was designed in commercial computer aided design (CAD) software, subsequently 3D printed and used for dual electrospinning. The polymers polycaprolactone (PCL) and polyurethane (PU) were electrospun from opposite sides onto a rotating collector (voltage = 15kV; flow rate = 3 ml/h; rotation speed for aligned fibers = 1520 rpm and 38 rpm for unaligned fibers). In a multistep approach scaffolds consisting of different layers with aligned or unaligned fibers were fabricated. Quality, morphology and orientation of the fibers were evaluated with fluorescence and scanning electron microscopy (SEM). Percentual porosity was assessed with gravimetric measurement. Biomechanical properties were determined by uniaxial tensile tests. Pseudomonas cepacia lipase was used for PCL degradation. Evaluation of the biocompatibility was achieved by static seeding of aligned and unaligned scaffolds with human fibroblasts. Cellular behavior was analyzed with SEM, histological and immunofluorescence microscopy.
Results
By CAD and 3D printing, it was possible to create an individual electrospinning collector, which precisely reproduces the macroscopic shape of a native heart valve. Thus, three-dimensional heart valve leaflets could be fabricated by using the collector in a dual electrospinning setup. To recreate the three layers (fibrosa, spongiosa, ventricularis) of the native valve, fibers were aligned circumferentially, randomly and radially. Homogenous, highly aligned (angle between fibers = 5.79 ± 1.61°) and unaligned fibers (no correlation possible) could be fabricated. Aligned fibers showed significantly higher tensile strength along the fiber direction than against it (15.72 ± 4.66 N/mm² vs. 1.83 ± 0.67 N/mm²; p<0.001). Unaligned layers had an overall tensile strength of 6.48 ± 2.3 N/mm². High percentual porosity (85.81 ± 1.59% for aligned and 83.49 ± 1.74% for unaligned fibers) in all layers of the scaffold could be demonstrated. Especially within the unaligned dual spun scaffolds the percentual porosity could be significantly enhanced (89.3 ± 2.95%; p<0.001) by dissolving the PCL using enzymatic degradation. A homogenous monolayer of adherent fibroblasts on the surface of the scaffolds was observed in SEM, histological and immunofluorescence staining. Furthermore, evaluation with SEM showed the formation of fibrin nets. This confirmed the biocompatibility of the material and its appropriate surface for cellular adhesion.
Conclusion
We established the development process of a biocompatible three-layered composite heart valve that replicates the fiber morphology as well as the geometry of a native aortic valve. The dual electrospun material was successfully seeded with fibroblasts, making it suitable for a regenerative approach. This method allows for individualized heart valve replacement by adjusting inserts of the 3D printed collector using personalized data e.g., CT scans.
94238121848
Cellular senescence is characterized by an irreversible cell cycle arrest and a pro-inflammatory senescence-associated secretory phenotype (SASP), which is a major contributor to aging and age-related diseases. Clearance of senescent cells has been shown to improve brain function in mouse models of neurodegenerative diseases as well as obesity. However, it is still unknown whether senescent cell clearance alleviates cognitive dysfunction during the aging process. To investigate this, we first conducted single-nuclei and single-cell RNA-seq in the hippocampus from young and aged mice. We observed an age-dependent increase in p16Ink4a senescent cells, which was more pronounced in microglia and oligodendrocyte progenitor cells and characterized by a SASP. We then aged INK-ATTAC mice, in which p16Ink4a-positive senescent cells can be genetically eliminated upon treatment with the drug AP20187 and treated them either with AP20187 or with the senolytic cocktail Dasatinib and Quercetin. We observed that both strategies resulted in a decrease in p16Ink4a exclusively in the microglial population, resulting in reduced microglial activation and reduced expression of SASP factors. Importantly, both approaches significantly improved cognitive function in aged mice. Our data provide proof-of-concept for senolytic interventions' being a potential therapeutic avenue for alleviating age-associated cognitive impairment.
41935603186
T.B.C.
41935604206
Senescent cells, induced by various stressors, present a heterogenous population of irreversibly cell cycle arrested cells. They release pro-inflammatory compounds into surrounding tissue, collectively known as senescence associated secretory phenotype (SASP). Higher frequency of senescent cells is present during the developmental phase, regeneration and in aged organisms. Their accumulation during the ageing process increases risk and severity of age-related pathologies and is associated with formation of chronic wounds. Skin is a heterogenous organ and markers of senescence have been detected in several types of cells including keratinocytes, fibroblasts and melanocytes. As drugs targeting senescent cells show specificity towards selected pathways, that are known to be more active in some types of skin cells over others, it is of primary importance to decipher common and differential signatures of senescence in different populations of skin cells.
Here we present our preliminary in silico results on defining and characterizing phenotypes of senescent skin cells. We generated a database consisting of 19 studies with publicly available 10x genomics single-cell-RNA-sequencing (sc-RNA-seq) datasets of mouse skin from the developmental stage (embryonic), regeneration (wounded/unwounded) and the aging process (young, adult, old). To our knowledge this is the biggest sc-RNA seq dataset of mouse skin generated to date. Our dataset was created by harmonising and clustering readouts from all the studies (78 samples comprising 406.563 cells) in order to identify specific cell types. This dataset was used to unravel the phenotypic characteristics related to cellular senescence of different populations of mouse skin cells. Moreover, by splitting the dataset based on the presence of a known senescence marker we generated a list of gene ontology terms associated with senescent cells in skin development, homeostasis, wounding and ageing.
94238140408
Mesenchymal stem/stomal cells (MSCs) are often studied for their possible tissue engineering applications. During in vitro expansion, however, MSCs enter a state of permanent growth arrest while remaining metabolically active; this phenomena is known as cellular senescence. Senescence can negatively affect tissue homeostasis and an increased number of senescence cells can be found in pathological tissues (e.g. osteoarthritic cartilage). Moreover, It has been shown that cellular senescence may alters the differentiation capacity of MSCs towards the osteogenic and adipogenic lineage, while little is known about the influence of cellular senescence on chondrogenesis. Therefore, the aim of this study was to determine the effect of senescence on chondrogenic differentiation capacity of MSCs.
Cellular senescence was induced in MSCs (N=4) either in monolayer prior chondrogenic differentiation, or at different time points during chondrogenic pellet culture (day-7 or day-14) using a 20 Gy gamma irradiation protocol. Senescence markers P16, P21 and IL16, and the β-galactosidase staining were used to confirm irradiation-induced senescence.
Chondrogenic differentiation capacity was induced by a standard TGFβ-based protocol for 21 days using a 3D pellet culture system, and evaluated by (immuno)histochemistry and RT-PCR. To investigate the paracrine effect of senescent cells on recipient cells, we treated chondrogenic pellets using 2-day conditioned media from senescent cells and treat chondrogenic pellets for 24h, which were then analyzed for the expression of chondrogenic (SOX9, COL2A1 and AGCN) and catabolic (MMP-1, MMP-3, MMP-13 and ADAMTS4) markers. Western blot analysis on phosphorylated SMAD2 (P-Smad2 monoclonal antybody) was performed to identify TGFβ signaling activation. Non senescent cells or conditioned media from non-senescent cells was used as control.
When cellular senescence was induced prior differentiation, it abolished the chondrogenic capacity of MSCs with more than 95% reduction of GAG and Collagen type-2 deposition, as well as for all the chondrogenic markers measured by RT-PCR, in all the donor tested. A similar trend but with a less significant reduction was observed when senescence was induced at day-7 of differentiation. Interestingly, no effect on chondrogenic differentiation was detected when irradiation-induced senescent was applied at day-14 of differentiation. Moreover, medium conditioned by pellets cultures made of senescent cells had no significant effect on the expression of catabolic and anabolic markers measured by RT-PCR in recipient chondrogenic pellets. This suggests the negligible paracrine effect of senescent cells in our model.
In order to better understand how senescence was able to interfere with the chondrogenic process, we analyzed the ability of senescent MSCs to respond to TGFβ, the main pro-chondrogenic factor for MSCs. Upon stimulation with TGFβ1, phosphorylated-SMAD2 levels (an intracellular TGFβ effectors) were strongly reduced in senescent MSCs compared to control.
In this study we showed that cellular senescence reduced the chondrogenic differentiation capacity of MSC, but only when senescence occurs early during differentiation, and likely by negatively impacting the ability of the cells to respond to the pro-chondrogenic factor TGFβ1. This is a step forward in the understanding of the molecular mechanisms governing cellular senescence in MSC, and towards better optimizing the use of MSC for tissue engineering applications.
41883640959
"INTRODUCTION: Burn injuries propound copious challenges to clinical care and leaves the patient traumatized for years with scars. Even so, scarring walls off foreign bodies and seals injured tissue, it curb the movement and cede the cosmetic appearance of the skin. This wound healing defect is coupled with impaired cytokine expression. Upon tissue damage, the injured skin residents release cytokines into the wound bed to attract immune cells. Dysregulation in cytokine expression dramatically ends in defective wound matrix. Cytokines exerts inflammatory response to enhance wound healing. On the contrary, prolonged episodes of inflammation deposits excessive dermal matrix as scars. Thereupon, designing the construct to act concurrently by modulating the expressions of inflammatory cytokines is inevitable. An art of tuning the scar derma would be of a great value in post-traumatic wound healing. Thus far, clinically effective therapy for scar-less burns remains unmet and highly desirable goal. The fabricated nano-construct administered with biological agents exerts external stimuli to facilitate skin regeneration with minimal scarring.
METHODOLOGY: Polymers of oxyethylene and hydroxyalkanoates were electrospun with preloaded bioactive compound to obtain the multi-component nanofibrous matrix. The efficiency was investigated for the modulation in the expressions of pro- and anti-inflammatory cytokines specific to dermal scars in rat. The tissue lysates were taken from burned lesions of the rat and quantified for the cytokine expressions. A glass slide was spotted with targeted antibody for all inflammatory mediators. The sample diluents were diluted with the reference standard solution and the protein concentration matched tissue lysates. The experiment was performed and quantified with the median of the measured intensities to draw the final cytokine concentration (pg/ml).
RESULTS: In line, we have demonstrated that the anti-inflammatory cytokine IL-10 with the autocrine effect down-regulates the pro-inflammatory cytokine burst in the experimental group compared to the controls by displaying the diminished intensity of TNF-alpha, IL-1 beta, IL-6 and IL-8. The key growth factor TGFbeta-3 is at its peak which was contradicted to the level of TGF-beta-1 that aids in dense collagen turnover. The EGF was up-regulated with the activation of epidermal cells. The potent angiogenic factor VEGF, exhibited a moderate intensity in all treated groups.
CONCLUSION: Expanding knowledge in bio-materials and connective tissue research has resulted in the development of novel materials for variety of pathological conditions. The extremely complex phenomenon of burn wound healing involves a number of well orchestrated events. Burns attracts a high interest exploration in the field of medicine due to the physiological note of the skin. To date, none of the medicaments are as favorable as surgery in ameliorating the survival impacts in major burns. Here, the developed in-situ network of nano-construct aids physical guidance that exactly mimics the natural extracellular matrix for the guided tissue regeneration with lesser possibility of excessive scars. This useful cost effective graft material pave the way to meet the requirements of the needy patients belong to the lower strata of the socio-economy where the expenditure towards is difficult to impossible.
REFERENCE: 1. Jovanovic J. et al., Glia 68, 574-588 (2020)."
20941811955
"INTRODUCTION
Osteoarthritis (OA) is the most prevalent degenerative joint disorder, but no reversing therapies are currently available [1]. This is mainly due to the disease complexity, that involves a failure of the entire joint, and to the disease multifactorial etiology [2]. Taking all this into account, a gap of knowledge still exists on initial disease mechanisms, linked to the unavailability of reliable human preclinical OA models [3]. In this scenario, organs-on-chip are promising candidates to deeply investigate tissues crosstalk in early OA stages. To this end, we developed a compartmentalized joint-on-chip model for the co-culture of cartilage and synovium, aiming at evaluating the disruption of the physiological cross-talk between these tissues that contributes to the pathogenesis of OA [4].
METHODOLOGY
The microfluidic platform consists of two separate culture areas, intended for synovium and cartilage 3D cultures, whose communication is controlled through valves that can be opened through vacuum application. An actuation layer allows to apply a mechanical compression to the cartilage compartment upon pressurization [5]. Human chondrocytes, and synovial fibroblasts and macrophages were separately cultured in the two compartments, respectively. To assess the effect of cartilage degeneration on triggering synovial inflammation, a hyperphysiological compression was first applied to the cartilage compartment to induce an OA phenotype [5]. Induction of synovial inflammation was then quantified upon valves opening through FACS analysis.
RESULTS
Culture conditions were first optimized to obtain mature synovium and cartilage microtissues separately, as demonstrated by deposition of extracellular matrix rich in Collagen type-I and lubricin, and Collagen type-II and Aggrecan, respectively. Then, an OA phenotype was successfully induced on cartilageupon application of a hyperphysiological compression, as proven by gene expression analysis, showing up-regulation of inflammatory markers (e.g. MMP13, IL8). Cartilage inflammation exerted a detrimental effect on synovial constructs, as the FACS analysis showed an increase in CD86+ and CD80+ macrophages upon valve aperture, indicating polarization towards M1 pro-inflammatory state.
CONCLUSIONS
The proposed compartmentalized microfluidic platform offers a solution to mature cartilage and synovial constructs and/or to induce OA traits in only one of the two compartments, by enabling a temporal control over chambers communications. The device was here exploited to prove that mechanically-damaged cartilage triggers inflammatory changes in the synovium, suggesting the possible role of cartilage as effector responsible for OA initiation. Furthermore, the platform is currently being used to elucidate the role of an inflamed synovium on cartilage degradation, and this will eventually allow to understand which of the two tissues has a predominant role in early OA stages.
REFERENCES
1. Martel-Pelletier et al, Nat Rev Dis Prim, 2, 16072 (2016).
2. Lories et al, Nat. Rev. Rheumatol., 7, 43-49 (2011).
3. Bartolotti et al, J. Clin. Med., 10, 1920 (2021).
4. Chou et al, Sci Rep, 10, 10868 (2020).
5. Occhetta et al, Nat Biomed Eng, 3, 545-557 (2019).
ACKNOWLEDGEMENTS
This work was supported by Fondazione Cariplo-uKNEEque - Rif. 2018/0551 and has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement No 841975."
41883623826
"Introduction: IL-1β and IL-17 are highly present in the synovial fluid after joint trauma and are plausible factors in the development of post-traumatic osteoarthritis (PTOA). Although a growing number of studies have identified a role for IL-17 in OA, the mechanisms underlining the pathophysiological role of IL-17A in early PTOA disease in healthy joint cells remains unclear. We performed a side-by-side study comparing the effects of IL-1β, IL-17A and their combination in synovial fibroblast (SFs) vs. chondrocyte (CHs) in vitro cultures to understand the contributing role of each of these cells in mediating early inflammatory and degenerative effects in healthy cells of the joint.
Methodology: Healthy bovine CHs (passage 1 [P1]) and SFs (P1 containing macrophages and P5 consisting only of SFs) were stimulated with IL-1β, IL-17A and their combination for 3 days to mimic acute inflammation, then cultured in medium without or with cytokines for 3 additional days to understand the post-acute (PA) vs. persistent/chronic (C) inflammatory response, respectively. Absolute quantification of gene expression was performed by ddPCR at d3, d6(PA) and d6(C) for stimulated as well as unstimulated cells (CN).
Results: In healthy CHs and SF(P1) and SF(P5), IL-1β+IL-17A synergistically and significantly decreased COL2A1 (CHs) and increased IL-6 and IL-8 in almost all cell types after acute (d3) and persistent [d6(C)] inflammation. Three days after cytokine withdrawal, IL-17A led to post-acute gene modifying effects in all cell types and significantly increased IL-6 [in CHs, SFs(P1,P5)] and IL-8 [in SFs(P1,P5)] vs. CN, suggesting post-acute contributing effects of IL-17A in inducing inflammation within the joint. SF(P1) > SF(P5) ≥ CHs cultures expressed the highest levels of IL-6 (d3), IL-8 (d3 and d6C), and MMP-2 [(d3, d6(PA), d6(C)] in response to all cytokines, which may be due to the presence of macrophage-like synoviocytes. In response to all cytokine treatments and in all conditions tested [d3, d6(PA), d6(C)], MMP-3 expression tended to equally increase in CHs vs. CN. Moreover, MMP-2 expression significantly increased in response to IL-1β, IL-17A and their combination vs. CN [d6(C)] in CHs.
Conclusion: IL-17A and IL-1β synergistically induce an early inflammatory and degenerative expression profile in healthy CHs and SFs. While both cell types (CHs and SFs) contribute to the early events following treatment, IL-1β and IL-17A treated healthy SFs [(SF(P1)>SF(P5)] contribute more than CHs in upregulating the expression of inflammatory cytokines and the cartilage matrix-degrading enzyme MMP-2, which can participate in the degradation of a wide range of ECM proteins found in cartilage tissue and may be due to presence of macrophage-like synoviocytes. This suggests that the combination of IL-17A and IL-1β synergistically induce early inflammatory and degenerative effects which could drive a chronic feedback loop of inflammation and degradation of cartilage tissue that involves cross-talk and feedback among chondrocytes, synovial fibroblasts and macrophage-like synoviocytes and thereby promote PTOA progression."
73296332844
"Introduction
Type 1 diabetes (T1D) is a disease, which affects milions of patients. Islet or pancreatic transplantation is a method of treating complicated T1D. The limitation of these methods is the lack of organs for transplantation. 3D-bioprinting using living cells could be a solution. We present results of bioprinted bionic pancreas on mouse and pig model.
Materials and Methods
Research was carried out on 60 mice (SCID) and 24 pigs. The mice were divided into 3 groups: control; IsletTx in which porcine pancreatic islets were transplanted under the renal capsule; 3D-bioprint in which bioink petals consisted of bioink A and porcine islets. The bioprinted petals were transplanted into the dorsal part of the muscles under the skin in mice. Daily glucose measurement was performed and the level of C-peptide was tested every 7-days.
The pigs were divided into 4 groups: control, diabetic group(pancreatectomy-T1D); with transplanted 3D-bioprinted bionic petals(TX-with previous pancreatectomy), pigs with transplanted 3D-bioprinted bionic organ with full vasculature. The animals were measured daily with blood glucose levels (from 5-20 measurements per day). 3D-bioprinted bionic pancreas were transplanted in some animals to the iliac vessels and in other subgroup to the aorta and vena cava.
Results and Discussion
The results obtained in mice initially showed no differences in the concentration of peptide-C and glucose between groups. However, as early as 7-days after transplantation, both parameters analyzed in the fasting state were significantly lower in the IsletsTx and 3Dbioprinted groups compared to the control group. On day 14, decreased values of C-peptide and glucose were observed only in the group with petals transplants.
Mean glucose levels were two times lower, compared to the period before petals transplantation. In addition, TX pigs required lower doses of insulin after petals implantation. After transplantation of 3D-bioprinted bionic pancreas, a stable flow through the organ was observed in vivo and after the excision of the organ.
Conclusions
Transplantation of bionic petals in mice and pigs resulted in a decrease in mean glucose levels. None of the animals died due to postoperative complications or the lack of biocompatibility with the bionic structure. Transplantation of fully vascularized organ created with 3D-bioprinting technology id feasible.
Acknowledgments
Project „3D – BIOPRINTING OF SCAFFOLDS USING PANCREATIC ISLETS OR INSULIN PRODUCING CELLS IN ORDER TO CREATE BIONIC PANCREAS.” funded by National Center for Research and Development in the framework of the programme „Prevention and Treatment of Civilization Diseases"" STRATEGMED. Timeframe: 01.01.2017 – 30.06.2021
Accomplished by the Bionic Consortium - Foundation of Research and Science Development, Medical University of Warsaw, Warsaw University of Technology, Nencki Institute, MediSpace sp. Z o.o."
20967801564
"Tracheal damage is associated with the narrowing, weakening and discontinuity of the conductive part of the lower respiratory tract. Extensive defects cannot undergo end-to-end anastomosis and current approaches present poor outcomes due to weak mechanical properties, poor re-epithelialisation and vascularisation of the implanted graft. Herein, we investigated the use of collagen-based tubular scaffolds reinforced with 3D-printed synthetic polymer architectures for tracheal repair. A collagen and hyaluronic acid film covered the inner lumen (IL) of the scaffold, using CHyA-B scaffolds to support the formation of a respiratory epithelium. The 3D printed reinforced collagen porous outer layer (OL) of the scaffold was designed to support the growth of underlying tissues including cartilage and connective tissues as well as the formation of a vasculature network around the graft. The mechanical strength and ultrastructure of the tubular scaffolds was characterised and an approach for cellular seeding of the different layers of the scaffold was developed.
Tracheal scaffolds showed a 10-20 compressive MPa Young modulus with no significant decrease in mechanical strength following cyclic loading used to mimic respiratory patterns. Scaffold characterisation revealed a porous microarchitecture using scanning electron microscopy imaging with mean pore size of 169.7±11.2 µm suitable for cellular proliferation estimated via toluidine staining. A seeding process with the support of a custom-made device and 3D printed accessory parts was developed to achieve targeted epithelial seeding (Calu-3 bronchoepithelial cells) on the IL while the outer layer OL of the scaffold was populated with Wi38 lung-derived fibroblasts using different seeding densities under rotation. The growth of Wi38 cells on the OL was monitored for 7 days, showing successful cellular growth on the OL with no cellular attachment and growth in the IL using 6x105cells/cm2. Calu-3 cells were grown on the tubular scaffolds for 10 days, showing optimal cellular growth on the IL of the scaffold using 1.25x105 cells/cm2 with little attachment and growth of Calu-3 cells in the porous OL. Immunofluorescence imaging and quantification of the film from the IL further demonstrated cellular growth on the film with an estimated epithelial coverage of the film >60%.
Reinforcement of CHyA-B scaffolds with 3D-printed polymer architectures represents a suitable approach for the development of tissue-engineered tracheal grafts, showing adequate mechanical properties and an optimal porous structure to support the formation and growth of tracheal tissues. Moreover, a seeding procedure using a custom-made device was developed allowing successful cellular attachment and growth in the different layers of the tubular scaffold. The establishment of this targeted cellular seeding procedure holds potential to enable the clinical translation of tissue engineered tracheal grafts by facilitating differentiated pre-seeding strategies prior to implantation."
83767216855
Extensive defects of the upper extremity cause significant patient burden, including disability and social stigma. Approximately, 500,000 bone defects are reconstructed annually in the USA alone at a cost of ~$2.5 billion, due to factors including donor site harvest and lengthy operative times. Bone defects >5cm are usually reconstructed with autologous vascularized bone transfer (bone from another region of the patient’s body is harvested to replace the defect), but limitations include donor site morbidity, infection, and delayed healing. These limitations drive innovation of biomaterial applications, but a tissue engineered approach to reconstruction remains elusive. The objective of this study was to assess the efficacy of 3D printed bioactive ceramic (3DPBC) scaffolds augmented with dipyridamole (DIPY), an indirect A2AR agonist known to enhance bone formation, to stimulate bone regeneration of a critical-sized defect of the radius in an in vivo translational model. A 3DPBC scaffold was utilized to repair critical sized long bone defects in-vivo. In this study, 3DPBC scaffolds were fabricated in a two-piece system. Following IACUC approval critical-sized full thickness (~7xm x full thickness) defects were created in the tibia diaphysis in sheep (N=8). The 3DPBC scaffold composed of β tri-calcium phosphate (β-TCP) was placed into the defect site, along with an intramedullary rod and animals were euthanized 24 weeks. The tibia were retrieved, for micro-CT, histological and mechanical analysis. Bone growth was assessed exclusively within scaffold pores and evaluated by microCT and advanced reconstruction software. Biomechanical properties were evaluated utilizing nanoindentation to assess the newly regenerated bone for elastic modulus (E) and hardness (H). Qualitative evaluation of the histological micrographs indicated directional bone ingrowth of bone, with an increase in bone formation toward the native bone morphology. Extensive bone formation with signs that scaffold has significantly resorbed, presenting areas of extensive structural discontinuity resorption was observed at both low and high magnifications. Histological micrographs at high magnification to better appreciate the features of the newly regenerated bone within the scaffold. Furthermore, qualitative evaluation did not yield any exuberant bone growth and the newly regenerated bone was limited to the defect and the scaffold regions. Our previous studies using a smaller preclinical model, rabbit, yielded favorable results, where with the implantation of a custom-fit 3D printed resorbable bioactive ceramic scaffolds into critical size radius defects resulted in bone morphology that remarkably resembled the original bone segment with a haversian cortical shell presenting cortical-like mechanical properties and associated marrow space. The application of this β-TCP scaffold has the potential for successful treatment outcomes while potentially minimizing the amount of surgery and less time spent in hospitals for individuals that would not fully recover from injury though current technology and treatment strategies available. Custom engineered, biocompatible and resorbable, β-TCP scaffolds treated with DIPY demonstrated to have an increased bone regeneration qualitatively and quantitatively. The custom approach and expedited healing has the potential to positively benefit the patient in terms of lowering health care costs and patient's quality of life, as well as returning to form and function.
73296301687
"Introduction. Irrespective of the several scaffold designs that have been investigated in the last 30 years, the actual number of scaffold guided bone tissue engineering (SGBTE) approaches that were able to reach clinical application are few. Most of these approaches fail translation into clinical settings firstly because outcomes of scaffold design properties and host immune responses results are poorly correlated, and secondly because accurate prediction on how they will behave in humans are mostly based on lower levels of organization animal model findings. To develop a de novo understanding of the biocompatible mechanisms of SGBTE processes upon implantation of 3D printed medical grade Polycaprolactone (mPCL) scaffolds, and the prospective of exploiting novel concepts and material design innovation, requires a rigorous pre-clinical experimental demonstration of therapeutic promise in clinically relevant animal models. Over the last twelve years, our research group has trialled a number of SGBTE concepts using our established sheep animal model as a pre-clinical tool for evaluating bone tissue reconstruction. Here we provide an overview of the pre-clinical segmental bone defect studies performed by our group in the last twelve years, as well as an overview of the SGBTE concepts that were able to reach clinical applications. Methodology. Studies used 3D printed mPCL scaffolds (Osteopore International, Singapore) in combination with a variety of mediators, including autologous bone grafts, autologous and allogenic mesenchymal bone marrow precursor, platelet rich plasma, and bone morphogenic proteins. Scaffold mechanical properties have been assessed. Merino sheep aged ≥ 6years old was the animal model used for all studies. Conventional X-rays, ex-vivo biomechanical testing, Micro computed tomography (µCT), histology, immunohistochemistry, scanning electron microscopy, and histomorphometry were used to monitor healing progression. Results. A total of eighteen pre-clinical and seven clinical studies were performed in the last twelve years. All animals recovered from surgical interventions and completed the experimental period uneventfully. Using state-of-art µCT, histological, immunohistochemical, image analysis techniques and innovative quantitative analysis, these studies have led to significant understanding of the bone biology, on the biocompatible mechanisms of SGBTE during the regeneration processes, as well as, on providing new insights into mimicking the natural bone tissue regeneration environment in large animal models. These studies were paramount in the development of a pre-clinical model protocol for assessing bone regeneration in large bone defects, instrumental on the world-first patient and largest segmental bone defect to be successfully reconstructed using a mPCL scaffold (not published) and on a femoral shaft critical-sized bone defect reconstruction.
Conclusion. While a lot of effort has been invested in optimization scaffold parameters, currently, there is a growing interest with much of the focus on profiling large animal model’s bone responses to 3D printed medical devices, with further emerging evidence suggesting that the scaffold architecture is a niche where adaptative immune cells are decoding scaffold features. As such, the in vivo evaluation of the bone responses through pre-clinical large animal models is an unavoidable component of translational research and should be used to justify and establish scaffold guided tissue engineering concepts in clinical settings."
94238123688
INTRODUCTION
With a growing demand for effective regenerative medicine therapies, more sophisticated tissue-engineered in vitro models are required for a better understanding of the fundamental biological processes that underlie regeneration. To tackle this need and further comprehend these processes, new technologies are emerging in the tissue-engineering field. The state-of-the-art technology of 3D bioprinting aims to achieve well-defined biological structures by printing cell- embedded hydrogels or bioinks in a layer-by-layer manner. A main challenge of 3D bioprinting is the lack of ""soft"" bioinks with a wide printability window, which offer adequate biofabrication properties as well as a cell-friendly extracellular matrix (ECM)-like microenvironment. Thus, allowing the encapsulation and culture of cells in complex in-vitro 3D tissue models1.
Self-assembling peptide hydrogels (SAPHs) are fully defined, semi-synthetic hydrogels, which are biocompatible and with tuneable mechanical properties. Therefore, SAPHs are believed to stand as a powerful option with unique properties that make them perfect candidates for this purpose2. Herein, this research aims to design and explore SAPHs as novel bioinks for extrusion-based 3D bioprinting.
MATERIALS AND RESULTS
To characterise subject hydrogels, rheological analyses, printability and cytocompatibility tests were carried out using oscillatory rheology, extrusion-based bioprinting and human Mesenchymal Stem Cells (hMSCs), respectively. Rheological analyses showed that our subject peptide-based hydrogels were shear thinning and recovered well under shear stress. Relaxation times fitting curves revealed the characteristic dynamic times in which our hydrogels recovered following a classical mechanical model3. All these rheological findings related to good printability in shape fidelity and integrity analyses. We investigated fibroblast and hMSC viability to assess the biocompatibility of the hydrogels. These studies resulted in SAPHs being promising printable and biocompatible biomaterials for extrusion-based 3D bioprinting with good biofabrication attributes.
CONCLUSION
We have successfully developed and tested SAPHs as bioinks and assessed cell viability over a 21-day culture period of bioprinted embedded-fibroblast and hMSC hydrogels. An application we are currently exploring, is to investigate if bone differentiation could be induced to determine how capable these constructs are to differentiate into physiologic bone phenotype4. Translated to real-world use, the biofabrication of bone and cartilage models through 3D bioprinting could result as a powerful tool for in vitro disease modelling and to treat bone conditions as osteoarthritis in early stages of the disease.
REFERENCES
83767212328
"INTRODUCTION
One of the main functions of guided bone regeneration (GBR) barriers are to preserve the bone graft and maintain its mechanical stability during the healing process. Personalized metallic meshes meet GBR demands as well as a good predictable tissue regeneration1. Although they offer good performance in terms of tissue regeneration, these metallic meshes present several drawbacks such as a second surgery to extract the mesh, autologous bone extraction from other anatomical locations, or mesh exposition. These problems cause extra pain and morbidity to the patient. Therefore, this project aims to substitute the use of metallic meshes with patient-specific biodegradable implants based on polycaprolactone (PCL), enriched with bioactive microparticles (MPs) to stimulate angiogenic and osteogenic processes.
METHODOLOGY
Different PCL scaffolds (PCLA, PCLB, PCLC) with bioactive MPs developed in our group were 3D printed (3D Discovery, RegenHU) with high interconnected porosity. The degradation behavior of scaffolds was evaluated in vitro under physiological conditions (HEPES 10mM, pH 7.4, 37ºC) for one year. The viability of human gingival fibroblasts (hGFib) and human mesenchymal stem cells (hMSC) seeded on scaffolds was assessed by Alamar Blue, and imaging. Cytotoxicity (LDH assay) and the expression of different proteins (ELISA) related with angiogenesis and osteogenesis were also evaluated. The in vivo performance of these scaffolds was studied using an in vivo subcutaneous mice model. In order to assess a correct fit of the personalized implants, 3D printed prototypes were tested with polyamide models kindly provided by AVINENT® Implant System.
RESULTS
SEM and MicroCT images showed homogeneous MPs dispersion and macroporosity of 3D printed scaffolds. Minimum scaffolds weight loss was observed after one year. Confocal images indicated complete colonization of scaffolds by hGFib. Similarly, good biocompatibility was observed in hMSC cultures. Analysis of protein expression by ELISA showed an increase in the levels of vascular endothelial growth factor (VEGF) which is related to neovascularization promotion. In the in vivo studies, an absence of acute inflammation and complete tissue integration were observed, indicating scaffold biocompatibility. Furthermore, blood vessels infiltration through scaffolds porosity was identified after one-month implantation. Moreover, personalized prototypes were successfully 3D printed from clinical cases and studied with polyamide bone defects models, obtaining a proper fit of the implant to the defect site.
CONCLUSION
This work shows a promising alternative to the use of metallic meshes, with bioactive and biodegradable materials to offer a personalized solution for GBR, avoiding their main drawbacks. Additional in vitro assays and an in vivo calvaria study are ongoing to support the results achieved.
REFERENCES
ACKNOWLEDGMENTS
This project is being developed in collaboration and with funding from AVINENT® Implant System. Authors would like to thank AGAUR for founding with “Doctorat Industrial” grant (2017DI076) and Dr. Elena Xuriguera from the University of Barcelona for her help in performing mechanical testing."
94238135204
Introduction: Contemporary reconstructive approaches for critical-sized bone defects carry significant disadvantages. As a result, clinically driven research has focused on the development and translation of alternative therapeutic concepts. Scaffold guided tissue regeneration (SGTR) is an emerging technique to heal critical-sized bone defects. However, issues synchronising scaffold vascularisation with bone-specific regenerative processes currently limit bone regeneration for extra-large (XL, 19cm3) critical-sized bone defects. To address this issue, we developed a large animal model that incorporates a corticoperiosteal flap (CPF) for sustained scaffold neo-vascularisation and bone regeneration. Methodology: A pre-clinical evaluation using a 3D-printed medical-grade ε-polycaprolactone b-tricalcium phosphate (mPCL-TCP) scaffold combined with a cortico-periosteal flap (CPF) was undertaken in ten sheep with a medium (M, 9.5 cm3) volume segmental defect of the tibia. Results: In ten sheep the efficacy of this approach for healing M volume segmental bone defects was demonstrated by plain radiography, micro-computed tomography, scanning electron microscopy, immunohistochemical and histological analysis. Furthermore, in two sheep we demonstrate how this approach can be safely extended to heal XL critical- size defects. Conclusion: This study presents an original CPF technique in a clinically relevant and well described pre-clinical model which can be used in conjunction with the SGTR concept to address challenging critical-sized bone defects in vivo.
94238129044
The knee meniscus plays an indispensable role in articular surface protection, shock absorption, and stress transmission. Meniscus injuries are extremely prevalent, with an annual incidence of 66 to 70 per 100,000 people. Due to limited vascularization, the regenerative capacity of the meniscus is relatively low and restricted to the most vascularized outer regions. The most commonly performed treatment involves suturing or removal by partial or total meniscectomy. However, meniscectomy significantly increases the incidence of osteoarthritis (OA) later in life by elevating the contact pressure on the tibial plateau. Approximately 50% of patients with meniscal injuries develop OA between 10 and 20 years after the injury. Therefore, optimal treatment options should preserve or mimic the mechanical properties of the meniscus.
3D bioprinting belongs to the family of additive manufacturing (AM) processes that utilize computer-aided design (CAD) for the generation of 3D models through layer-by-layer deposition. The constructs are printed with bioink comprised of viable cells, biomaterials, and additional biological substances. These artificial, cell-laden scaffolds promote and support new tissue formation by providing a suitable environment for cell migration, proliferation, differentiation, and ensuring a proper extracellular matrix (ECM) secretion.
The presentation is focused on a 3D bioprinting-based approach to regenerative medicine of the meniscus. It will also highlight the process of an ECM-based bioink formulation utilizing supercritical CO2 extraction, and a custom-made bioprinting tool on a 6-axis robotic arm.
Acknowledgments: This work was supported by the National Centre for Research and Development TECHMATSTRATEG-III/0027/2019-00 grant.
20941803339
"Cellular models are needed to study human development and disease in vitro, and to screen drugs for toxicity and efficacy. Current approaches are limited in the engineering of functional tissue models with requisite cell densities and heterogeneity to appropriately model cell and tissue behaviors. This talk will describe how spheroid bioprinting in suspension baths can be used to engineer high-cell density microtissues of prescribed spatial organization. Example applications of this technology will include bioprinting induced pluripotent stem cell-derived cardiac microtissue models with spatially controlled cardiomyocyte and fibroblast cell ratios to replicate the structural and functional features of scarred cardiac tissue that arise following myocardial infarction, including reduced contractility and irregular electrical activity. It will also describesd how these models can be used for screening miRNA therapeutics targeting cardiomyocyte proliferation for cardiac regeneration. Recent advances in bioprinting high-cell density tissues with controlled cellular organisation will also be described. These methods are useful for a range of biomedical applications, including the development of precision tissue models with advanced physiological relevance."
31451706447
Introduction: Despite the crucial role of the muscle extracellular matrix in the organotypic organization and the transmission of mechanical force, most 3D muscle models do not mimic its specific characteristics, namely its biochemical composition, stiffness, anisotropy and porosity. In vivo, muscle extracellular matrix possesses specific characteristics such as a high amount of aligned collagen I to create an anisotropic structure with a significant porosity and a suitable stiffness. Recent approaches of muscle models used non porous hydrogels fabricated from low concentrated collagen to encapsulate muscle cells. Hence, the in vivo properties are not reproduced. Here, we developed a 3D printed collagen hydrogel that mimic the muscle extracellular matrix, i.e collagen anisotropy, adequate stiffness and two ranges of porosity (one to ensure nutrients and oxygen diffusion and the other for cell cultivation).
Methodology: Dense collagen solutions (30 mg.ml-1) were printed through the 23G flat bottom needle inside a buffer bath. The extrusion process aligned the collagen molecules along the axis of extrusion. The buffer bath played two major roles: it “froze” the collagen alignment and triggered collagen gelling. The printing process was performed unidirectionally for each layer to create an intrinsic porosity between the different collagen filaments. After a rapid period of collagen gelling, needles were introduced within the hydrogel to generate large pores. An additional gelling period was performed to tune the mechanical properties. C2C12, murine skeletal muscle cells were then seeded within the printed hydrogels to evaluate the cell colonization, myotube formation and organotypic organization.
Results: By tuning the extrusion speed and the gelling process, a 3D printed hydrogel with aligned collagen fibers was obtained. A combination of two gelling strategies (24h PBS 5X + 24h NH3) was optimal to obtain both anisotropy and adequate mechanical properties (E=10 kPa). Scaffold anisotropy was obtained at two different scales: all filaments were printed in the same direction (macroscopic) and collagen fibers were aligned inside printed filaments (microscopic). Concerning the porosity, changing the height between two successive layers allowed to create an intrinsic porosity from 50 to 150 µm. Interestingly, the generation of 100 µm pores preserved the scaffold cohesiveness. This porosity is suitable for nutrients and oxygen diffusion in the whole scaffold, thereby favoring cell viability. Larger pores created by needles molding generated straight channels of 600 µm in diameter. These were easily colonized by C2C12 cells mixed with Matrigel® to create a suitable 3D environment. After 4 days of differentiation, aligned multinucleated myotubes were formed. Immunostaining with sarcomeric heavy chain myosin revealed the cell commitment into mature myotubes.
Conclusions: In this study, we developed a 3D printing technique to create a biomimetic muscle extracellular matrix suitable for muscle cell differentiation and cultivation. Our approach focused on the extracellular matrix and its key parameters since it is deeply involved in muscular functions. Hence, this model could be used with patients cells to study and have a better understanding on muscular dystrophies.
20941802324
INTRODUCTION: Embedded 3D bioprinting is a promising approach to engineer complex tissues such as patterned or pre-vascularized tissue constructs1,2. However, the resulting tissue constructs are often mechanically weak, unable to form mechanical or chemical gradients, and lack on-demand tunability. Here, we report on dual crosslinkable dextran-based hydrogel as a hydrogel bath for embedded bioprinting, which uniquely allows for local on-demand functionalization as well as formation of spatially controlled chemical and mechanical gradients within printed tissues.
METHODS: Dextran was functionalized with tyramine and biotin moieties to create a dual crosslinkable polymer3. Physically crosslinked embedding baths were created via biotin/avidin protein/ligand interaction. A gelatin or PEG based sacrificial bioink was extruded into the hydrogel using an Inkredible+ 3D printer. Covalent enzymatic or photo-initiated crosslinking of the printing bath was used to create mechanically robust tissues. The tissue’s biotin moieties were subsequently used for on-demand biochemical functionalization of the bulk and/or the channel surfaces.
RESULTS & DISCUSSION: Rheological characterization of the physically crosslinked bath revealed shear-thinning and self-healing properties that were highly suitable for embedded bioprinting. Covalent crosslinking resulted in a three-fold increase of the storage modulus of the hydrogels, which enabled the stabilization of printed channel networks, while diffusion of crosslinking agents from the ink resulted in controllable stiffness gradients in the bulk. Without photocrosslinking the bulk, tubular structures were created via enzymatic inside-out crosslinking. Here, tube diameter and channel wall thickness could be independently controlled through variation of the printing speed and crosslinker concentration, respectively. Furthermore, the ink/bath interface allowed for one-step functionalization by loading the ink with biotin-coupled cell bioinstructive moieties.
CONCLUSION: We report on a novel and dual crosslinkable hydrogel suitable for embedded bioprinting, which offers mechanical stability, mechanical tunability, with on-demand biochemical functionalization of tubular and pre-vascularized engineered tissues.
ACKNOWEDGEMENTS: Financial support was received from the European Research Council (ERC, Starting Grant, #759425) and the Dutch Research Council (NWO, Vidi Grant, #17522).
REFERENCES:
1 Lee, A. et al., Science 365, (2019).
2 Highley, C. B. et al., Adv Mater 27, (2015).
3 Kamperman, T. et al., Nat Commun 10, (2019).
94238106505
An open source extrusion bioprinter based on the E3D motion system and tool changer to enable FRESH and multimaterial bioprinting
Adam Engberg1, Christina Stelzl1, Olle Eriksson1, Paul O’Callaghan1 & Johan Kreuger1
1 Department of Medical Cell Biology, Uppsala University, Uppsala, Sweden.
Bioprinting is increasingly used to construct complex 3D cell models and tissue constructs for in vitro studies, and its capacity to produce transplantable tissues is being intensely explored. However, progress in these fields could be further accelerated by increasing the access to easy-to-use open source bioprinters.
Here we describe an open source extrusion bioprinter based on the E3D motion system and tool changer which enables high resolution multimaterial bioprinting.
The E3D motion system and tool changer was adapted to control the position of a custom 3D-printed syringe pump extrusion tool, equipped with a stepper motor to actuate the plunger and regulate bioink extrusion. The bioprinter is housed in a polycarbonate enclosure equipped with a HEPA filter to reduce the risk of contamination during printing. The versatility of the bioprinter was demonstrated by creating collagen constructs using the freeform reversible embedding of suspended hydrogels (FRESH) method (1), as well as printing multimaterial constructs composed of distinct sections of laminin and collagen bioink. Image analysis of cell viability dyes was used to evaluate the capacity of the bioprinted constructs to support survival of breast cancer cells following direct seeding onto printed constructs or after printing in cell-laden bioinks, and was assessed after short-term (24 h) and long-term (1 wk) incubations (2).
The syringe pump extrusion tool is compatible with different syringe volumes, and needles and nozzles of different calibres, and we determined that narrow linear features could be accurately reproduced (100 ± 12 mm) when extruding bioinks through a 50 mm needle. The dimensions of FRESH and multi-material printed constructs proved faithful to their intended designs, and a high degree of cell viability was seen for cells seeded onto collagen or dispersed in laminin bioinks following both short- and long-term incubations. Furthermore, cell death could effectively be studied in cells grown on FRESH printed collagen constructs following exposure a known apoptosis-inducing agent.
This open source bioprinter is easily adapted to the specific needs of various bioprinting applications; for example, creating small-scale 3D tumor tissue constructs for the purpose of drug-screening. The bioprinter solution presented here is versatile, easy-to-use, and the motion system is already supported by open source data, which together offer an accessible entry point to the novel and rapidly expanding field of bioprinting.
References:
1 Lee A, Hudson AR, Shiwarski DJ, et al. Science 365(6452):482-487 (2019)
2 Engberg, A., Stelzl, C., Eriksson, O. et al. Sci Rep 11, 21547 (2021).
31412732346
3D printing has revolutionized the manufacturing of volumetric components and structures in many areas. Several fully volumetric light-based techniques have been recently developed thanks to the advent of photocurable resins, promising to reach unprecedented short print time (down to a few tens of seconds) while keeping a good resolution (around 100 microns). However, these new approaches only work with homogeneous and relatively transparent resins so that the light patterns used for photo-polymerization are not scrambled along with their propagation. Herein, we propose a method that takes into account light scattering in the resin prior to computing projection patterns. Using a tomographic volumetric printer, we experimentally demonstrate that implementation of this correction is critical when printing objects whose size exceeds the penetration depth of light. To show the performances of the scattering corrections we fabricate cell-laden hollow constructs that would be difficult to print otherwise because of light scattering by the cells. Bioprinting cm-scale hollow constructs is therefore challenging but also crucial because hollow channels allow for the inflow of nutrients and oxygen to the cells deep inside the hydrogel. Based on a fine characterization of the scattering process, the proposed scattering correction spatially redistributes light to avoid over-polymerization and clogging the channels, more light is sent to the fine features of the edges while less light is sent to the bulk of the construct. As an example, this technique allowed us to fabricate in 36 seconds a cm-scale construct with four millimetric channels unclogged and a solid core in a soft hydrogel loaded with 4 million HEK 293 cells mL-1. As a comparison, the same printer without the scattering correction yielded clogged channels and a void core. Previous reports have demonstrated the fabrication of similar structures under concentrations of only 10 000 or 1 million cells mL-1 using similar printing technologies. To conclude, this scattering correction extends the capabilities of conventional light-based volumetric printing and opens up promising perspectives in printing inside turbid materials with particular interesting applications for bioprinting cell-laden constructs.
83767218366
Introduction:
Tendon tissues have highly-anisotropic physical properties that are responsible for its biomechanical performance and biological organization. The recreation of its 3D extracellular matrix (ECM) and cellular patterns in bioengineered constructs remains challenging. The concept of magnetically-assisted 3D bioprinting with magnetic hydrogel bioinks can be exploited to fabricate anisotropic scaffolding materials with 3D architectures that resemble the organization of tendinous ECM and to modulate biophysical/biochemical cues that influence the fate of encapsulated cells. Moreover, magnetic nanoparticles (MNPs) remote response enables their use as magnetomechanical actuators to control cellular/tissue behavior. However, a main challenge hindering the implementation of this concept is how to control the 3D organization of magnetic elements during layer-by-layer printing without compromising the fidelity and resolution of printed constructs. To overcome this dichotomy, here we combine the concepts of magnetically and matrix-assisted 3D bioprinting technologies. This strategy enables to fabricate high-resolution constructs with magnetic bioinks that remain liquid for long enough before gelation to allow the orientation of magnetic elements, thus building 3D fibrillar patterns resembling the microstructure of tendon tissues.
Methodology:
Monodisperse iron oxide-based MNPs displaying extremely-high magnetization values were synthesized through thermal decomposition. These MNPs were then incorporated into electrospun polycaprolactone meshes, which were subsequently cryo-sectioned at different lengths to produce dispersed magnetic microfibers. Magnetically-responsive bioinks were prepared by mixing the magnetic short fibers with gelatin solutions and human adipose-derived stem cells (hASCs). The 3D extrusion bioprinting steps were performed under the presence of fairly uniform external magnetostatic fields produced by a two parallel magnets setup. Agarose and cellulose nanocrystals (CNCs)-based fluid gels (supplemented with transglutaminase for gelatin crosslinking) were tested as support baths.
Results:
Zinc-doping demonstrated to be the most efficient approach to increase the magnetic power of superparamagnetic iron oxide-based MNPs. Zn-Fe3O4 MNPs were used to prepare magnetically-responsive electrospun polycaprolactone microfibers with 20-100 µm of length. The incorporation of these microfibers and hASCs in gelatin solutions resulted in bioinks that enabled the fabrication of high-resolution 3D-printed constructs when using CNCs as suspension baths, but not when with the respective granular agarose gels. Exploiting the high magnetic power of the MNPs, very low particle concentrations and weak magnetic field strengths were enough to align the fibers during the layer-by-layer extrusion printing steps. The anisotropic microstructure of the biomimetic constructs induced elongated growth and phenotypic commitment of the encapsulated cells.
Conclusions:
Our strategy allows the 3D manufacturing of biomimetic magnetic constructs that replicate the architecture of native tendons ECM. We established the design of MNPs with extremely-high magnetic power as a key factor to fabricate bioink hydrogels that can be manipulated using low contents of magnetic material and weak magnetic fields, minimizing the toxic/safety risks associated with these factors. The combination of magnetically-assisted 3D bioprinting strategies with the use of CNCs support baths has demonstrated to be essential for enabling the proposed concept. The resulting anisotropic 3D fibrillar microstructure of the printed constructs revealed effective on directing encapsulated cell fate. The effects of remote magnetomechanical actuation on cellular constructs is currently under investigation.
83767224366
Introduction: Osteochondral (OC) disorders like osteoarthritis (OA) and rheumatoid arthritis (RA) damage the joint's cartilage and subchondral bone. Their treatment remains a significant challenge for both researchers and orthopedics. In vitro models of OC tissue have become an essential tool to help investigate pathogenesis, develop drug screening, and test potential therapeutic approaches. This study aims to create a bio-printed OC construct recapitulating the bone and cartilage compartment as drugs testing platforms.
Methodology: Two different hydrogels, including a blend composed of gelatin methacrylate (GelMA) with nanosized hydroxyapatite (nHA) and tyramine-modified hyaluronic acid (THA), were selected for the bioprinting of bone and cartilage tissue mimics. The composition of GelMA hydrogel (10% w/v) with different concentrations of nHA (1-10% w/v) and THA with concentrations of 2.5-5% w/v were characterized by rheology and their cytotoxicity was assessed via live-dead assay. Later, the pre-differentiated osteoblast and endothelial cells were encapsulated into GelMA-nHA and micropellet chondrocytes into THA hydrogels for bioprinting osteochondral construct. After 2 weeks of culturing, the successful generation of OC tissue was confirmed by real-time RT-PCR and histology.
Results: The storage modulus (G') of all GelMA/nHA hydrogels was significantly higher than GelMA, however, there was no significant difference in G' values for the GelMA/nHA as a function of added nHA. Due to the know temperature sensitivity of GelMA, a rheological temperature sweep and series of printing tests were performed to establish a suitable printing temperature, which was confirmed to be 20°C, independent of the addition of nHA. Calcein-AM (Ca-AM) and Ethidium Homodimer-1 (EthD-1) staining for GelMA (10% w/v) with three concentrations of nHA (1, 3, and 5% w/v) at 2, 24, 72, and 168 h after printing showed the percentage of living cells after 72h in GelMA containing 3 and 5% (w/v) nHA was less than 50%, while in GelMA with 1% (w/v) nHA it remained high (>95%) even after 168h. Therefore, this formulation was chosen for the subsequent generation of bone tissue mimic.
Shear flow curves of THA hydrogels showed an increase in viscosity as a function of THA concentration. The damping factor, which is a ratio between the loss modulus G'' and the storage modulus G', has been shown to be directly related to the extrudability[1]. The calculated damping factors for each concentration of THA (%w/v) (THA 2.5%= 0.4947 ± 0.038, THA3.5%= 0.5935 ± 0.012, and THA5%= 0.7391 ± 0.039), indicated that THA3.5%(w/v) was in the printable range. Cell viability assays for THA hydrogels showed a high percentage of living cells for THA 3.5% (w/v) compared to THA 5% (w/v) after 168 h. Based on cell viability assay, viscosity, and printability, a 3.5%(w/v) concentration of THA was selected for generating cartilage tissue mimic part.
Conclusion: We developed GelMA-nHA and THA hydrogels for bone and cartilage parts respectively. We also optimized printing parameters based on printability and shape fidelity and cell density according to cell viability for bioprinting OC constructs.
[1] Petta et.al, 2018, ACS Biomater. Sci. Eng, DOI: 10.1021/acsbiomaterials.8b00416. </div>
31412756349
TBA
INTRODUCTION
Myocardial infarction (MI) leads to a significant loss of cardiomyocytes followed by the progressive formation of a non-contractile fibrotic scar. Recently, the use of microRNA (miRNA) has emerged as a promising strategy for cardiac regeneration. Paoletti et al. demonstrated that the transient transfection with four microRNA mimics (termed “miRcombo”) can induce the direct reprogramming of human cardiac fibroblasts (CF) into induced cardiomyocytes (iCMs)[1]. To overcome the limitations of in vivo miRNAs administration, proper nanocarriers are required. Moreover, alginate-based injectable hydrogels can be exploited for controlled in situ release of miRNAs-loaded nanocarriers [2]. However, alginate presents some limitations such as low degradability in vivo and limited cell adhesion.
In this work, new lipoplexes (LPs) loaded with miRcombo were developed to obtain more efficient encapsulation and release of miRcombo to CFs compared to a commercial agent. Then, LPs were combined with an optimized alginate dialdehyde (ADA) hydrogel for potential in situ controlled release of miRNA in the damaged tissue.
METHODS
LPs containing negmiR (negative control) or miRcombo based on a mixture of cationic and helper lipid were prepared at different N/P ratio (N/P 3 - 0.35) and physiochemically characterized [3]. Adult human CFs (AHCFs) were treated with miRcombo-loaded LPs to assess transfection efficiency and ability to promote direct reprogramming of AHCFs into iCMs through gene expression analysis at 15 days culture time. ADA was prepared by ALG oxidation using sodium metaperiodate [2]. ADA-based hydrogels with different compositions were characterised for their physicochemical properties. Preliminarily, siRNA-Cy5-loaded LPs were encapsulated into ADA-based hydrogels with selected composition and Cy5-siRNA release was studied.
RESULTS
MiRcombo-loaded LPs with optimal N/P ratio of 3 were selected based on stability studies (evaluated in different media by DLS analysis), showing high encapsulation efficiency (99%). In vitro cell test of LPs with AHCFs showed high biocompatibility and miRNA cellular uptake. Moreover, treatment of AHCFs with miRcombo-loaded LPs favoured their direct reprogramming into cardiomyocyte-like cells, evaluated through the expression of cardiomyocyte markers such as cardiac troponin C (cTnT).
ADA was prepared with an average yield of 68% and an oxidation degree of 23%. ADA concentration and ionic crosslinking were optimised to develop injectable hydrogels with cardiac-like viscoelastic properties. Model Cy5-siRNA-loaded LPs were physically entrapped within ADA-based hydrogels, completely releasing Cy5-siRNA within 24h.
CONCLUSION
A novel miRNA-delivery system, consisting of miRNA-loaded LPs encapsulated in an ADA-based injectable hydrogel, was developed for cardiac regenerative medicine. Cy5-siRNA release data suggested the need for tailoring the surface charge of miRNA-loaded LPs by proper coating, to minimize the electrostatic interactions between ADA and the positively charged miRNA-loaded LPs. This activity is currently in progress and preliminary data showed an enhancement in the stability of hydrogel-embedded LPs by tailoring their surface charge.
1 Paoletti C, et al. Front Bioeng Biotechnol. ,8,529 (2020). 2 . Sarker, B et al., J. Mater. Chem. B, 2(11), 1470-1482 (2014). 3. Nicoletti et al., Nanomed.: Nanotechnol. Biol. Med., under submission.
BIORECAR is supported from the European Research Council (ERC) under the EU H2020 research and innovation programme (GA N° 772168).
62825434605
Spheroids are one of the well-characterized 3D cell culture approaches for drug screening and therapeutic studies. Magnetic levitation (MagLev) is a newly developing approach to form 3D cellular structures and spheroids [1,2,3]. Magnetic levitational assembly of cells provides rapid, simple, cost-effective 3D cell culture formation while ensuring scaffold-free microenvironment. Here, our efforts are summarized in designing new magnetic levitation platform and biofabrication of 3D cellular entities via magnetic levitation for tissue engineering. Magnetic levitation and guidance of cells were provided by using a paramagnetic agent to fabricate scaffold-free 3D cellular structures. The parameters of cell density, paramagnetic agent concentration, and culturing time were optimized to obtain 3D cardiac cellular structures with tunable size, circularity, and high cell viability. Cellular and extracellular components of the 3D cellular structures were demonstrated via immunofluorescent staining. Also, 3D cardiac cellular structures showed more resistance to drug exposure compared to 2D control. In conclusion, MagLev methodology offers an easy and efficient way to fabricate 3D cellular structures for drug screening studies.
References
52354564359
AN INDUCED PLURIPOTENT STEM CELL-BASED MODEL TO STUDY THE MECHANOBIOLOGY OF MYOCARDIAL FIBROSIS
Francesco Niro1,2, Daniel Pereira De Sousa1,2, Jorge Oliver-De La Cruz1, Soraia Fernandes1, Stefania Pagliari1, Marco Cassani1, Vladimir Vinarsky1,2 Ece Ergir1, Giancarlo Forte1,2
Cardiac fibrosis is the consequence of chronic insults on the myocardium, and it is characterized by the abnormal accumulation of extracellular matrix (ECM). The transdifferentiation of cardiac fibroblasts (cFbs) into myofibroblasts drives pathological ECM remodeling, a process highlighted by biochemical and structural changes which compromise cardiomyocytes (CMs) contractile activity and eventually lead to heart failure [1]. Here, we adopted bioengineering tools and induced pluripotent stem cells (iPSCs) to investigate how fibrotic ECM affects the structural and functional properties of CMs.
Thus, we derived cFbs from induced pluripotent stem cells (iPSCs-cFbs) and optimized a protocol based on biochemical and mechanical stimulation to induce their transdifferentiation to myofibroblasts. Next, we obtained fibrotic ECM (dECM) by implementing a decellularization procedure of the activated iPSCs-cFbs and analyzed the pathological changes occurring during the deposition of cardiac diseased ECM. The results generated through this analysis were confirmed by studying dECM of cFbs isolated from heart failure patients and their healthy counterparts. Then, we generated iPSCs-CMs and cultured them either on healthy or fibrotic dECM. Morphological and functional analyses were implemented to study how the biomechanical properties of pathological ECM affect CMs physiology and function.
Finally, we established a 3D in vitro culture system, which entails the co-culture of isogenic iPSCs-CMs and -cFbs that better reproduces the cellular complexity and functionality of the human heart and represents a powerful tool for personalized medicine applications.
By capitalizing on this approach, we might be able to recapitulate the accumulation of fibrotic tissue occurring during heart disease and investigate the contribution of pathological ECM to the progression of heart failure.
References
[1] Frangogiannis, N.G., J Clin Invest.127(5), 1600-1612 (2017)
62825441237
Introduction
With continued progress of wearable sensor technology and drug-screening in-vitro models, there is a need for more advanced biomaterials and scaffolds to enhance electrical performance for stimulation and recording.1 Poly(3,4-ethylenedioxythiophene):poly-styrenesulfonate (PEDOT:PSS) is an electroconductive polymer often applied within biosensors and more recently as scaffold in tissue engineering.2 In this context, its conductive properties are hypothesized to enhance the effect of electrical stimulation; known to play a potent role in differentiation of progenitor stem cell sources into cardiomyocytes and in the maturation of cardiac engineered organoids.3
In this project, PEDOT:PSS was engineered into tunable, aligned, three-dimensional (3D) porous sponge-like structures. Scaffolds were functionalised via a crystallisation treatment to enhance their properties for tissue engineering. Afterward, we conceptualized and fabricated a bioelectric pacing bioreactor to deliver electrical stimulation to 3D scaffolds and an ad-hoc rig for in-vitro contraction-tracking, validated in-vitro using C3H10, primary rat cardiomyocytes and induced pluripotent stem cell derived cardiomyocytes.
Materials and methods
PEDOT:PSS was covalently crosslinked using glycidoxypropyl-trimethoxysilane (GOPS) and fabricated via lyophilisation. Crystallisation was achieved with incubation in pure sulphuric acid to improve conduction networks and remove excess PSS. Morphology of constructs was evaluated qualitatively using scanning electron microscopy (SEM) and quantitatively through image analysis of scaffold microtomed sections. Ethanol intrusion provided quantification of the overall porosity. Mechanical properties were determined using a Zwick-Roell uniaxial testing apparatus, while electrical features were simultaneously obtained from a Keithley sourcemeter. Via X-ray diffraction (XRD) it was possible to confirm the crystallisation of PEDOT:PSS. In-vitro studies determined the material biocompatibility and effectiveness of custom designed bioreactor. Viability, proliferation via DNA quantitation, metabolism and cell orientation were chosen as performance indicators. Bioreactor designs were generated with Solidworks® and rapid-prototyped with either Prusa-i3 or Formlabs SLA printers. Matlab® was adopted for the writing of scripts and the analysis of datasets, such as piezoresistivity, pore size, stress-relaxation, cell-directionality.
Results & Discussion
Controlled freeze-drying parameters achieved highly porous structures with either isotropic or aligned architectures. Crystallised scaffolds exhibited 1000-fold higher conductivity compared to untreated ones, while preserving stiffness and biocompatibility in a range matching to induce myogenic differentiation.4 We designed and prototyped both a pacing bioreactor that can fit standard 6-well plate and a chip with flexible anchorage for tracking of contraction (R3S), that is reusable and easy to manufacture. A 7-day study applying electrical pacing to C3H10 cells, showed that pacing does not decrease cells viability, and that it also promotes metabolism and alignment of cells, synergistically with the aligned topography of the scaffolds. Studies on primary rat cardiomyocytes and induced pluripotent stem cell derived cardiomyocytes further corroborated the use of these scaffolds for in vitro models.
Conclusion
Overall, PEDOT:PSS scaffolds provided an asset for the production of versatile platforms for tissue engineering applications.
References
1. Nezakati T, et al. Chemical Reviews. 118(14). 6766-6843. 2018.
2. Guex AG, et al. Acta Biomater. 62(91-101. 2017.
3. Solazzo M, et al. APL Bioengineering. 3(4). 041501. 2019.
4. Solazzo M, et al. Biomater Sci. 9(12). 4317-4328. 2021.
52354501244
Purpose: Each year, 15 million people suffer from a myocardial infarction and heart failure, resulting in one of the leading causes of death worldwide. Owing to the fact that the adult mammalian heart lacks a regenerative capacity, the ischemic cardiac muscle is replaced by scar tissue. While the mechanisms involved in fibrotic tissue formation are still elusive, the immune system is known to play a critical role. Therefore, modulating the immune response after cardiac injury is becoming a promising strategy to prevent scar formation and improve heart function after myocardial infarction.
Methods: Using a permanent left coronary artery ligation mouse model, we assessed the cardiac repair after myocardial infarction, in two conditions. The first condition involved depletion of a specific immune cell subtype with diphtheria toxin, using a genetically engineered model. On the other hand, the second condition involved the adoptive transfer of these cells in a clinically relevant manner, one day post ischemia, in wild type mice. The reparative cardiac capacity was assessed with echocardiography four weeks post ischemia, an angiogenesis assay, and Mason’s trichrome staining to assess the fibrotic area. To investigate the mechanisms of cardiac repair, we performed an EdU proliferation assay and a TUNEL survival assay on cardiomyocytes. Moreover, we performed flow cytometry and RNA sequencing on various immune cell subsets isolated from the infarcts to determine changes in the inflammatory response.
Results: Upon depletion of this particular immune cell subset, there was a significant increase in infarct size and a reduced left ventricular contractility (reduced ejection fraction and increased end diastolic volume), as evidenced with echocardiography. On the other hand, the adoptive transfer of the immune cell subset improved cardiac repair by reducing fibrosis and increasing the left ventricular contractility. This functional improvement was accompanied by enhanced angiogenesis in the infarct area, reduced cardiomyocyte cell death and moderately increased cardiomyocyte proliferation. In addition, the immune cells delivered by adoptive transfer accumulated at the site of cardiac injury and in secondary lymphoid organs post myocardial infarction. Mechanistically the pro-repair effect of this therapy was attributed to the differences in accumulation and the inflammatory phenotype of T cells and macrophages in the infarcts.
Conclusion: Taken together, we found that the delivery of this immune cell subset post ischemia diminished scar tissue formation by acting on multiple target populations and cellular processes, resulting in long-term improvement of cardiac function. This study demonstrates the potential of using this immune cell subset as a therapy for patients with myocardial infarction and potential heart failure.
41883604207
Introduction
Cardiovascular diseases remain the leading cause of death worldwide. Our research focuses in building cardiac microtissues that resemble the native heart as closely as possible, in terms of both structure and function. Across its thickness, native myocardium is built of several thin tissue sheaths, and the cardiomyocytes (CMs) in each of the layers are aligned, and thus contract, in a specific direction. Herein we aim at mimicking the laminar architecture of the myocardium, by producing tissue engineered constructs which show a preferential direction of contraction, and can be assembled to achieve relevant tissue thickness.
Methodology
Fibrous scaffolds with a diamond pattern were manufactured by melt electrospinning writing (MEW) of medical grade polycaprolactone, mechanically characterized and subsequently seeded with a mixture of 90% CMs and 10% cardiac fibroblasts, both obtained from the differentiation of human induced pluripotent stem cells. Samples were kept in culture and fully characterized, and their beating compared to that of other pore geometries.
Results
Myocardial tissue from porcine samples was histologically characterized in order to determine the variation of the fiber orientation from epicardium to endocardium, showing an angle variation of 6.39°/mm. A design with diamond-shaped pores was predicted to produce an in-plane contraction. MEW proved to be a reproducible and accurate method for printing these scaffolds, which showed adequate levels of compliance when mechanically tested. Engineered cardiac microtissues exhibited relevant cardiac-like features, including beating rate, sarcomere length and gene expression, as well as good viability and metabolic activity. When compared to other pore geometries, diamond-patterned scaffolds not only contracted along a preferential direction we had anticipated (that of lower mechanical resistance, i.e., the short axis of the diamonds), but also displayed greater magnitude and velocity of contraction than squared and rectangular scaffolds. Furthermore, optical mapping of the constructs showed better electrophysiological properties for the diamond-patterned samples, with values closer to native human cardiac tissue. Individual constructs could be stacked to a total thickness of ≈800 µm, and showed good cohesion of the distinct layers as well as more complex beating patterns.
Conclusions
In this work we developed a diamond patterned, melt electrospun scaffold, and show how this particular architecture favours the biomimetic contraction of seeded CMs along the short axis of the diamonds. By subsequently stacking several scaffolds with distinctly oriented diamonds, we obtained cardiac microtissues with increased biological representativity, in terms of their thickness, their multi-layered structure, and the varying principal orientation of each of the layers. Constructs demonstrated an adequate performance in vitro and were also tested in vivo, and show great potential for cardiac tissue engineering and regenerative medicine applications.
94238132466
The human gut microbiota constitutes the most bountiful and divergent community of organisms compared to the other areas of the body [1]. Growing attention is devoted to the bacterial equilibrium and constitution in human intestines, being highly plastic over time [2,3]. Bacteria present a significant part in response to immunotherapy in cancer [4]. Meanwhile, predominant cell cultures and animal models encounter substantial limitations [5].
3D-multi-compartment microfluidic cultures may overcome met obstacles, mimicking elaborate multicellular architectures and niches, while maintaining high control. Among the numerous gut-on-chip designs, the differentiation between 2D and 3D cellular microenvironments is possible, either mechanically stimulated or not. Mechanobiological actuation and 3D niche recapitulation proved integral in maturing and translating models from in vivo to in vitro [6,7,8].
Here, we present the intestine-on-chip PDMS-based device with endothelium captured in 3D under mechanobiological stimulation. As a proof-of-concept, the device integrates actuation on a three-cell types coculture, which includes human micro-endothelial cells and two human intestinal epithelial cell lines endowed with different adsorbing and secretory properties, respectively. A collagen-based extracellular-matrix compartment connects the intestinal and the vascular spaces. Multi-parametric assessment of cell viability and function at different time points describes the influence of mechanobiological stimuli on the maturation of the gut endothelium. Transcriptional profiling of the epithelial cells and functional characterization of different primary immune cells illustrate the suitability of the system to dissect complex interactions between components of the tumour microenvironment.
The biomechanically stimulated 3D intestine-on-chip provides an elegant platform to study how microorganisms inflect the crosstalk between epithelial and endothelial compartments in the gut and portray a relevant alternative for preclinical studies. The acquired model allows dissecting the trans-endothelial migration of immune cells in health and disease.
20941828449
"INTRODUCTION: Myocardial infarction (MI) is an ischemic and inflammatory event majorly orchestrated by macrophages from infiltrating monocytes. These macrophages play a critical role in deciding the fate of the heart post-MI. However, there is no cardiac disease model in existence that incorporates an immune response. Hence, the aim of this project is to develop a humanized model of MI, using induced pluripotent stem cell (iPSC) derived cardiomyocytes together with inflammatory cytokine stimulation, to model the disease environment.
PURPOSE: Despite the advances in developing effective engineered heart tissue (EHT) models for MI that can recapitulate intricacies of the native myocardium, such as contractile properties and ability to respond to different chemical stimuli, there is still a need to make these models physiologically relevant. We hypothesize that the addition of macrophage-derived inflammatory cytokines can aid in making EHT models of MI more humanized.
METHODS: The first objective of this project is to obtain conditioned media obtained from immune cells for stimulating cardiomyocytes. In order to achieve this, iPSCs were differentiated to obtain macrophages (iMacs). Their expression of general macrophage (CD14, CD11b) and resident macrophage (CX3CR1, CCR2 and HLADR) markers were assessed, in addition to their phagocytic and polarization potentials. Next, iPSC-derived cardiomyocytes were obtained (iCMs) and the experssions of general cardiomyocyte (cTnT, cx43) and maturation (sarcomeric actinin) markers were assessed. Finally, polarizsation of iMacs were analyzed within a collagen/matrigel hydrogel system.
RESULTS: iMacs were found to be 93.6% CD14highCD11bhigh. Compared to blood-derived macrophages, CCR2 was downregulated and CX3CR1 and HLADR were upregulated in iMacs showing a resident macrophage phenotype. Additionally, iMacs also showed phagocytic potential (39.9%) and ability to be polarized to pro-inflammatory (on stimulation with LPS and IFN-gamma) and anti-inflammatory (on stimulation with IL4) states. iCMs were found to be positive for cTnT, cx43 and sarcomeric actinin. Additionally, they were found to be positive for MLC2a, denoting an atrial cardiomyocyte phenotype.
iMacs were found to not polarize within the collagen/matrigel hydrogel system, which will be used as the final model to develop the EHT.
CONCLUSIONS: Macrophages with a higher resident phenotype and have been generated. In the future, conditioned media from these iMacs stimulated with pro-inflammatory factors will be used to treat cardiomyocytes, to understand its effects on cardiomyocyte function."
62825406088
"RECONSTRUCTION OF FUNCTIONAL GRADIENTS USING MELT ELECTROWRITING
Frendion F.S.J. Marchena1,2, Magdalena Gladysz1,3, Malgorzata K. Wlodarczyk-Biegun1,4
1Polymer Science - Zernike Institute for Advanced Materials, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands
2Hanze University of Applied Science in Groningen, Zernikeplein 7, 9747 AS Groningen, The Netherlands
3Pharmaceutical Analysis, Groningen Research Institute of Pharmacy, University of Groningen, Deusinglaan 2, 9713 AW Groningen, The Netherlands
4Biofabrication and Bio-Instructive Materials, Biotechnology Center, The Silesian University of Technology, B. Krzywoustego 8, 44-100 Gliwice, Poland
Contact: Malgorzata.wlodarczyk-biegun@polsl.pl
Introduction
Melt Electrowriting (MEW) is a biofabrication approach that combines principles of electrospinning and 3D printing. It allows obtaining the porous scaffolds with well-defined fibers in the range of a few micrometers with unprecedented precision. In our group, we use this approach to reconstruct the structure of different native tissues and model their functions in vitro.
This project aims to develop a functional reconstruction of human trabecular meshwork (HTM). HTM is a mesh located in the eye, responsible for the drainage of liquid for the anterior chamber and, therefore, maintaining the proper pressure in the eye. When dysfunctional, it often leads to glaucoma development.
Methodology
Different designs of porous scaffolds, closely mimicking the structure of native HTM, were designed and printed using MEW. Polycaprolactone was used for printing, and the scaffolds were coated with poly-L-lysine for cell culture studies. Primary HTM cells were seeded, and their performance was characterized in 21-days culture. Mechanical properties of the scaffolds were analyzed in tensile testing and tuned to obtain values matching healthy and glaucomatous eyes. To analyze the functionality of the proposed models, the permeability test was performed on cell-seeded scaffolds at the physiological liquid pressure.
Results
The scaffolds with the architectures resembling three distinctive zones of native HTM were successfully obtained. HTM cells cultured on the prints revealed high viability and expression of cell-specific proteins. Tensile test results revealed that mechanical properties close to healthy or glaucomatous tissue could be obtained by varying the scaffolds’ design. Cell-seeded scaffolds showed permeability values relevant for native tissue.
Conclusions
The MEW approach allowed to obtain structures closely mimicking native HTM regarding the design, mechanical properties, and function (i.e., permeability). We envision that these scaffolds will find applications as in vitro testing platforms for glaucoma drugs and, after further optimization, as implants for patients that require removal of dysfunctional HTM. This is an especially impactful finding as adequate HTM in vitro models, and biomimetic HTM implants are currently missing.
"
83767214567
"Introduction
Osteosarcoma (OS) is the most frequent malignant bone tumour with a survival rate of less than 25% in metastatic disease. Treatment is a combination of surgical resection and systemic chemotherapy with doxorubicin (DOX). However, this can cause serious long-term adverse effects. To overcome this limitation, we have developed an advanced rat OS model that converges tissue engineering and regenerative medicine principles to improve treatment outcomes for OS patients.
Methodology
A humanized bone microenvironment was created by implanting an orthotopic tissue engineered bone construct (ohTEBC) fabricated from melt electrowritten medical grade polycaprolactone coated with calcium phosphate (mPCL-CaP) together with fibrin glue (Baxter) and 45 µg rhBMP-2 (Medtronic) around the femur of an immunocompromised rat. A primary OS tumour was then created by injecting human SaOS-2-luc human OS cells into the humanized bone niche. Bone volume was measured with in vivo CT (Molecubes µPET-CT) and tumour formation and metastasis was monitored using bioluminescent imaging (BLI) (IVIS Spectrum, Perkin Elmer). The humanized bone niche and primary OS tumour was characterised by ex vivo histology and immunohistochemistry.
Results
A humanized bone microenvironment formed within 6 weeks of implantation, with µCT confirming increased bone volume around the femur. Following SaOS-2-luc injection, BLI confirmed primary OS tumour growth and development of lung metastases over a 14-week period. µCT revealed pathological increase in bone volume within the OS tumour. In future studies, the humanized rat OS model will be used to investigate the efficacy of scaffold-mediated local DOX delivery following surgical resection of the primary OS tumour, in comparison to systemic DOX chemotherapy delivery. The model will also be used to study the regeneration of post-DOX-treated critical-sized segmental bone defects using scaffold-guided tissue engineering and regenerative medicine approaches.
Conclusion
Here, we have created an orthotopic OS tumour model that recapitulates the hallmarks of human disease within an immunocompromised rat. This model will allow to study complex surgical interventions and regenerative medicine techniques never before possible in previous model systems. The outcomes of this study will improve the chemotherapeutic and limb sparing surgery options for those affected by OS."
83767220867
"Introduction: Chronic ulcerative oral mucosal inflammatory diseases, including oral lichen planus and recurrent aphthous stomatitis, are painful and highly prevalent, yet lack effective clinical management. These conditions are usually treated with either topically applied or systemically-delivered corticosteroids depending on severity with varying degrees of success. In recent years systemic biologic therapies, including monoclonal antibodies that block the activity of proinflammatory cytokines, have increasingly been used to treat similar immune-mediated inflammatory conditions, such as rheumatoid arthritis and psoriasis. The ability to deliver these therapies locally to the oral epithelium could radically alter treatment options for oromucosal inflammatory diseases, where cytokines, in particular tumour-necrosis factor-a, are a major driver of pathogenesis.
We previously developed a dual-layer electrospun mucoadhesive patch with high patient acceptability and a long in vivo residence time (Colley, HE et al., Biomaterials. 178:134-146 (2018)). We have incorporated small molecules drugs such as corticosteroids and anaesthetics into these patches and have demonstrated their release and activity (Clitherow KH, et al., Mol Pharm 16:3948-56 (2019)). Recently, we showed that small proteins and peptides can also be incorporated whilst retaining their biological activity (Edmans JG. et al., Mat Sci Eng 112:110917 (2020)). Here, we investigate the inclusion and delivery of therapeutic anti-TNF-α F(ab) antibody fragments for the treatment of oral inflammatory disease.
Methodology and results: Biotinylated F(ab) fragments was incorporated into an electrospun mucoadhesive membrane and were found to be retained within the fibres in aggregates when visualised by confocal microscopy. These F(ab) were rapidly eluted from the patch without loss of antigen binding activity (97 ± 5% released within 3h). Neutralising anti-TNF-α F(ab) fragments were generated from whole IgG by papain cleavage, as confirmed by SDS-PAGE, then incorporated into patches. Antibody-containing patches were found to have TNF-α neutralising activity, as shown by the suppression of TNF-α-mediated CXCL8 release from oral keratinocyte grown as monolayer cultures. Patches applied to lipopolysaccharide stimulated immune-competent oral mucosal ulcer equivalents that contained primary macrophages, led to a statistically significant reduction in the levels of biologically active TNF-α, suggesting successful delivery of a therapeutically relevant dose. Moreover, inhibition of TNF-α by patch-released anti-TNF-α F(ab) fragments also resulted in a significant decrease in the levels of T-cell chemokines produced, indicating that patch-delivered neutralising antibody therapy impacts on downstream immunological events such as leukocyte recruitment that will further prevent pathogenesis.
Conclusion: Electrospun oromucosal patches can deliver active biologics such as therapeutic antibodies topically to diseased oral mucosal sites in vitro. These patches have the potential to change the way these debilitating oral diseases are treated in the future."
83767225105
"Breast cancer (BCa) is the most common cancer amongst women worldwide and the leading cause of cancer related death. The triple negative BCa (TNBCa) subtype is associated with a particularly aggressive clinical behavior including an early peak of fatal distant metastasis, predominantly to bone. Routine systemic chemotherapeutic treatment with doxorubicin (DOX) is limited due to its severe side effects, especially cardiotoxicity. Novel (targeted) nanotherapies are a promising breakthrough to enhance treatment efficacy and specificity of known chemotherapeutics while at the same time decreasing their systemic toxicity. Therefore, we studied the therapeutic effect of non-targeted and targeted DOX-containing nanoparticles as a novel treatment approach against TNBCa bone metastases using a humanized tissue-engineered mouse model.
A humanized bone microenvironment was created in NSG mice by subcutaneous implantation of humanized tissue-engineered bone constructs (hTEBCs) consisting of tubular biodegradable medical grade polycaprolactone scaffolds seeded with human osteoblasts and an inner vascular bone marrow niche. After 13 weeks of in vivo bone formation, TNBCa primary tumor was induced by injecting MDA-MB-231BO-Luc cells into the mammary fat pad and the tumor was allowed to grow and metastasize to humanized bone. Subsequently, treatment with DOX-loaded hyper branched methoxy polyethylene glycol (mPEG) (HBP) nanoparticles was performed. The DOX-HBP nanoparticles were either non-targeted or targeted with a Thomsen-Friedenreich (TF)-mPEG bispecific antibody (BsAb) and administered once per week over a duration of 3 weeks.
Implantation of the hTEBCs resulted in the formation of a chimeric bone organ in vivo containing human-derived extracellular matrix, bone marrow and showing evidence of ongoing complex bone formation through endochondral ossification. HBP nanoparticles predominantly accumulated at the primary tumor and hTEBCs. The non-targeted HBP-DOX nanoparticles were able to slow primary tumor growth and reduce metastasis compared to the targeted HBP-DOX nanoparticles and non-DOX containing control groups. Additionally, the non-targeted nanoparticles reduced systemic toxicity effects (cardiotoxicity, hepatotoxicity, hematological toxicity) and prolonged survival compared to free DOX treatment. Further targeting with the BsAb did not improve treatment outcome, most likely because of enhanced clearance (accumulation in liver and spleen). However, the targeted HBP-DOX nanoparticles lead to increased lung metastases and tended to increase metastasis to the liver and hTEBCs compared to the saline control.
In conclusion, this study is an exciting example of complex pre-clinical disease modelling including a humanized bone niche in the mouse. Furthermore, it highlights the great potential of nanomedicines in cancer therapy, but also demonstrates how changed nanoparticle properties can alter their treatment efficacy in vivo."
52354539126
"Introduction
Patients with chronic kidney disease (CKD) experience multiple comorbidities, among which mineral/bone disorders (MBD) contribute to high mortality due to increased facture risk [1]. CKD affects the quality of bones which become weaker and easily break. Recently, endogenous metabolites, such indoxyl sulfate (IS), were reported to have an active role in the development of uremic bone as they accumulate in blood due to poor kidney function [2]. However, the effects of these metabolites on the bone matrix is not well understood, due to lack of appropriate models. For this, we established an in vitro 3D model of human bone to investigate the activity of osteocytes, master regulators of bone remodeling, under exposure to IS. We investigate the osteocyte phenotypic signature (gene and secretion of remodeling molecules) accompanied by analysis of the quality and quantity of the deposited bone matrix.
Materials and Methods
We encapsulated human adipose-derived stem cells (hASCs) into fibrin hydrogels and differentiated them into osteoblasts- and osteocytes-like cells. The osteogenic differentiation was performed either under standard conditions or under treatment with IS. To assess the effect of IS treatment on the newly formed bone-like matrix, we examined the differential expression of the major osteoblastic (Opg, Col1a1, Opn, Osteonectin, Tnfsf11) and osteocytic genes (Fgf23, Col1a1, Mepe, Sclerostin, Opg, Osteonectin), and evaluated the secretion of soluble key regulators of bone remodeling (OPG, FGF23, SOST). The composition and mineralization of the bone matrix was also analyzed by staining for calcium (Alizarin Red) and collagen (Sirius Red and immunοstainings), accompanied by mechanical testing of the bone-like construct.
Results
HASCs successfully differentiated into osteoblasts and osteocytes-like cells in standard osteogenic differentiation conditions, as evidenced by a highly mineralized and collagen enriched matrix. IS treatment was shown to significantly downregulate the expression of bone remodeling genes. Osteocytes-secreted bone remodeling mediators were also significantly lower (p<0.05) when compared to healthy cultures. Even more, the quality and the quantity of the deposited bone were impaired. The amount of collagen, as well as the degree of mineralization were reduced by 43.9%, p<0.001 and by 29.7%, p<0.05, respectively. Via X-ray imaging, we could identify a poor calcium-containing matrix while under exposure to IS. These changes were reflected in the decrease (p<0.05) of the mechanical properties of the uremic bone-like construct.
Conclusion
Our results suggest that IS impairs the osteogenic differentiation of hASCs and induces alterations of the bone extracellular matrix, in terms of collagen deposition, amount of inorganic matter and mechanical properties. Furthermore, exposure to IS during the transition of osteoblasts to osteocytes affects the acquisition of key regulatory features of bone remodeling.
Acknowledgments
This project received funding from the 3Rs stimulus Funds of the Utrecht University.
References
[1] Covic A, et al., Lancet Diabetes Endocrinol. 2018 6(4):319-331, PMID: 29050900
[2] Kamprom W, et al., Int J Med Sci. 2021 18(3):744-755. PMID: 33437209."
52354528084
"Introduction: Folding is a crucial process that modulates the function of proteins:[1] non-covalent intramolecular interactions between amino acids give rise to a defined three-dimensional structure with minimal free energy known as protein native state. This is an error prompt process that often results in misfolding and the formation of off-pathway aggregates associated with pathological conditions. There are different cellular mechanisms to control this process, e.g., post-translational modifications that change the free energy of the protein. An example is glycosylation that affects the protein folding and aggregation.[2] The thermodynamics and kinetics of these processes are still poorly understood and often rely only on in silico models. Herein, we show that O-glycotripeptides are extremely useful reductionist models to study the involvement of glycosylation in these processes at molecular and microscopic levels.[3]
Methodology: Minimalistic glycoproteins were designed using glycosylated serine (S) or threonine (T) flanked by phenylalanine (F). The glycosylated S and T are characteristic structural components of O-glycoproteins, while the aromatic F was introduced to augment the aggregation propensity of the glycopeptides. The aggregation of these glycotripeptides was compared to their respective non-glycosylated analogues using in silico all-atom molecular dynamics simulations and in vitro by circular dichroism (CD) and X-ray diffraction (XRD). The morphology of the generated aggregates was visualized by scanning (SEM) and transmission (TEM) electron microscopies and their mechanical properties were measured by atomic force microscopy (AFM).
Results: We were able to assess the distinct contributions of F, S or T and glucose to the glycopeptides’ stereochemistry and aggregation. Although S and T differ only by a methyl group, this subtle variation affects the inter- and intramolecular CH-pi interactions between F and S or T: F/S << F/T. S to T substitution also induced alterations in the morphology of the generated supramolecular aggregates as shown for the non-glycosylated peptides. O-glycosylation introduced changes in the pi-interactome by establishing additional CH-pi interactions, i.e., Glc/F. The aggregates of the glycopeptides have reduced stiffness and increased thermal stability when compared to their non-glycosylated counterparts. These changes were more prominent for the S analogues when compared with the T ones.
Conclusions: We demonstrate that simple glycotripeptides are a useful model for revealing the mechanism(s) of the aggregation processes at the molecular level. The generated assemblies can be also used as functional biomaterials acting as biomimetics of glycoproteins.
Acknowledgements: We acknowledge the EU's H2020 program (Forecast 668983) and the Portuguese FCT (BD/113794/2015; PTDC/BTM-MAT/28327/2017 CARDIOHEAL; CEECINST/00077/2018) for the financial support. Part of this research was supported by National Science Foundation (NSF) grant CHE-1808143 and a grant of computer time from the City University of New York High Performance Computing Center under NSF grants CNS-0855217, CNS-0958379 and ACI-1126113.
References:
[1] Shental-Bechor, D. et al. PNAS. 24, 8256-8261 (2008).
[2] Xu, C. et al. Nat. Rev. Mol. 16, 742-752 (2015).
[3] Brito, A.B. et al. JACS, 143, 19703-19710 (2021)."
31412705605
Whey protein isolate (WPI) is an inexpensive byproduct from the dairy industry which can be processed into autoclavable hydrogels with high compressive strength which support the adhesion, growth and differentiation of cells which can be applied in bone regeneration. Furthermore, WPI hydrogels can solubulized hydrophobic molecules and hence serve as a delivery vehicule for hydrophobic molecules with biological activity (e.g. antibacterial agents.) WPI can also be processed into flibrillar coatings for biomaterials, which also support the adhesion, growth and differentiation of cells. This presentation will review the aforementioned applications of WPI.
41883636486
INTRODUCTION
Creating biofunctional scaffolds could potentially meet the demand for patients suffering from bone defects without having to rely on donors or autologous transplantation. 3D printing has emerged as a promising tool to fabricate scaffolds with high precision and accuracy by computer design using patient-specific anatomical data1. Among other relevant key points for 3D-printed bone scaffold selection, to achieve controlled degradation profiles is an essential feature to consider. Thus, the importance of a deep characterization of the biomaterial degradation under physiological conditions is needed2.
METHODOLOGY
50:50 blend made of PCL-PLGA was created to fabricate cylindrical scaffolds by 3D printing. The blend was fabricated by dissolving PCL pellets and PLGA powders in dichloromethane, by casting and evaporating the solvent. PCL-PLGA filaments were extruded with a mechanical extruder. Cylindrical scaffolds were finally printed with a 7mm diameter, 2mm height, 400µm pore sizes. Their hydrolytic degradation under different conditions was quantified.
Static buffer medium and flow perfusion were applied to the samples inside an in-house fabricated bioreactor, which contains four main individual chambers. Perfusion tests were done inside the bioreactor thanks to a roller pump which imposed a PBS flow rate of 4 mL/min. Samples were incubated in normoxia (21% O2 and 5% CO2) for two and four weeks. Degradation under static conditions was also conducted inside the bioreactor with no flow. During both conditions, PBS in the wells was exchanged every two days and the pH was measured periodically.
Several techniques were used to characterize the degradation of the polymers by the end of the incubation period including chemical changes on the surface by x-ray photoelectron spectroscopy, polydispersity index by gel permeation chromatography, surface inspection by scanning electron microscopy, mechanical properties decrease weight loss and medium acidification over time.
RESULTS AND CONCLUSIONS
In this work, we have thoroughly characterized the hydrolytic degradation of the final samples at different incubation periods, achieving different outcomes in agreement with our initial hypotheses. Results confirm a faster degradation of PCL-PLGA scaffolds when flow is forced through the samples. Besides, it was also confirmed the quicker degradation of PLGA in the blend. In addition, time is also a key factor and we obtained significant differences for both incubation times: 2 and 4 weeks.
ACKNOWLEDGEMENTS
Curabone project received funding from ITN-MSCA0 (grant No. 722535), the use of SAI and LMA (Universidad de Zaragoza), Spanish Ministry of Economy and Competitiveness through Projects DPI2017-84780-C2-1-R and PID2020-113819RB-I00 and the Government of Aragon in the form of grant awarded to PAD (Grant No. 2018-22).
REFERENCES
1. Roseti, L. et al. Scaffolds for Bone Tissue Engineering: State of the art and new perspectives. Mater. Sci. Eng. C 78, 1246–1262 (2017).
2. Li, C. et al. Design of biodegradable, implantable devices towards clinical translation. Nat. Rev. Mater. 5, 61–81 (2020).
41883601764
"Introduction
In present times, implant development focusses on optimizing biocompatibility, mechanical strength, and reproducibility. Although cardiovascular implants currently available on the market are medically established and therefore widely used, they have a limited lifetime which also impacts the mechanical properties and functionality. Additional issues are the formation of blood clots in mechanical implants (heart valves), the formation of bacterial biofilms (dental implants), and the degradation of bioprotheses. Also, implant rejection by the patient’s body is still a challenge. A solution for these issues can be the use of biohybrid hydrogels. Especially fibrin-based hydrogels are very promising.
Methodology
The aim of this research is the synthesis and characterization of fibrin-based hydrogel-matrices for Tissue Engineering applications with the research focus on cardiovascular implants, to be precise on heart valve replacements. We have reinforced fibrin by the addition of linear functional copolymers, specific fibrin-binding peptides, and functional microgels. Our goal is to establish an innovative functional tool-box for fibrin-based biohybrid hydrogels that allow also for patient-specific individualisation of implants. The effect of incorporated functional additives in the hydrogel-matrices is analyzed in regard to their mechanical properties, fiber-network morphology and biocompatibility.
Results
In terms of our research, we could already demonstrate that the addition of linear poly(N-vinylpyrrolidone)-copolymers with functional epoxide groups can enhance the mechanical behavior of the fibrin-based hydrogels due to covalent crosslinking, resulting in higher storage moduli, thicker fibers, and a decreased degradation rate compared to pure fibrin-hydrogels. The obtained hydrogels additionally possess a high biocompatibility as proven by cell viability experiments.
In addition, specific fibrin-binding peptides were applied, which exhibit supramolecular interactions within the fibrin-matrix. A combination of supramolecular and covalent interactions by mixing linear polymers and fibrin-binding peptides in various ratios can enhance the strain-stiffening behavior of the hydrogel matrix. Also, the fiber thickness could be increased.
As a third modification of fibrin-based hydrogels, functional thermoresponsive microgels were used instead of linear copolymers and specific peptides. Just like the linear copolymers, the N-vinylcaprolactam-based microgels include glycidyl methacrylate as a functional comonomer to enable covalent attachment to the fibrin by epoxide groups. We can demonstrate that the use of microgels as colloidal crosslinkers resultes in hydrogels providing a temperature-dependent increase in storage modulus, which is not present in pure fibrin-gels. As microgels are widely studied for their possible application in drug delivery, owing to their ability to encapsulate active substances, their use is of high interest in Tissue Engineering applications.
Conclusion
We could develop a functional tool-box for the reinforcement of fibrin-based hydrogels. The mechanical and morphological properties can be tailor-made by selecting the respective type of additive. Regarding the Tissue Engineering of materials mimicking native heart valves, compartments with different mechanical properties are needed, which is exactly what our tool-box allows us to create."
20941818306
"Introduction
Cell infiltration is essential for the repopulation of dense materials in tissue engineering. During that process, several factors, such as scaffold topography, mechanical properties or porosity play a key role. These acquire special importance when designing the materials to substitute and regenerate articular cartilage. Because of their similar structure and composition, decellularized cartilage scaffolds represent an ideal candidate. Our group previously developed a biomaterial from auricular cartilage, which allowed us to study cell migration [1]. Removing the elastic fibres and depleting the glycosaminoglycans (GAGs) leaves a network of open channels. This process reduces the matrix density, altering their mechanical properties and enhances the elastic fibre removal process. In this study, we aimed to investigate the influence of GAG removal on the mechanical properties and cellular ingrowth on decellularized auricular cartilage scaffolds.
Methodology
Scaffolds were harvested from bovine ears by punching 8mm diameter discs as described previously [1]. Briefly, scaffolds were cut to a standard thickness and subjected to several freezing-thawing cycles. Afterwards, an enzymatic treatment comprised of pepsin and elastase was used. Because pepsin is the enzyme that removes the GAGs, a concentration series from 0 to 1 mg/mL was used. Elastase, the enzyme that depletes the elastic fibres was maintained constant at a concentration of 0.03 U/mL. First, GAG content was quantified by histology (Alcian blue staining) and blyscan assay. Second, fluorescent-labelled adipose-derived stromal cells (ASCs) and human articular chondrocytes were seeded on the scaffolds and cultured under dynamic conditions for one week. Cell infiltration was monitored by confocal microscopy and immunohistochemistry. Furthermore, the mechanical properties were measured by a stepwise compressive test using a Zwick uniaxial testing machine. The viscosity and elasticity of the samples were further computed using a mathematical model. For comparison of two groups unpaired t-test or Mann–Whitney test was chosen according to distribution.
Results
A concentration series of pepsin was used to modify the GAG content, which was inversely correlated. Residual GAG concentration after high and low pepsin concentrations differered significantly (1 mg/mL pepsin: 0.058 0.014 mg/µg vs. 0.2 mg/mL pepsin: 0.15 0.042 mg/µg; p = 0.0022). Cellular infiltration, however, followed the inverse trend. Higher concentrations of pepsin increased cellular infiltration. Nevertheless, even the lowest tested concentration (0.2 mg/mL) maintained adequate levels of cell migration into the open channels. Interestingly, cells were unable to penetrate the scaffolds treated without pepsin, forming a monolayer on the surface. In the slower infiltrating chondrocytes, the effect of matrix treatment was even more visible than for ASCs. Additionally, the mechanical properties followed a similar pattern. Native scaffolds and those treated with 0.2 mg/mL pepsin showed a viscoelastic behaviour and higher stiffness. On the contrary, concentrations above 0.4 mg/mL led to a more viscoplastic and fluid-like behaviour.
Conclusion
Auricular cartilage scaffolds are a suitable tool to study cell infiltration. Pepsin reduces considerably the GAG content, leading to reduced stiffness. Cell migration into the scaffolds is highly dependent on both parameters, which are crucial for scaffold development.
References:
1. S. Nürnberger et al., Acta Biomater. 86, 207–222 (2019)"
83767224905
"Introduction -
The development of novel biocomposite formulations has significantly contributed to the biomedical field- specifically within translational and clinical applications. This study centres on the polyhydroxyalkanoates-based copolyester superfamily of materials, with a particular emphasis on the poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] copolymer as a novel biomimetic substrate. Both 3HB and 4HB monomers have been recognised as natural metabolites in mammalian systems and this copolymer can be synthesised using a microbial-based production system. Crucially, it has been accepted that the 4HB monomer molar fraction determines the physical characteristics of the final copolymer which, ultimately, defines its endpoint application. This project compares two different fermentation techniques used for the synthesis of P(3HB-co-4HB) in terms of yield, purity and composition. Importantly, the synthesised material is characterised in terms of its physical, mechanical and biological profiles before being further enhanced/functionalised with 45S5 bioactive glass and graphene.
Methodology -
P(3HB-co-4HB) copolymer, from Cupriavidus sp. USM1020, was extracted from a shake flask cultivation or bioreactor inoculation system before being purified to obtain crude copolymer. 45S5 bioactive glass was prepared using a sol-gel technique [ratio of 45SiO2-24.5CaO-24.5NaO-6P2O5 (wt%) precursors]. Graphene monolayers were fabricated using liquid exfoliation of graphite. The biocomposite mixture was prepared via facile blending by dissolving P(3HB-co-4HB) crude copolymer in chloroform followed by ultrasonication with the bioactive glass and graphene. Casting was achieved by either solvent or emulsion freeze drying techniques to assess the differences in their porosity. Characterisation was performed using SEM, FT-IR and DSC. Disc diffusion antibacterial assay was employed with Escherichia coli and Staphylococcus aureus. Biocompatibility was assessed using human dermal fibroblasts (HDF) and murine osteoblastic (MC3T3-E1) cells, and standard cell culture assays: cell attachment (SEM imaging, live/dead), proliferation and viability (metabolic activity/MTS, LDH-release), and differentiation activities (cytokine expression/western blotting). Preliminary in vivo studies were performed using male Sprague-Dawley rats.
Results –
The shake flask cultivation system generated a higher percentage (~69%) of 4HB compared to the bioreactor system. Miscibility of the biocomposite was further improved via facile blending. Morphological analyses showed that the emulsion freeze drying technique resulted in a more porous structure compared to solvent casting and the associated change in wettability confirmed with water contact angle measurements. The mechanical profiles of the biocomposite and antibacterial activities were enhanced following incorporation of graphene. Optimised composition of both bioactive glass and graphene within the biocomposite is vital in ensuring optimal level of cells adhesion, which resulted from the observed attachment and proliferation of both cell lines. The animal studies (i.e. skin flap and bone defect) demonstrated good biocompatibility and favour interaction between P(3HB-co-4HB)-bioactive glass-graphene biocomposite and native tissues with enhanced presence of nuclei and neo-vascularisation, and minimal immune response.
Conclusions –
Taken together, this study illustrates the crucial optimisation parameters of the novel formulation P(3HB-co-4HB)-bioactive glass-graphene, which includes processing techniques that affects the final morphology and behaviour of the biocomposite. Owing to the individual benefits of each prominent material utilised in this study, increased potential in translational biomedical applications especially as therapeutic dressings and non-load bearing scaffold for bone regeneration can be considered."
73296328205
"INTRODUCTION:
Extracellular matrix (ECM) protein is often used in cell culture to provide developmental cues for cells such as skin cells. This is especially important for the generation of epidermal models as without a dermal compartment the keratinocytes are reliant on the ECM coating to provide them with a foundation to adhere to and for pro-developmental cues. A major limitation of most commercially available ECM is its composition, as the majority of commercial/scientific suppliers only provide a single ECM component (such as Collagen I). This is a major limitation as this confers reduced physiological relevance in comparison to native human ECM which contains a plethora of ECM components. In addition, commercially available products are often not of human origin which severely limits their physiological relevance and therefore their overall effectiveness in cell culture. As a result, we have developed a methodology of obtaining and purifying a broad-spectrum ECM derived from primary human fibroblasts, which will confer numerous benefits not only to cell culture, but also for topical cosmetic applications and wound healing.
METHODOLOGY:
Primary human fibroblasts were expanded and seeded into a microporous Alvetex® scaffold, which provides a 3D microenvironment to allow for replication of the native conditions found in skin that serves to promote increased cells attachment and ECM deposition. In order to stimulate maximum ECM production, the skin models were cultured in a 54mm diameter Alvetex® scaffold with Dulbecco’s modified eagle medium (DMEM) with 10% non-heat treated foetal bovine serum (NHT-FBS). Once the dermal model had reached maturity after 4-weeks of culture time, the ECM was extracted from our in-house generated dermal models by a process of mechanical homogenisation of the dermis followed by re-solubilisation of the extracted ECM components using an extraction solution. This was followed by centrifugation to remove any pieces of the supporting scaffold and cellular debris/insoluble components, leaving the ECM suspended in the supernatant, which was then collected and lyophilized to produce the final purified ECM product.
RESULTS:
Analysis of extracted ECM by Western blotting and total collagen assay showed that the composition of extracted ECM contained predominantly Collagen type I as expected from native human skin. The purified ECM was then used as a coating solution for the generation of epidermal models using primary human keratinocytes in comparison to a range of currently commercially available coating matrixes It was found that this solution was capable of supporting the growth and development of a complex and stratified epidermis.
CONCLUSIONS:
Extraction of ECM from our in-house generated dermal models has been shown to be a viable method for the production of physiologically relevant human derived ECM that is capable of supporting the growth and development of a multi-layered and stratified epidermis. Overall, this has implications in cell culture model generation to provide more physiologically relevant cell culture conditions and developmental cues. Including further uses in downstream applications in the cosmetic industry whereby human collagen can be incorporated into topical cosmetics to help improve the appearance of skin aging."
20941865528
"Introduction Tissue engineering (TE) is an interdisciplinary field that creates functional biologic substitutes for the repair of damaged tissues or organs. One major challenge when generating a functional TE model is its vascularization. Indeed, nutrient and oxygen supply, as well as metabolic waste products collection, are essential for the survival of the engineered tissue after transplantation. Hence, the development of a stable and functional vascular network within the constructs is essential. Using materials that include angiogenic cues as scaffolds for TE constructs may be a potential solution. Understanding this, the stromal vascular fraction (SVF) of adipose tissue has been proposed as a tool for in vitro pre-vascularization1. SVF is a heterogeneous cell population that has shown spontaneous vasculogenesis when cultured in vitro, in the absence of added growth factors. The extracellular matrix (ECM) produced by SVF cells is a key component to this capability. Many recent reports detail the use of ECM-derived hydrogels as TE scaffolds able to support cellular activities due to their similarity to native tissue’s ECM. We herein report the development of an angiogenic hydrogel derived from the ECM of SVF cell sheets.
Methodology SVF cell sheets were subjected to a decellularization protocol by a combination of freeze-thaw cycles and a nuclease treatment. Then, the samples were freeze-dried and digested with an acidic pepsin solution, and hydrogel polymerization occurred after pH neutralization with 0.5 M NaOH and temperature increase up to 37ºC for 1h. DNA quantification allowed to assess decellularization efficiency, while circular dichroism (CD) allowed to verify protein secondary structure maintenance. Regarding the ECM-related protein content, SDS-PAGE and Western blot were used. ECM-derived hydrogels were also stained for the presence of nuclei and collagen by using, respectively, Hematoxylin and Eosin (H&E) and Sirius Red/Fast Green staining.
Results DNA quantification and H&E staining confirmed decellularization efficiency. Through CD technique, it was possible to detect the triple helix conformational structure typical of collagen, confirming conservation of protein structure after the extraction protocol. Protein analysis with SDS-PAGE and Western blot revealed high protein variety within the ECM extract, with type I collagen being the predominant one. This was also verified by Sirius Red/Fast Green staining.
Conclusions Overall, these results show that we were able to isolate ECM proteins from SVF cell sheets and successfully create an ECM-like hydrogel with a Freytes solubilization protocol. Ongoing studies are focused on the proteomic characterization of the hydrogel as well as on in vitro cell culture studies to confirm the angiogenic potential compared with commercial collagen hydrogels. If effective, the use and development of regenerative strategies based on angiogenic ECM-like hydrogels can lead to promising advances in the TE and regenerative medicine fields.
Acknowledgements: EU Horizon 2020 research and innovation program under the ERC grant CapBed (805411); IF/00347/2015.
62825420164
Introduction: Due to their biocompatibility, biodegradability and cell-interactivity, gelatins are widely used in the biomedical and tissue engineering fields. Gelatin can be chemically crosslinked to originate constructs that are stable at body temperature. However, if modified gelatins cannot be produced with consistent molar mass and degree of modification (DoM), these advantageous biomaterials cannot be used in a reliable manner. To achieve predictable hydrogel strengths, consistent GelMA molar mass, degree of modification and the associated photo-kinetics need and can be tightly controlled.
Methodology: Six GelMA types (90 kDa and 160 kDa at DoM of 40, 60 and 80 %), were dissolved at concentrations of 5, 10 and 15 w/v% in PBS. The photo-initiator LAP was added to the GelMA resins. The photo-crosslinking kinetics of the GelMA resins were studied at 20 °C using a TA Discovery Hybrid Rheometer HR-2 (TA Instruments, the Netherlands) that was equipped with an Hönle - Bluepoint 4 - UV-A light (7.4 mW cm-2). The samples were in situ irradiated from the bottom through the quartz plate using a parallel plate setup (Ø 20 mm, gap 0.300 mm). A shear frequency of 1 Hz and an amplitude (strain) of 1 % were selected as they were within the linear visco-elastic range of the GelMA materials. The storage moduli (G’) were monitored over time.
Results: The herein presented data confirm that with increasing degree of modification (DoM) the storage modulus of the GelMA hydrogels will increase too. The increase in DoM can be correlated to an increase in storage modulus with an exponential component of e~0.6x for the 160 kDa GelMAs and an exponential component of e~0.54x for the 90 kDa GelMAs. With increasing GelMA concentrations (w/v%) the storage modulus of the hydrogel will increase. This statement was confirmed for the 90p GelMAs with a near perfect power-correlation (x~3.3). Interestingly, the 160 kDa GelMAs do not show a power correlation, but a linear one, between hydrogel strength and GelMA concentration. As a result of these different correlations, a convergence is seen between molar mass affected storage modulus, and concentration (w/v%) affected storage modulus. At low GelMA concentrations a clear difference is seen between molar mass and storage modulus, but as concentration increase this difference in storage moduli becomes smaller and smaller.
Conclusion: With consistent GelMA molar mass and degree of modification, hydrogel strength becomes predictable, as evidenced by the exponential-correlations found for the 160 kDa GelMA (e~0.6x) and 90 kDa GelMA (e~0.54x). Regarding hydrogel strength in function of GelMA concentration, a power-correlation was evinced (x~3.3) for 90 kDa GelMAs and a linear correlation for 160 kDa GelMAs, showing a convergence in hydrogel strength across GelMA molar mass and GelMA concentration.
Acknowledgement: This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 964497.
94238130728
"Image-based analysis of cells is a powerful modality to measure and record the high content information which reflects the cellular status during their culture. Although morphology has been known for a long to contain significant information to monitor the transitions of cellular status, their analysis has been limited to experience-based interpretations. However, by the recent rapid development of image processing and AI technologies, we now have tools and platforms to tackle for understanding the real-time cellular status in a more quantitative manner. However, image-based analysis has been extremely limited to analyzing “labeled-images,” and rare applications have been challenged to measure and utilize the heterogeneity information of cells for predicting cellular quality.
Our group has been reporting the label-free morphology-based analysis approaches for developing enabling technology for cell quality monitoring and control for maximizing the efficiency and reproducibility of cellular research and manufacturing [1-3]. Practically, from the time-course microscopic images, our data processing extracts not only the morphological descriptors of individual cells, but also the populational transition information, and tags them to the experimentally obtained cell quality ground-truth data. The key point of this technology is to combine the right combination of imaging hardware, automation technology, image processing, and data processing for objective performance. It should be noted that such a deep neural network structure is not always the best solution for such AI-based quantitative analysis.
In this talk, we will show the practical successful examples to apply such image-based label-free analysis for cell quality maintenance applications in (1) mesenchymal stem cell allogenic cell bank establishment, (2) single-cell morphological analysis for detecting senescence in mesenchymal stem cells, (3) novel optical challenge for label-free evaluation of spheroids. Our results show a high potential of image-based morphological analysis to enhance the quantitative understanding of the status of cells and their culture conditions. We also discuss the limitations and technological difficulties of AI-based image analysis compared to other image-based achievements in other fields for sharing the key points to enable the image-based cell evaluation successful.
94355102706
We will discuss the influence of cell shape and mechanical load on cell adhesions, cytoskeletal structure, cell signaling and transcription as determinants of cell fate and function.
83871204866
Introduction: Chondrocytes beneath the joint surface display a distinct superficial chondrocyte spatial organization (SCSO), which is a marker of tissue ultrastructure and function that undergoes a proliferative remodeling in early osteoarthritis [1,2]. Cellular filamentous actin (F-actin) and cell volume, two proliferation cofactors, change with osteoarthritis and might contribute to proliferation in early osteoarthritis. We asked whether chondrocyte proliferation rate (PR) can be controlled by controlling cell shape, and whether cell shape, the macroscopic tissue disease state (MTDS), or cytoskeletal F-actin intensity, density, or distribution has the strongest impact on the PR. To answer this question, we established a quantitative biology approach, combining single cell analyses with partial least square (PLS) analyses and random forest (RF) classification and regression predictive modeling.
Methodology: Chondrocytes were isolated from two MTDSs, macroscopically intact (MIA) or osteoarthritic-lesional areas (MOA), and cultured on custom-designed micro-patterned adhesion sites (MPs) for 1 or 7 days. Fixed chondrocytes were stained with phalloidin iFluor488 and DAPI and imaged using intensity-calibrated fluorescent beads for calculating F-actin intensity per cell and F-actin density. Fiji was used for textural analyses to quantify F-actin distribution, cell shape, and PR. PLS and RF were used to identify the most relevant factors for controlling and predicting PR.
Results: The PR of chondrocytes varied on different MP designs from 0.25 to 8.37, demonstrating that MP geometry allows minimizing and maximizing chondrocyte proliferation. PLS revealed that the highest impact on PR (in descending order) had cell shape (day_7), cell shape (day_1), and MP shape. The PR correlated negatively with cell solidity and roundness (day_7) and positively with cell area, length and aspect ratio (day_7), cell length being the most crucial factor. Interestingly, the PR of MOA chondrocytes was always higher than that of MIA chondrocytes. Using circular and H-shaped MPs in two sizes, we characterized single cell F-actin content, density, and distribution over time, revealing that F-actin density on day 1 was largely determined by MP size but not geometry or MTDS, whereas at day 7 F-actin density was determined by MTDS but not by MP size, geometry, or cell shape. PLS revealed that F-actin density (day_7), MTDS, and F-actin amount (day_7) had the highest impact on the PR, whereas other factors were relatively unimportant for controlling PR. Using RF regression with cell shape and actin parameters as input, the MTDS was predicted with an accuracy of 84.27 %, confirming the relation of these parameters to the PR of human chondrocytes.
Conclusion: The PR of chondrocytes is not only controlled by MP or cell shape, but to a greater extent by F-actin density and MTDS. In cell culture, cytoskeletal F-actin density was initially dependent on MP size but this was superseded over time by F-actin density being determined by MTDS. Thus, these data suggest that in situ proliferation leading to loss of SCSO in early osteoarthritis appears to be more controlled by MTDS-associated changes in F-actin than MTDS-associated changes in cell shape.
References: 1:Rolauffs et al. Arthritis Rheum. 2010 62(2):489-98; 2:Felka et al. Osteoarthritis Cartilage. 2016 Jul;24(7):1200-9.
20941884069
The expansion culture of cells is an essential process for manufacturing cells for therapeutic use. However, it is also an activity with a huge dilemma. This is because for clinical cell therapy treatment, and also for preparing cells for establishing cell bank, it is strongly required to expand cell number by passage culture to prepare a sufficient number of cells for applications, however at the same time, it is known that such over passage culture critically damages cell quality, especially in human mesenchymal stem cells (MSCs), therefore there is always a risk of establishing cell bank with quality decayed cells. Such balancing of production efficiency and cell quality is a critical issue to produce high-quality MSCs, however, the decision of such balance has long been relying on human experiences. To maximize the efficiency of obtaining high-quality MSCs for various applications, a more practical but efficient and quantitative method to enable non-invasive continuous monitoring of MSC’s condition has been expected.
Our group has been developing “morphology-based cell quality prediction method (= morphometry)” by combining the recent image processing technology together with machine learning techniques [1,2]. We here propose the high detection performance of such morphology-based cell quality prediction method applied to evaluate senescence status in the expanded mesenchymal stem cells. In this work, we have intentionally prepared the over-passaged mesenchymal stem cells and measured their morphological descriptor profiles from the time-course microscopic images and their total expression profile by RNA-seq. From the machine learning of passage numbers and their morphological profiles, especially their population heterogeneity information, we succeeded in clearly discriminating the “over-passaged MSCs” which lost their growth potency and differentiation status. Moreover, our expression profile analysis indicated some novel marker gene expression profiles to target for understanding the quality decay in over-passaged MSCs. We also developed a novel image-based single-cell cytometry analysis method, to profile the heterogeneous cell populations in expanded MSCs, and show that there are several morphological categories to be detected for understanding the senescence type in MSCs.
20941851177
Using a machine learning-supported approach for assessing and predicting the susceptibility of articular cartilage to mechanical trauma-induced changes in cellularity
M. Selig1,2, Laura Saager, Klaus Böhme, Bodo Kurz2, and B. Rolauffs1
Presenting Author: Mischa Selig, mischa.selig@uniklinik-freiburg.de
1G.E.R.N. Research Center for Tissue Replacement, Regeneration & Neogenesis, Dept. of Orthopedics and Trauma Surgery; 2Albert-Ludwigs-University of Freiburg, Hugstetter Straße 55, 79106 Freiburg, Germany; 3Institute of Anatomy, University of Kiel, Otto-Hahn-Platz 8, 24118 Kiel, Germany;
INTRODUCTION: The diagnosis and potential prevention of early post-traumatic osteoarthritis (PTOA) remain shot topics in orthopedic research because PTOA is among the leading causes of worldwide disability. In clinical routine macroscopic tissue damage after trauma is being diagnosed using imaging methods such as MRI or x-ray. Besides macroscopic damage, post-traumatic tissue regeneration vs. progressive degeneration to full PTOA depends also on the extent of cell death and survival, which can currently not be assessed clinically. We used here a clinically applicable score, the superficial chondrocyte spatial organization (SCSO) as a surrogate marker for articular cartilage ultrastructure and nanoscale functionality. We asked whether (i) the SCSO determines cell survival after simulated injury and (ii) can be used to predict the extent of cell death and survival. The ability to quantitatively assess a given patient’s cell population after trauma and even predict his / her susceptibility to increased post-traumatic cell death would help improving diagnostic capability.
METHODS: Discs from human OA articular cartilage (AC) explants were fluorescently-labeled with Calcein AM (Thermo Fischer), a cell viability indicator, and Propidium iodide (Sigma- Aldrich), a cell dearth indicator. Each disc’s SCSO was classified as chondrocyte string, double string or cluster organizations. One group of discs was subjected to mechanical injury (50%/0.5 s/0 s). Injured and control discs were stained for cell viability and cellularity, single cell morphology (area, length, width, circularity, roundness, and solidity), and multiple quantitative SCSO (qSCSO) parameters (nearest neighbor cell-cell distance (NNDs), cell intensity, and measures of cell grouping) were calculated for each disc. Both the morphological and the qSCSO data of viable cells were then used for training a random forest regression model (RF, R2: 0.955) to predict the extent of cell death and survival. Statistical analyses were performed with SigmaPlot (α<0.05).
RESULTS: A significantly higher number of chondrocytes organized as strings and double strings survived injury than chondrocytes organized in cell clusters and, conversely, a significantly lower number of chondrocytes organized as strings and double strings died after injury (p<0.001), demonstrating an SCSO-dependent susceptibility to trauma-induced cell death and survival. Chondrocyte morphology and qSCSO parameters were significantly different between SCSO stages and between injured vs. control discs (p<0.001). The RF model excellently predicted the amount of injury-surviving cells after trauma (R2: 0.946).
DISCUSSION & CONCLUSIONS: The extent of chondrocyte cell death after trauma depends on the SCSO, revealing SCSO-dependent trauma susceptibility. Furthermore, qSCSO and single cell morphology can be used as machine learning predictors to predict SCSO-dependent trauma susceptibility with excellent precision.
41883633849
Introduction: Macrophages are a heterogeneous population of cells. In response to microenvironmental cues macrophages shift their polarization state, alter their phenotype and adopt pro- or anti-inflammatory functions, promoting tissue inflammation or contributing to its resolution. Using a novel high throughput image-based single cell morphology and protein intensity machine learning approach, we aimed to distinguish macrophage M1, M2a vs. M2c subtypes and determine whether cell shape could predict a cell’s IL-10 immunogenic profile. This would aid in developing methods for assessing a cell population’s inflammation modulating potential using machine learning.
Methodology: Blood-derived human CD14+ monocytes were isolated and matured into macrophages using GM-CSF followed by GM-CSF/TNF-α/IFN-γ (M1 macrophages) or M-CSF followed by either M-CSF/IL-4 (M2a macrophages) or M-CSF/IL-10 (M2c macrophages). Cells were then actin stained with phalloidin and DAPI for quantification of cell area, length, width, aspect ratio, roundness, circularity, and solidity. Cells were additionally immunostained for anti-inflammatory markers IL-10 and CD163 and the pro-inflammatory marker CD80. The resulting phenotypes were confirmed via a cytokine ELISA. The image-derived single cell morphology descriptors were then used to train a machine learning algorithm to determine how accurately cell phenotype could be predicted. We then asked whether machine learning could be used to predict IL-10 production potential, quantified as IL-10 intensity per cell.
Results: A number of classification models were generated based on cell shape and immunostaining data. When only shape parameters were included, macrophage phenotype was determined with an accuracy of <50% when comparing all groups (M0, M1 control, M1, M2 control, M2a, and M2c). The accuracy increased to >80% when only assessing M1 vs. M2 macrophages. Incorporation of staining intensity and density measurements for CD163+CD80 and IL-10+CD80 improved the accuracies to 84% and 79% for M1, M2a and M2c classes, respectively. For distinguishing M2 subtypes (M2a and M2c), an accuracy of 91% was obtained with the addition of CD163+CD80 co-staining and 88% for IL-10+CD80 co-staining. Importantly, a random forest regression model was able to predict IL-10 intensity based on only cell shape data with R-squared metrics of >95% for M2c, M2a, and M1-M2a-M2c datasets. Macrophage phenotypes were confirmed by release of TNF-α by M1 and IL-10 by M2 (M2c > and M2a) macrophages into the culture supernatants.
Conclusion: The use of single cell morphology and phenotype marker expression data was able to reliably predict M1, M2a, and M2c phenotypes when applied to a machine learning model. This is the first study to use cell morphology data to accurately predict M1, M2a, and M2c macrophage phenotypes. Moreover, the incorporation of machine learning regression analysis showed, for the first time, that cell morphology is sufficient to predict IL-10 production by macrophages at the single cell level. A number of cell shape descriptors were strong indicators of IL-10 staining intensities, which point to a means of predicting IL-10 production and thereby relevant anti-inflammatory properties, based only on cell morphology. This may provide the foundation for a generalizable strategy for identifying functional subpopulations of cells and predicting the functional response of IL-10-producing cells.
52354515605
"Introduction
Layer-by-layer (LbL) coating is a method for surface modification based on the electrostatic interactions between two polyelectrolytes. LbL coatings are used for multiple biomedical applications, because natural polyelectrolytes presenting good biocompatibility can be used for LbL film build-up. It is possible to develop antibacterial surfaces, smart healing materials, and coatings for tissue engineering. Moreover, LbL coatings can be used for loading drugs or other bioactive molecules, which allows their local delivery. Even though the mechanisms of LbL film development are well-established, the empirical manner of polycation/polyanion selection is an impediment on rapid new coating development, while the current health crisis has shown the importance of accelerated development of biomedical solutions such as antiviral coatings.
Methodology
In this work, we hypothesize that using the current state of the art data science techniques, we can determine how different parameters affect coating thickness and predict the thickness of the new coatings. To do so, we used historical and generated data for predictive model development using machine learning, an approach which uses algorithms that improve upon training on large datasets and is able to find complex patterns, make predictions and decisions.
Results
Using literature data and newly generated experimental results, we first analyzed the relative impact of 23 coating parameters on the coating thickness. Next, a predictive model has been developed using aforementioned parameters and molecular descriptors of polymers from DeepChem library. Model performance was limited because of insufficient number of data points in the training set, due to the scarce availability of data in the literature.
Conclusion
We demonstrate, for the first time, utilization of machine learning for prediction of LbL coating properties (1). It can decrease the time necessary to obtain functional coating with desired properties, as well as decrease experimental costs and enable the fast first response to crisis situations (such as pandemics) where coatings can positively contribute. Besides coating thickness, which was selected as an output value in this study, machine learning approach can be potentially used to predict functional properties of multilayer coatings, e.g. biocompatibility, cell adhesive, antibacterial, antiviral or anti-inflammatory properties.
References
1. Gribova, V. et al., Sci. Rep. 11, 18702 (2021)"
94238154728
Brain organoids represent the 3D tissues that recapitulate the structure and function of the developing human brain. Much efforts have been made to advance the regionalization and to utilize the brain organoids to study human diseases. We developed region-specific cortical organoids and used them to study Rett syndrome. Since neuroectoderm differentiation of the human embryonic stem cells (hESCs) is an essential first step in brain organoid formation, the majority of cells in brain organoids are of the neuroectoderm origin. However, the cells that comprise the blood vessel in brain, and the residential immune cells in brain are from the mesoderm. In order to implement the mesoderm cells in brain organoids, we genetically engineered the hESCs to express the transcription factors that facilitate the formation of vascular-like structure and microglia-like cells. The vascular-like structures highly improved the quality of the brain organoids, dramatically decreasing the cell death, and increasing the neural maturation. The microglia-like cells demonstrated the innate immune function such as phagocytosis. Overall, our engineered brain organoids provide the essential mesoderm-derived cells in neuroectoderm-oriented brain organoids that play critical roles in brain function.
62903406129
The human brain is unique in size and complexity, but also the source of some of the most devastating human diseases. While many of these disorders have been successfully studied in model organisms, recent experiments have emphasized unique features that can not easily be modeled in animals. We use cerebral organoids to recapitulate those features in vitro and to test their role in human disease.
While the human brain is subdivided into distinct functional areas along its rostro-caudal and dorso-ventral polarity axes, brain organoids are currently limited by the lack of such polarity. To overcome those problems, we have used two approaches. First, we have introduced localized engineered sources of morphogens that can induce polarity in elongated organoids. Second, we have fused organoids resembling distinct brain areas to recreate polarity axes. We will present approaches to recreate functionally integrated multi-part organoid systems and their application to model neuro-developmental and neurodegenerative brain disorders.
62903404146
"Introduction
In silico methods integrate physical and biochemical models with computational tools, and are a powerful support for tissue engineering. They are particularly relevant for studying organoids and assembloids: the multiplicity of parameters which condition organoid growth and morphology can be explored in virtual models, facilitating experimental design, and enabling prediction and extrapolation of behaviour and function. Here we use statistical physics and evolutionary algorithms to predict how cells in cerebral constructs cooperate to share resources (oxygen) and generate functional forms.
Methodology
Kleiber’s Law (KL) is a universal law of biology, stating that the metabolic rate of an organism scales with its body size according to a quarter-power law 1. Its pertinence for designing human-relevant in vitro models 2–4 has been highlighted. However, KL is formulated as a deterministic framework, although fluctuations and heterogeneity are inevitable, and known to shape the response of biological systems to external perturbations (nanoparticles, viruses). We generated joint distributions of construct masses and metabolic rates, developing new statistical tools to test whether and in which organoid size range a generalized stochastic formulation for KL 5 applies. KL is combined with physical, metabolic and mechanical constraints to generate in silico models of organoids with different shapes and sizes to identify optimal design criteria for functional models.
Results
We found that stochasticity significantly restricted the range of construct sizes complying with KL, implying that to date many cellular models may lack translatability 6. Evolutionary algorithms based on the optimization of a cost function which incorporates resource uptake, surface energy and cooperative metabolic effort have enabled the generation of model datasets. These studies are used to assess to what extent morphometry or metabolic phenomena affect organoid formation and growth and to identify experimental design specifications to obtain constructs with translational value.
Conclusions
In silico models enable the definition of criteria for designing brain organoids and assembloids with translational value and hence useful for robust in vitro to in vivo extrapolation, paving the way towards predictive and precision medicine and reducing animal tests. Ongoing experimental validation suggests that three dimensional constructs manifest cooperative behaviour and that rather than being discarded, variability and fluctuations in organoids confer robustness.
References
1. Savage, V. M. et al. The predominance of quarter-power scaling in biology. Funct. Ecol. 18, 257–282 (2004).
2. Ucciferri, N., Sbrana, T. & Ahluwalia, A. Allometric Scaling and Cell Ratios in Multi-Organ in vitro Models of Human Metabolism. Front. Bioeng. Biotechnol. 2, 74 (2014).
3. Ahluwalia, A. Allometric scaling in-vitro. Sci. Rep. 7, 42113 (2017).
4. Magliaro, C., Rinaldo, A. & Ahluwalia, A. Allometric Scaling of physiologically-relevant organoids. Sci. Rep. 9, (2019).
5. Zaoli, S. et al. Generalized size scaling of metabolic rates based on single-cell measurements with freshwater phytoplankton. Proc. Natl. Acad. Sci. U. S. A. 116, 17323–17329 (2019).
6. Botte, E. et al. Scaling of joint mass and metabolism fluctuations in in silico cell-laden spheroids. Proc. Natl. Acad. Sci. 118, e2025211118 (2021)."
31412737644
"Introduction: Mitochondrial dynamics and metabolic alterations play a pivotal role in neuron maintenance and differentiation during early human neurodevelopment. Brain organoids (BO) derived from human induced pluripotent stem cells (hiPSC) provide a unique model to study developmental stage-specific sensitivity of mitochondrial dynamics to different microenvironmental cues. Our previous experiments performed on hiPSC-derived neuronal cultures showed that physiological normoxia (5% O2) impacts neural to glial cell fate by increasing expression of the astrocytic markers and lowering expression of the neuronal markers. In this work, we used a brain organoid model from hiPSCs grown in two different oxygen conditions – 5 and 21% O2 - to decipher the influence of mitochondrial dynamics on neural cell fate.
Methods: BO were generated from hiPSCs and cultured either in 21% (control) or 5% O2 (physiological normoxia). Then, 11-day neurospheres (11D-N), 44-days (44D-BO) and 4-month brain organoids (4M-BO) were collected. BO metabolism was evaluated by monitoring the ATP levels and through Alamar Blue assay. Changes in expression for markers specific to neural stem cells (Nestin, Pax6), neuronal cells (MAP2, bTubIII), glial cells (GFAP), proliferation (Ki-67), mitochondria (MAB1273) were determined by immunofluorescence labelling. To determine the effect of low oxygen on 44D-BO at the genomic level, RNA-seq experiment was performed.
To assess changes in mitochondrial networks, organoid sections were immunostained with anti-MAB1273, a mitochondrial surface protein. Confocal and structured illumination microscopy images were acquired on a Zeiss LSM 780. Image processing and quantitative analysis of the mitochondrial morphology were determined using ZEN software, FIJI plugins (Mitochondrial Analyzer plugin, MorpholibJ plugins and 3D Manager) and ad hoc Matlab routines.
Results: Our mitochondria analysis framework revealed changes in mitochondrial network parameters throughout brain organoid development and show that physiological normoxia affects key parameters of mitochondrial morphology in a developmental stage-specific manner. The most noticeable effect of low oxygen conditions on mitochondrial shape, connectivity and size predictors was observed at the stage of 44-day brain organoids therefore most of the analyses were performed at this stage. Metabolic assays showed a lower rate of metabolism, which is accompanied by a noticeably smaller diameter of 44D-BO in 5% O2 compared to 21% O2 controls. Furthermore, 3D analysis of segmented mitochondrial objects from the super-resolution microscopy images revealed significant alterations in mitochondrial volume, surface area, equivalent diameter, sphericity in the cortical zone of BO grown in 5% O2 with respect to 21 % O2 -cultured ones. Transcriptomic analysis revealed upregulation of genes involved in: HIF-1 signaling pathway, glycolysis/gluconeogenesis, central carbon metabolism and pyruvate metabolism in 44D-BO grown in 5% O2. Our results suggest that in physiological normoxia, glycolysis prevails over oxidative phosphorylation (Oxphos). This is accompanied with decreased expression of neuronal markers (βtubIII, MAP2) and increased level of glial marker (GFAP) in brain organoids grown in low oxygen conditions compared to controls, confirming the influence of low oxygen on neural to glial cell fate transition.
In summary, this study shows that oxygen conditions influence neural fate by inducing changes in glycolysis/Oxphos ratio and mitochondrial dynamics in a stage-specific manner during brain organoid development."
62825466227
In this seminar, I will provide a basic overview of the developmental mechanisms underlying the emergence of functional areas in the cerebral cortex. I will start by providing an overview of studies performed in rodents. Subsequently, I will transition to describe our studies in human, highlighting similarities and differences in progenitor cell diversity between mice and humans, I will subsequently describe how single cell transcriptomics and single cell epigenomics have helped us to identify modules of co-regulated and co-functional genes during human cortical development. Finally, I will describe our latest work on deciphering the role of developmental signaling pathways in promoting cortical arealization in the human brain.
31451707929
Three-dimensional (3D) printing is already routinely used in the clinic, e.g. for pre-operative models or intra-operative guides. However, this does not involve the generation of living 3D structures, i.e., biofabrication of tissues and organs. This automated approach holds potential to advance the field of regenerative medicine as outer shapes can be personalised and organised constructs can be produced when printing with multiple bio-inks. Recent developments have now resulted in the availability of a plethora of bioinks, new printing approaches, and the technological advancement of established techniques. Nevertheless, mimicking the functional properties of the tissues and clinical translation of the technology are two important remaining challenges. In order to achieve this, we urge that the field now shifts its focus from materials and technologies towards the biological development of the resulting constructs. Moreover, there is an urgent need for more specialized production facilities to move this technology towards the patient.
62903404977
Introduction: Mesenchymal stromal cells (MSC)-based therapies for inflammatory diseases rely mainly on the paracrine ability to modulate different cell populations involved in the advance of the disease, such as macrophages. These immune cells possess a broad spectrum of inflammatory responses. In addition, previous data have shown that the MSC secretome influences macrophage phenotype and functional capacities. Furthermore, culturing MSC with physiomimetic cues from the extracellular matrix (ECM) have shown to improve their repairing actions upon transplantation. Physiomimetic culture of cells relies mainly on ECM-derived biomaterials, such as, decellularised scaffolds and lung ECM hydrogels which provide a similar biomechanical milleau to the organ. Despite the recent advances in MSC-based therapies, there is scarce information regarding the changes on the secretome content attributed to these culture platforms, and especially, how the secretome profile could influence macrophage activity in favour of therapy. In this setting, the aim of this study was to assess the macrophage activity exerted by the secretome isolated from physiomimetically cultured lung-resident mesenchymal stromal cells (LMSC).
Methodology: LMSC from human donors were cultured on in-house developed devices that enable lung-mimetic strain. Medium from LMSC cultured in either lung ECM scaffolds and in lung ECM hydrogels whilst subjected to cyclic stretch, and on tissue culture plates (TCP) was analysed for typical cytokines, chemokines and growth factors. RNA was analysed for the gene expression of relevant mechano regulators CTGF and CYR61. Human monocytes were differentiated to macrophages by adding PMA and assessed their phagocytic capacity of bioparticles in the presence of LMSC secretome. Macrophages were also polarized to M1 and M2 phenotypes by adding LPS or IL-4 plus IL-10, respectively. M0 (quiescent), M1 and M2 macrophages were exposed to the medium of LMSC from the different culture conditions and analysed for surface markers by flow cytometry.
Results: CYR61 gene expression showed decreases when cultured on the aforementioned lung-mimetic environments compared to TCP. Furthermore, CTGF and CYR61 displayed a marked reduction when cultured in lung ECM hydrogels. The secretome content was plotted in UMAPs where the scaffold clusters mostly to itself while there is a large overlap between the hydrogel and the TCP samples. Additionally, stretch elicited different changes on HGF, MCP-1, IL-6 and TNF-α according to the environment where LMSC had been cultured. Similarly, phagocytosis showed a differential increase on TCP due to the stretch which was not observed in the physiomimetic culture.
Conclusion: Mechanical features of the lung ECM orchestrate key outcomes on LMSC, hence providing new insights into preconditioning of MSC for therapy.
52354545999
Introduction: Recent bone tissue engineering strategies propose recapitulating the endochondral ossification process for an effective repair. To this end, primary human mesenchymal stromal cells (hMSCs) can be primed in vitro towards hypertrophic cartilage (HyC) formation. While holding promises, limits arise from the performance variability associated with the use of primary cells. Recently, we developed a customized hMSC line, the MSOD-B, over-expressing BMP-2 and capable of reproducible cartilage formation in vitro. Following devitalization, the tissue exhibited remarkable osteoinductive properties. Here, we aim to move one step closer to clinical translation by investigating the possibility of decellularizing our cartilage graft and assessing its in vivo regenerative capacity in a rat critical-sized femoral defect.
Method: Our graft consists of in vitro engineered cartilage tissue produced by the MSOD-B. Subsequently, the tissue is decellularized by a combination of hypertonic/hypotonic, detergent (SDS), and DNase washes. The osteoinductivity of decellularized constructs was assessed through subcutaneous implantation in immunodeficient (ID) mice. To evaluate their repair capacity, , decellularized constructs were implanted in a critical-sized femoral defect (5-mm) in Sprague Dawley rats. Repair was assessed 6- and 12-weeks post-implantation through histological, micro-computed tomography (µCT), and mechanical analyses.
Results: We demonstrated the reproducible engineering of decellularized cartilage by exploiting a mesenchymal line. Decellularization resulted in a drastic reduction of DNA (<100ng/construct) with a minimal impact on tissue structure and composition (collagen, GAGS, and embedded growth factors). Remarkably, the capacity to instruct bone formation by endochondral ossification of our decellularized cartilage was not affected. This was validated both at ectopic site and in a critical-sized femoral defect in an immunocompetent rat model, with full-bridge after six weeks and complete bone repair observed 12 weeks post-implantation.
Conclusion and discussion: Our study illustrates the capacity of exploiting customized human lines to produce osteoinductive decellularized extracellular matrices (dECM). The strategy offers both standardization of performance and unlimited tissue availability, opening new avenues for the manufacturing of dECM for bone repair.
31412740986
Since synthetic vascular prosthesis perform poorly in small diameter revascularization, biological vascular substitutes are being developed as an alternative. Although their in vivo results are promising, their productions involve tissue engineering methods that are long, complex and expensive. To overcome these limitations, we propose an innovative approach that combines the human amniotic membrane (HAM), which is a widely available and cost-effective biological raw material, with a rapid and robust textile-inspired assembly strategy. [1] Fetal membranes were collected after cesarean deliveries at term. Once isolated by dissection, HAM sheets were cut in ribbons that could be further processed, by twisting, into threads. Characterization of HAM yarns (both ribbons and threads) showed that their physical and mechanical properties could easily be tuned. Since our clinical strategy will be to provide an off-the-shelf, allogeneic implant, we studied the effects of decellularization and / or gamma sterilization on the histological, mechanical, and biological properties of HAM ribbons. Decellularization had little effect of HAM yarn mechanical properties other than a small increase in strain at failure. However, gamma sterilization of the dried and decellularized HAM caused a decrease in rehydrated yarn diameter, an increase in ultimate tensile strength and a decrease in strain at failure. Gamma irradiation of hydrated (and decellularized) HAM largely avoided these mechanical changes and the process did not interfere with the ability of the matrix to support endothelium formation in vitro. Finally, HAM-based, woven, tissue-engineered vascular grafts (TEVGs) showed clinically relevant mechanical properties with a burst pressure of over 8000 mmHg (at a diameter of 4.4 mm), suture retention strength of over 5 N, and a transmural permeability of 1 ml·min-1·cm-2 Thus, this study demonstrates that human, completely biological, allogeneic, small diameter TEVGs can be produced from HAM, thereby avoiding costly manufacturing strategies based on cell culture and complex bioreactors.
[1] Magnan, L., Labrunie, G., Fenelon, M., Dusserre, N., Foulc, M.P., Lafourcade, M., Svahn, I., Gontier, E., Velez, V.J., McAllister, T.N., and L'Heureux, N. Human textiles: A cell-synthesized yarn as a truly "bio" material for tissue engineering applications. Acta Biomater, (105), 111-120, doi: 10.1016/j.actbio.2020.01.037 (2020).
94238145786
Introduction
To overcome organ shortage, designers develop engineered livers: devices/methods aiming to temporally assist or permanently replace it. As a complex organ with more than 500 functions, the design of engineered livers is one of the greatest challenges of the field. Since the mid-20th century, multiple pathways have been taken using diverse materials such as charcoal or cells. We argue that establishing a design strategy to engineer a liver is not entirely a technical issue[1]. Our research aims to highlight all the factors and thus should give new design directions to engineered organ designers.
Methodology
We undertook a philosophical analysis guided by the literature and starting from the field. We conducted participant observation in the lab experiments and 24 semi-structured interviews with 19 international actors of this engineered organ field. A thematic method[2] was employed to analyze the data by distributing the interview’s material into groups such as design strategy or regulation.
Results
We identified two major designers’ sources of inspiration. First, their vision of the liver, which could be defined by its functions and/or structure. Secondly, the technologies/methods that the designer mastered. In addition, several constraints such as budget or regulation had an impact. Based on those influences, designers developed design strategies leading to different engineered livers such as artificial, bioartificial, or hybrid. Each designer had the same mantra “keep it simple” meaning “find the shortest path to the patient”. Hence, if liver detoxification functions and artificial kidney were designers’ inspirations, the simplest strategy was to avoid living components that complicated and lengthened the research and the industrialization process. Such a path led to an artificial device, which couldn’t improve patients’ survival. Also inspired by cellular culture, some designers changed their strategy: turn the dialysis filter into a bioreactor by adding cells, betting that the hepatocytes will perform their functions as in vivo. Such a path led to a bioartificial device, which was so far not more successful. Organ decellularization/recellularization mobilized the same betting strategy but designers' vision of the liver was more structural, speculating that if the structure was right, the function should follow. Recellularization remained however complex for large scale organs.
Conclusion
Highlighting the influences behind the technical choices and the resulting strategies can open up designers to innovative engineered livers. Grasping a device’s nature is the first step towards understanding its ethical impacts, thus helping the legislators adapt the regulatory categories with requirements suited. Even if this research focuses on the liver, its conclusions could be valid for other organs.
[1] Daston L. The Moral Economy of Science. Osiris 10, 2, 1995
[2] Blanchet A., Gotman A. L’entretien. Broché. 2015
41883621846
Introduction
In vitro liver models allow investigation of the cell behavior in disease conditions or in response to changes in the microenvironment and are therefore valuable tools for basic research, drug screenings or toxicological analyses. Mimicking the tissue-level complexity of liver to achieve functional constructs is a major challenge, however, 3D bioprinting technologies open novel options to recreate the liver microarchitecture. Previously, we have demonstrated the high potential of coaxial extrusion-based 3D bioprinting to establish patterned co-cultures of hepatocytes and fibroblasts in core-shell fashion [1]. The aim of the present study was to develop a liver sinusoid-like model consisting of a vascularized core compartment which is surrounded by a hepatocyte-laden shell compartment. A suitable core bioink was developed, bioprinting of the triple culture model was established and cell-cell interactions were observed.
Methodology
The shell bioink consisted of 3 wt% alginate and 9 wt% methylcellulose dissolved in fresh frozen plasma (plasma-algMC) as described [2]; HepG2 were added immediately prior to printing. The core bioink was prepared by mixing collagen, fibrinogen and gelatin to achieve a CFG blend with final concentrations of 1.33 mg/ml C, 5 mg/ml F and 4 wt% G. Human endothelial cells (HUVEC) and fibroblasts (NHDF) were mixed into the ink immediately prior to printing. Core-shell bioprinting was conducted using a Bioscaffolder 3.1 (GeSiM) equipped with a coaxial needle as described [1]; crosslinking of the bioprinted constructs was done in 100 mM CaCl2 supplemented with 0.3 U/ml thrombin solution. Cell viability was examined by live/dead staining, cell proliferation was investigated by cell number quantification and EdU staining. Immunostaining was conducted to visualize endothelial tube formation in the core and to prove hepatocytes biomarker expression in the shell. Albumin secretion was analysed by ELISA.
Results
Core-shell constructs consisting of the CFG bioink as core and the plasma-algMC bioink as shell were nicely printable and, after dual crosslinking, stable during further cultivation over at least 21 days. Plasma enhanced viability and supported proliferation, cluster formation and biomarker expression of HepG2 in the alginate-based shell compartment. Using NHDF as supportive cells, HUVEC formed a pre-vascular network in the CFG core. This network formation was also visible in the presence of HepG2 in the shell compartment, however, a competition of the HepG2 for the matrix-forming fibroblasts in the adjacent compartment was observed if a high number of HepG2 was used. The presence of HUVEC in the core compartment resulted in an increased albumin secretion of HepG2 in the shell compartment.
Conclusion
Based on core-shell bioprinting, a patterned triple culture model has been established which can be further developed towards a more physiological liver sinusoid model.
References
1 Taymour, R. et al., Sci Rep, 2021, 11, 5130
2 Ahlfeld, T. et al., ACS Appl Mater Interfaces, 2020, 12, 12557
Acknowledgements
The authors thank the European Social Fund ESF and the Free State of Saxony for financial support in the course of TU Dresden based Young Researchers Group IndivImp and the microscopy facility CFCI of TU Dresden for providing equipment and support in cell imaging.
73296369939
Introduction: Mortality caused by liver disease and its complications is on the rise, representing a significant global health issue. Transplantation is the only efficient treatment for end-stage liver disease but is limited by the shortage of organ donors. Bioengineering represents a promising option, with researchers aiming at developing suitable organ replacements for transplantation. Tissue engineered organs rely (i) on a proper source of cells, able to support organ’s functionality long term, and (ii) on bioreactors, for the culture of whole-organ constructs. Amnion epithelial cells (AECs) can be isolated from full-term membrane with no ethical concerns. AECs can mature into hepatocyte-like cells (HLC), representing a promising source of hepatocytes for liver regenerative medicine and toxicological evaluations. Extracellular matrix (ECM) proteins promote cell maturation and long-term function; organ-specific ECMs can be obtained using decellularization, which allows eliminating cells from a tissue while maintaining ECM composition and 3D-architecture.
Here, we induced maturation of human AEC into functional HLC by culturing the cells into a 3D decellularised liver construct in a custom-made bioreactor, and evaluated differentiation and functionality of the HLC obtained.
Methodology: 40-50 million human AEC (isolated from full-term amnion membrane and characterized via FACS and qPCR) were seeded into decellularised rat liver scaffolds obtained via established detergent-enzymatic treatment. Constructs were cultured in custom-made bioreactors for up to 40 days (10 in expansion and 30 in hepato-specific culture conditions), with static cultured scaffolds used as control. Metabolites (e.g. lactate and glucose) and hepatic activities were monitored at different time points via NMR, ELISA and EROD assays, and compared to primary human hepatocytes. Transcriptome and proteomic analysis at intermediate and final time points were used to confirm the functional analysis.
Results: Freshly isolated AECs were positive for epithelial markers but negative for mature hepatocytes markers. AEC seeded in ECM-scaffolds adhered and proliferated to some extent when exposed to proliferative conditions. After 2 weeks in hepatic maturation media, AEC expressed immature hepatocyte marker alpha-feto protein, while 2 additional weeks generated CK18+-HLC characterized by secretive (Albumin) and functional (CYP3A4) protein expression. Albumin and urea concentration in the culture media increased in bioreactor-cultured constructs in respect to static culture, and NMR showed a shift in metabolite production over the course of the maturation. Finally, phase-1 hepatic metabolism was quantified via EROD assay at different time points.
Conclusion: Here we show that liver ECM-scaffolds efficiently supported the maturation of AEC into functional HLC in a 3D liver model. Remarkably, both cell distribution and hepato-specific activities and functionality were enhanced when human AEC were cultured in bioreactor than in static ECM scaffold. The bioreactor technology may provide an advantage for cell differentiation thanks to a more even distribution of oxygen and nutrients in comparison to static conditions. The technology here presented can serve as a paradigm for hepatic maturation in a 3D model of the liver composed by natural ECM and can help to investigate the role of ECM-specific protein in cell maturation and functionality.
20941830879
Introduction: Dynamic culturing systems can overcome challenges of in-vitro fabrication and maintenance of complex 3D tissues, however, the unique physiological conditions to which each tissue is subjected has been hampering noteworthy developments for many engineered tissues. Here we report the in-house development (design, manufacturing, and validation) of a glass slide size microbioreactor for the preparation, maintenance, and/or conditioning of human multilayer tissues or multi-tissue structures, evidencing applications in skin tissue-engineered analogs and ex-vivo human skin.
Methodology: The autoclavable bioreactor comprises a sandwich modular structure of hard undeformable layers of 3D printed medical grade polycarbonate intercalated with soft deformable layers of silicone. When compressed, the soft layers expand laterally against the sample sealing the layers between fluid streams avoiding their intermixing [1]. The bioreactor is modular, each module being an independent fluid circuit for one tissue, and up to three modules can be assembled to enable multi-tissue structures cultured in dynamic conditions. Each fluid stream is regulated by an independent piezoelectric pressure controller coupled with a flow sensor that allow measuring the dispensed media.
Results: The bioreactor was capable of holding skin tissue samples of 8mm in diameter and of providing, without mixing, different culture media corresponding to the three layers of the tissue, the outmost epidermis, the underneath dermis, and the innermost adipose tissue. By changing the thickness of the soft layers, it could easily be adapted to accommodate samples from different anatomical regions and with varied thicknesses. The bioreactor allowed nourishing each cell type/tissue layer with a specific cell culture medium, increasing the maintenance time of the native structure in the ex-vivo skin. Furthermore, the bioreactor permits establishing an air-liquid interface for epidermis turnover in the skin explant, while still maintaining the separation of the culture media underneath.
Conclusions: This dynamic culture system contributes to diminishing the time of preparation of complex tissues or multi-tissues and prolonging the viability and use of in vitro and ex-vivo tissues being, therefore, a valuable tool for drug discovery, personalized medicine, and cancer development studies.
Acknowledgments: Consolidator Grant Project “ECM_INK” (ERC-2016-COG-726061).
1] L. Gasperini et al., “Bioreactor for tissue engineering of multi-tissue structure and manufacturing method,” Provisional Patent Application 116901, (2020).
31412722057
Organ- and tissue-level biological functions are intimately linked to microscale cell-cell interactions and to the overarching tissue architecture. Advances in biofabrication technologies offer unprecedented opportunities to capture salient features of tissue composition and thus guide the maturation of engineered constructs into mimicking functionalities of native organs. Light-based bioprinting techniques enable superior resolution and ability to generate free-form architectures, compared to conventional extrusion technologies. These rely on the spatio-selective polymerization of a bioresin, a photo-responsive hydrogel laden with cells, in response to user-defined, cell-friendly 2D or 3D light fields. In this lecture, the design of new photoresponsive biomaterials for light based 3D printing will be discusses, together with their application for lithographic, layerwise bioprinting and the most recent advances in the development of layerless volumetric bioprinting techniques inspired by optical tomography, capable of processing cm-scale objects in less than 20 seconds. In particular, applications in musculoskeletal as well as soft (liver) tissue engineering are discussed. Notably, as in light-based printing cells are processed in absence of extrusion nozzles, in a contactless fashion, mechanically fragile organoids can be easily introduced as building blocks in the printing process, and shaped into complex and customized, cm-scale 3D tissue analogues. In this way, the ability of organoids to self-assemble enables to generate multi-scale constructs, in which the engineering of the cellular microenvironment is delegated to the (stem) cells that compose the organoid, leveraging their innate capacity for tissue morphogenesis, while at the same taking advantage from the environmental cues influenced by the macroscale milieu provided by the printed hydrogel. With such nozzle and shear stress-free, highly rapid cell processing approach a variety of hydrogel-based constructs can be assembled into hydrogel-based actuators for potential applications in soft robotics, or as platforms to enhance cell viability and maturation post-printing, including the shaping of large networks of hepatic epithelial organoids into defined 3D perfusable structures which exhibit biosynthetic and metabolic functions. This technology opens up new possibilities for regenerative medicine and personalized drug testing, and for the production of new in vitro models for fundamental biological research.
41935602166
Various 3D Printing and Bioprinting approaches have proven useful for tissue engineering applications. The achievable spatial resolution of the most widespread technologies, such as for example extrusion, is usually in the range of hundreds of micrometers, limited by the intrinsic attributes of these methods. However, light-based technologies and in particular multiphoton lithography (MPL) can produce features much smaller than a single mammalian cell [1]. Among other things, it has recently enabled realization of highly porous biodegradable microscaffolds capable of hosting individual cell spheroids [2]. The resulting tissue units can be used for bottom-up self-assembly of larger tissue constructs with very high initial cell density [3]. Furthermore, we have recently demonstrated that it is possible to embed living cells using MPL of photosensitive hydrogels, placing this technology in the domain of high-definition bioprinting, as well as fabrication of microstructures directly inside microfluidic devices [4].
MPL opens exciting perspectives for the engineering of advanced microscaffolds and 3D biomimetic cell culture matrices. In this contribution, our recent progress on MPL development will be presented. Current state of the art, challenges and future perspectives will be discussed.
References:
1. Multiphoton Lithography: Techniques, Materials, and Applications, J. Stampfl, R. Liska, A. Ovsianikov (Eds.) John Wiley & Sons (2016), [ISBN: 978-3-527-33717-0]
2. A. Arslan et. al, Polymer architecture as key to unprecedented high-resolution 3D-printing performance: The case of biodegradable hexa-functional telechelic urethane-based poly-ε-caprolactone, Materials Today (2021)
3. O. Guillaume et. al, Hybrid Spheroid Microscaffolds as Modular Tissue Units to Build Macro-Tissue Assemblies for Tissue Engineering
4. A. Dobos et. al, On-chip high-definition bioprinting of microvascular structures, Biofabrication, 13 : 015016 (2020)
The exceptional properties of natural structures with density gradients (e.g. bone, sponges, bamboo) have stimulated the interest in reproducing such complex architectures harnessing biopolymer functionality. However, the possibility to generate a hierarchical structure comprising multiple density gradient has not yet demonstrated, mainly due to the lack of technological advancements in engineering of new emulsion materials and rapid fabrication platforms.
In the current work, we reported the 3D printing of porosity-controlled dextran methacrylate (DexMA) oil-in-water (o-w) emulsions using a microfluidic circuit and a fluid-gel support bath. The fabrication of density gradient scaffolds within a supporting gel overcomes the problems associated with low-viscosity bioink extrusion in 3D printing, supporting density gradient structures that would be otherwise impossible to print in-air. The density gradient was engineered using a flow-focusing printhead. The characterisation of the emulsions demonstrated how the regulation of the continuous and dispersed phases by using microfluidic pumps allowed the controlled and automated tuning of the material final porosity. Therefore, we proved that a higher droplet diameter is obtained by increasing the flow rate of the oil phase with a direct and significant proportionality between the diameter and the volume fraction of the dispersed phase (p<0.0001). The rheological characterisation of the emulsions revealed a decrease in viscosity as the applied shear rate increased. The continuous phase of DexMA and Pluronic F-68 exhibited a Newtonian fluid-like trend, while the emulsions presented an increasingly pseudoplastic behaviour with expanding dispersed phase volume fraction.
To show the effectiveness of the developed methodology, we realised complex geometries consisting of porous biopolymer fibres, as well as porous scaffolds with axial (two, four and alternate) and radial density obtain differential regions within a single construct. The inclusion of photo-radical initiators in the outer phase of the inks enabled the crosslinking of the structure, following printing, directly into the supporting fluid-gel medium.
The 3D printed porous scaffolds exhibited high-end mechanical properties and elastic response to applied strains. Furthermore, morphological characterisation allowed the observation of the hierarchical internal porous architecture of the scaffolds using X-ray computed micro-tomography (μCT), scanning electron (SEM) and laser scanning confocal microscopy (LSCM), confirming the ability of the novel bioprinting platform to deposit high-resolution density gradient constructs in 3D.
Moreover, we demonstrated the possibility to print highly complex density gradient structures (e.g. free-standing stairs, inverted pyramids, hollow structures) with extremely low viscosity using an agarose fluid-gel. Furthermore, we investigated the printing of a combination of materials (DexMA and GelMA; DexMA and nHA) by a multi-inlet flow-focusing printhead, resulting in density gradient structures with hierarchical mechanical properties and swelling ability.
Altogether, this work outlines the potential of combining microfluidics and rapid prototyping techniques with the use of a suspending medium, providing a viable alternative for optimally 3D printing of biphasic systems with low viscosities and controlled densities.
41883609524
Introduction
While the two most-commonly applied approaches in tissue engineering (TE), namely the scaffold-based and the scaffold-free approach come with individual advantages but also drawbacks, Ovsianikov et al. proposed a third strategy for tissue engineering which combines the advantages of both approaches [1].
We propose here to utilize this third strategy to fabricate millimeter-size tissue constructs by fusion of multiple spheroids encaged within microscaffolds. Our approach offers great perspective in regenerating osteo-chondral tissue in vitro, by loading the microscaffolds with human adipose derived stem cells (hASCs) and differentiating them towards osteogenic and chondrogenic phenotypes.
Methodology
The highly porous structures, inspired by the chemical structure of fullerene (C60) referred to as buckyball (BB), were printed using a custom-built Two-Photon polymerization (2PP) system. The 2PP system consisted of a pulsed femtosecond laser operated at 800 nm, that resulted in writing speeds of 600 mm⸱s-1 at an intensity of 85 mW using a 10x objective. The biocompatible and biodegradable, prepolymer called hexa-acrylate-endcapped urethane-based poly-ε-caprolactone (UPCL-6) [2], in combination with 0.5 wt% of photoinitiator M2CMK served as photosensitive resin for 3D-printing. The use of low-adhesive 96-well plates hosting single microscaffolds in each well allowed seeding (4000 cells/well) of hASCs. After 2 days, spheroid formation within the buckyball scaffold was detectable and spheroids were differentiated towards the osteogenic and chondrogenic lineage for 21 days. After differentiation, the microscaffold-reinforced spheroids, referred to as tissue units (TUs) were harvested and merged together in a custom-made cylindrical agarose mold with a diameter of ~1.8 mm and a height of 8 mm for 7 days. 50 TUs differentiated towards the osteogenic lineage were placed at the bottom of the mold, while 50 TUs differentiated towards the chondrogenic lineage were seeded on top of the osteogenic ones to result in an osteochondral interface.
Results and Discussion
The successful differentiation of the spheroids within the microscaffolds towards the osteogenic lineage was verified by calcium deposition quantification, fluorescent calcein green staining, while the successful chondrogenic differentiation was verified by quantification of sulfated glycosaminoglycans and total protein amount. The formation of larger tissue constructs in the range of several millimeters was possible, using these differentiated spheroid-laden BB as “building blocks”.
Conclusion:
Our results indicate that this third TE method could be a promising approach with wide applicability, as the microscaffold can be used for instance to tailor the nature of the final tissue. In addition, the utilization of different cell sources or differentiation into further lineages could pave the way towards a variety of different TE applications.
References:
[1] Ovsianikov, A. et al., Trends in Biotechnology, 2018, doi.org/10.1016/j.tibtech.2018.01.005.
[2] Arslan, A. et al., Mater. Today, 2021, doi: 10.1016/j.mattod.2020.10.005.
Acknowledgements:
This research work was financially supported by the European Research Council (Consolidator Grant 772464 A.O.)
52354537368
Introduction
Bone graft substitutes are typically provided as ceramic granules. Whilst they have undergone successful clinical implementation, they are not without limitations. These include brittleness, variable resorption rates and a lack of control over the microarchitecture, all of which can lead to poor integration of the graft and fibrous tissue formation at the interface. Porous polymer microparticles can overcome some of these limitations and have seen increased research interest recently due to their potential to deliver cells and therapeutics in a minimally invasive manner. As there is evidence that pore size and shape can be used to control mesenchymal stem cell (MSC) fate, defining the microarchitecture of a particle to include beneficial geometries may be a route by which a reliance on co-administration of exogenous growth factors can be reduced. Tightly controlling microparticle architecture at a length scale relevant to bone cells can only be achieved through high-resolution additive manufacturing techniques such as two-photon polymerisation (2PP). Therefore, here we describe the fabrication of multiple defined-geometry microparticles via 2PP from a range of materials and their subsequent evaluation for cellularisation and osteogenesis.
Method
By cross referencing data from a microarray screen of polymers that promote MSC attachment and the number of acrylate moieties on the precursor monomers, potential photosensitive materials for 2PP were ranked. Processing parameters of the top three candidates were optimised to permit rapid fabrication of complex structures with robust structural integrity. Using designs based on mathematical solids to allow precise definition of geometry, these materials were then fabricated into six different designs of 100 μm diameter defined-geometry porous microparticles, and a solid-sphere microparticle control. To assess amenability to cellularisation, each geometry/chemistry combination (21 total) was fabricated into tessellated 1×1 mm arrays to mimic the close packing of particles that would occur in vivo. Arrays were seeded with three different donors of human bone marrow-derived MSCs (N=3, n=4) and cultured for five days before examination by confocal microscopy. By integrating fluorescence intensity across the height of the arrays, cellularisation could be quantitatively compared.
Results and Discussion
Quantitative analysis of array cellularisation revealed variation in pore shape can modulate cell infiltration even with a constant material chemistry. Particular geometry/chemistry combinations outperformed the solid particle controls, and importantly, certain geometries were observed to have a high degree of cellularisation across all chemistries investigated, indicating an architecture with utility in regenerative medicine. Microparticles which encouraged cell infiltration are currently undergoing screening for synergistic osteogenic effects that may further enhance their performance as bone graft substitutes. In parallel, fabrication of 3D arrays (e.g. 1×1×1 mm) of microparticles for assessment in a small animal model are underway.
In summary, 2PP allows us to incorporate a defined, cell-scale internal geometry within porous polymer microparticles. This work revealed a mathematically definable geometry that promotes MSC infiltration in comparison to a solid polymer microparticle which may be of interest clinically and is currently undergoing further evaluation. </div>
Acknowledgements
We acknowledge funding from the United Kingdom Regenerative Medicine Platform 2 (UKRMP2) [MR/R015651/1] and Next Generation Biomaterials Discovery [EP/N006615/1].
31412750166
Introduction
Microfluidic droplet-based bioprinting offers several advantages over conventional extrusion-based bioprinting methods such as (i) high-precision spatial patterning of the biologics (including cells, molecules, drugs and bioinks) and (ii) ease of their compartmentalization. These advantages, combined with high reproducibility of the generated microdroplets, facilitate high-throughput fabrication of well-defined 3D tissue constructs with complex inner architecture which could be used, e.g., in drug- or biomaterial-screening.
Most of the techniques currently availabe in the literature rely on the use of simple W/O emulsion droplets [1]. In this context, double-emulsion W/O/W core-shell droplets consisting of an aqueous ‘core’ encapsulated by a (biocompatible) oil ‘shell’ in an external aqueous phase could offer additional benefits. The shell phase could serve as a selective permeable barrier allowing the transport of small molecules and oxygen from the external environment while ensuring that the cells and bioinks contained in different droplets remain compartmentalized and develop into separate microtissues.
Here, we establish the possibility of printing of single-file chains of double emulsion aqueous core-droplets onto a glass substrate under external aqueous media. This strategy allows generation of ordered arrays of droplets for future use as microfluidic biomaterial- or drug-testing assays. In particular, we also demonstrate printing of hydrogel (GelMA) droplets.
Methodology
Double emulsion droplets are generated using an aqueous solution of GELMA 6% w/v + 0.2% w/v photoinitiator (LAP) as the inner phase, NOVEC 7500 + 3 % PFPE-PEG-PFPE surfactant as the shell/intermediate phase and distilled water as the external phase. The substrate is a glass slide treated with a fluorophilic coting NOVEC 1720.
GelMA droplets are encapsulated in NOVEC 7500 using a microfluidic T-junction micromilled in a polycarbonate chip. The generated droplets are then directed towards a substrate through a 25G needle immersed in an external aqueous media. The spacing between the tip of the needle and the substrate is precisely adjusted (<200 micrometers) to allow immediate deposition of the droplets at the substrate via wetting by the oil phase while leaving enough space for the droplets to remain stable upon extrusion.
Results
We show that the GelMA droplets can be printed onto a substrate in the form of a line of liquid ‘cores’ encapsulated by a thin oil ‘shell’ under external aqueous media. The presence of the surfactant-rich oil phase not only prevents coalescence of the droplets but also leads to adhesive capillary forces between them which stabilizes the printed lines. We demonstrate printing of hundreds of droplets at various substrates and in particular find optimal printing conditions using rough or porous substrates which facilitate rapid droplet deposition (prevent droplet ‘sliding’ at the substrate).
Conclusion(s)
We present a microfluididc-bioprinting platform that could, in the future, serve as a novel tool for high-throughput reproducible production and printing of thousands of compartmentalized microtissues. The technology could be developed towards, e.g., high-throughput biomaterial-screening via incorporating different hydrogel in each droplet followed by the deposition of the droplets into ordered arrays and their long-term culture.
References:
1 Zhou, L. et al., Adv. Mat. 32, 2002183, 2020
20941850106
INTRODUCTION. Volumetric bioprinting (VBP) is a recently developed light-based biofabrication method enabling the rapid generation of complex 3D structures within seconds. Short printing times combined with freedom of design allow for the advancement of novel in vitro models and physiologically relevant constructs. However, a more in-depth understanding of the effects of light-based bioprinting techniques is needed. During printing, the bioresin volume is illuminated, triggering the production of free radicals needed for common photocrosslinking reactions (i.e. chain growth and thiol-ene). Albeit safe light doses have been identified, radicals include reactive oxygen species, which concentration needs to be kept below supra-physiological values in the proximity of cells to avoid DNA damage. Moreover, in light-based bioprinting, unreacted bioresin volumes (from the reaction vat) are typically discarded, resulting in the loss of valuable cells. Unraveling the impact of cell-light interactions in bioprinting is key for the design of clinically-viable constructs for biomedical applications. Herein, we investigated the cell response after VBP via single-cell transcriptomics and analyzed stress and health markers. Further, we evaluated the usability of recycled mesenchymal stromal cells (MSCs), retrieved from the excess bioresin in the printing vat.
METHODOLOGY. Viability, metabolic activity, and H2O2 production were assessed upon printing human bone marrow-derived MSCs with 10% wt GelMA and 0.1% wt lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP). Casted gels (crosslinked with 365nm or 405nm lamps), and enzyme-crosslinked, non-photoexposed gelatin were used as controls. Samples were printed with increasing light dosages (375-625 mJ/cm2). DNA damage was assessed through immunostaining of apoptosis and double-strand breaks markers (Caspase-3, gH2AX, and TP53BP1). To determine inter-cellular variability and screen in-depth for light-based cell effects, single-cell RNA analyses were performed with a library of ≥75000 target genes. Cells were sequenced without undergoing VBP, 1 hour post-printing, and after 7 days in culture post-printing. Finally, MSCs retrieved from the uncrosslinked GelMA were collected and re-plated after printing, to assess proliferation rate and differentiation ability (osteogenic, adipogenic, and chondrogenic lineages).
RESULTS. Viability was unchanged over seven days of culture for the printed structures, compared to the casted gels (>90%), and higher light dosages correlated with higher metabolic activities. The first assessment of H2O2 concentration showed no significant increase post-printing. No significant DNA-damage was measured with the different markers. Re-plated cells had increased metabolic activity post-printing. Initial results from the single-cell sequencing data have shown a mild activation of stress-related genes, mainly involved in the TNF-a and KRAS signaling pathways, 1 hour post-printing. Results from the sequencing of cells cultured 7 days post-printing will shed light on the effect of the maturation process on their transcriptional state, and on the time-dependent response in the transcriptome.
CONCLUSION. Volumetric bioprinting is a fast biofabrication method that allows for the generation of tissue constructs without hampering cell viability and functionality post-printing. High-throughput, single-cell transcriptomic assays have great potential to elucidate the safety and risks related to bioprinting technologies. These results give valuable insights on cell behavior post-printing, which are needed to develop the next generation of bioprinted in vitro models and patient-specific grafts.
41883633448
Musculoskeletal tissue engineering (MTE) has proven to stimulate survival and differentiation of myoblasts towards tissue regeneration both in vitro and in vivo (1). In this field, different polymeric biomaterials have been employed to provide a biomimetic environment where cells can proliferate and differentiate into muscle tissue.
Polycaprolactone (PCL) is a synthetic aliphatic biodegradable polymer with remarkable mechanical properties, thermal stability, and controllable degradation. It is also approved by the FDA as a biomaterial for biomedical applications, being used in a wide range of biomedical applications, such as tissue engineering, dental implants, and drug delivery. (2,3, 4). PCL-based muscular grafts are promising tools in MTE (3).
Musculoskeletal tissue is well known for its electrosensitive cells since it is innerved by motoneurons that stimulate muscle myofibers to contract. Therefore, it is of great interest to obtain electrically active cell substrates to assess their influence in cell differentiation with and without external electrical stimulation. Different polymeric nanocomposites with conductive particles have been developed in recent years (5). Graphene (G), a polycyclic aromatic hydrocarbon with excellent conductive properties, has been incorporated as a filler to obtain electrically active biomaterials for MTE applications (6,7).
In addition, the role of different bioactive factors in combination with polymeric scaffolds have been deeply studied for MTE (1). Therapeutic inorganic ions, such as calcium (Ca2+) and zinc (Zn2+), are being studied in applications of tissue regeneration since they induce regeneration avoiding the drawbacks of growth factors (immunogenicity problems, risk of cancer and alterations in cellular homeostasis). In particular, Zn2+, a relevant metallic element in the human body, has been shown to induce proliferation, differentiation, and migration of cells, accelerating in vitro muscle formation (8). It was also proved that extracellular Zn2+ enhances myogenic differentiation by the activation of the Akt signaling pathway (9).
In this study, we hypothesise that bioactive cell environments based on electroactive nanohybrid biomaterials together with Zn2+ ions can synergistically stimulate myogenic differentiation. Conductive PCL/G nanocomposites were prepared with different amounts of G nanoparticles and a non-cytotoxic concentration of extracellular Zn2+ (40 µM) was chosen to analyse its effects in myogenic differentiation with murine myoblasts. The results show that the combination of conductive substrates and extracellular Zn2+ ions (PCL/G/Zn) increases myogenic differentiation in a significant way. However, further studies are needed to explore their full potential in MTE.
Acknowledgments
Financial support from the Spanish Ministry of Science and Innovation (MCINN, AEI/FEDER funds) through the project RTI2018-097862-B-C21 is acknowledged.
References:
[1] Langridge et al. Journ. Mat. Scie. 2021, 32, 15.
[2] Heang Oh et al. Biomaterials. 2007, 28, 1664.
[3] Siddiqui et al. Mol. Biotech. 2018, 60, 506.
[4] Chang et al. Biomacromolecules 2018, 19(6), 2302.
[5] Smith et al. Nano Mat. Sci. 2019, 1, 31.
[6] Palmieri et al. Front. Biotech. 2020, 8, 383.
[7] Patel et al. Ann. Biom. Eng. 2016, 44, 2036.
[8] Ramalingam et al. , J. Ind. Eng. Chem. 2020, 83, 315.
[9] Mnatsakanyan et al. Nature Sci. Rep. 2018, 8, 13642.
52354529355
T cell activation is modulated by signaling molecules on the surface of antigen-presenting cells (APC); however, in recent years, it has become increasingly clear that cellular forces have a crucial role in T cell activation and subsequent effector responses. Therefore, understanding mechanical modulators is critical in advancing current immunotherapy approaches. To address underlying questions, we engineered a biomimetic system to recapitulate the immune synapse, which is the interface of APC-T cell interaction, using polyacrylamide hydrogels with a defined stiffness range comparable to APC stiffness. The hydrogels were functionalized with different ratios of immobilized anti-CD3 (aCD3) and anti-CD28 (aCD28) antibodies. Our results showed that T cell proliferation, cytokine secretion, and intracellular signaling were all reduced at lower gel stiffness. We observed similar results in our cells’ models, in which APCs with reduced cell stiffness induced lower T cell activation. To enhance the physiological relevance of the biosystem, we fabricated cell-sized microbeads of varying stiffnesses, then embedded them in 3D collagen matrices. Overall, our biosystem allows decoupling of biophysical and biochemical interactions in T cells activation in a physiologically relevant microenvironment.
62825416877
"[Introduction] Extracellular matrix (ECM) plays a critical role in the control of cell adhesion and growth as in vivo scaffold material, therefore the design of ECM-like or ECM-mimicking scaffold has been one of the powerful concepts to provide a successful culture platform for tissue engineering. However, by their molecular size, it has been a challenge to obtain highly purified ECM molecules. Moreover, it is also extremely costly to obtain ECM molecules to be feasibly used for scaffold material. As a result, such ECM-molecular which can be easily obtained and used for scaffold coating has been limited to several bulk-produced proteins, such as collagens. From the aspect of obtaining pure and highly designable functionalization performance, peptides are advantageous with their synthesis and purification feasibility and their designability for effective surface modification applications. Our group has been investigating short peptides, 3-mer peptides, which can control not only cell adhesion but also “cell-selective adhesion”, to design ECM-mimicking surfaces on cell culture scaffolds [1,2]. The enhancement of cell-selective adhesion is expected to improve the regeneration process on the surface of implantable medical devices because they can provide a material surface to be attractive for “objective cells for treatment”, and be inhibitory for “non-objective cells that disturb treatment”. In this research, we tried to screen novel peptide sequences and combinations to enhance such cell-selective adhesion using peptide array-based direct cell assay [3].
[Methods] On the cellulose membrane, a peptide array was designed using Fmoc peptide synthesis. On the solid-bound peptide array, target cells (endothelial cells, fibroblasts, neural cells, smooth muscle cells) were seeded directly, and their cell adhesion rates were evaluated by a plate reader. We designed various short peptide libraries, containing control RGD cell adhesion peptides, and combined their sequences with various amino acid linker sequences.
[Results and conclusions] By the combinatorial direct screening on peptide array, we found that several short 3-mer peptides can be found to control the cell-selective adhesions to relatively discriminate objective cells and non-objective cells. Moreover, from the data, we found that the short peptide-based cell adhesion effect can be greatly influenced by the linker motifs, the neighboring sequence which provides a physicochemical effect for better or worse cell-peptide interaction. Our results indicate the effective performances of short peptides and their effective surface modification methods to provide a cell-selective effect on cell culture scaffolds. Moreover, our data also suggest that it is important to control the cell-surface interaction mechanism which affects apart from the integrin-mediated adhesion.
41883653837
Introduction
Infection is the major cause of implant failure after breast reconstruction surgery [1]. Medical-grade polycaprolactone (mPCL) scaffolds designed and rooted in evidence-based research offer a promising alternative to overcome the limitations of clinically routinely used silicone implants for breast reconstruction [2-3]. Nevertheless, as with any implant, biodegradable scaffolds are susceptible to bacterial infection, too. Especially as bacteria from the skin can rapidly colonize the mPCL surface during scaffold implantation and form subsequently biofilms. Biofilm-related infections are clinically challenging to treat and can lead to chronic infection and persisting inflammation of the implant host interface [3]. We hypothesize that scaffold guided breast reconstruction combined with an antibacterial implant coating allows to prevent bacterial infection while promoting, at the same time, implant integration and subsequently tissue regeneration.
Methodology
Macroporous scaffolds of a mPCL composite containing 45%(w/w) of sucrose particles with crystals size ranging from 20 to 50 µm, were additively manufactured using a BioScaffolder 3.1 (GeSiM mbH, Germany). The printed scaffolds were immersed in ultrapurified water (AriumR pro UF Ultrapure Water System, Germany) for 15 days in order to leach out the sucrose particles and create microporosity on the surface and within the scaffold struts. Fabricated scaffolds were sterilized by exposure to 70%v/v ethanol followed by evaporation. Scaffolds were then incubated in 1% and 5% human serum albumin (HSA) solutions overnight, at room temperature and under agitation. Resulting coatings of HSA were subsequently stabilized/crosslinked by incubating with 10% or 1%TA. Microporosity of scaffolds, as well its influence on the mechanical properties of clinically relevant large scaffolds was characterized by scanning electron microscopy, microcomputed tomography and uniaxial compression testing. Moreover, 3D in vitro assays were used in order to investigate the stability of the newly developed antibacterial coating and its efficacy against two of the most commonly found bacteria in breast implant-infections, S. aureus and P. aeruginosa.
Results
The physical immobilization of 1% and 5%HSA onto the surface of 3D printed macro- and microporous mPCL scaffolds, resulted in a reduction of S. aureus colonization by 71.7± 13.6% and 54.3± 12.8%, respectively. Notably, when treatment of scaffolds with HSA was followed by tannic acid (TA) crosslinking/stabilization, uniform and stable coatings with improved antibacterial activity were obtained. The HSA/TA-coated scaffolds were shown stable when incubated at physiological conditions in cell culture media for 7 days. Moreover, they were capable of inhibiting the growth of S. aureus and P. aeruginosa, two of the most commonly found bacteria in breast implant infections. Most importantly, 1%HSA/10%TA- and 5%HSA/1%TA-coated scaffolds were able to reduce S. aureus colonization on the mPCL surface, by 99.8± 0.1% and 98.8± 0.6%, respectively, in comparison to the non-coated control specimens.
Conclusion
This study presents the first set of results for a new biomaterial strategy designed for the prevention of biofilm-related infections on implant surfaces to be used in scaffold-guided breast reconstruction.
References
Visscher, L. E. et al., Tissue Eng. Part B Rev. 23, 281–293
Cheng M. et al., Tissue Eng. Part C Methods. 6, 366-377
Janzekovic J. et al., Aest Plast Surg, 2021
83767204924
Introduction: Excessive immune response and development of bacterial infections are two major problems accompanying organ replacement and implant surgeries. Our group aims to develop bioactive coatings to address these issues. Poly(arginine) and hyaluronic acid (PAR/HA) layer-by-layer films are supramolecular thin films (thickness about 1µm) that are easy to build, with promising immunomodulatory and antimicrobial properties 1-4. Here, we investigated the film behavior on CAVI-T intranasal balloon. CAVI-T (Dianosic, France) is a CE-marked medical device which addresses the treatment of intranasal bleeding and which can be used after nasal reconstruction. The device is composed of an inflatable balloon made of polyurethane used for compression of the nasal cavity. A feasibility study was performed with (PAR/HA) film onto polyurethane, looking for film characterization and its antimicrobial activity once deposited onto the balloon, after sterilization, storage and balloon handling, including balloon inflation. As an inflatable medical device CAVI-T is a good testing bed for the modification of mechanically active biomaterial structures.
Methodology: To construct (PAR/HA) multilayer films, polyurethane was alternatively dipped in PAR and HA solutions with intermittent rinsing steps. Coating construction on the substrate was characterized through observation by fluorescence microscopy after film staining with fluorescein isothiocyanate-conjugated PAR. For antimicrobial assays, coated polyurethane was incubated with a Staphylococcus aureus suspension for 24 hours. Planktonic bacteria were quantified by OD600nm measurement in the supernatant. Adherent bacteria were observed at the polyurethane surface after a fluorescent staining of healthy bacteria. Antimicrobial assays were repeated on the device after ethylene oxide sterilization, after accelerated aging and after 10 cycles of inflation/deflation of the balloon.
Results: The best coating construction parameters were defined according to the film homogeneity and thickness. (PAR/HA) films deposited onto polyurethane show a strong bactericidal activity. No bacteria were detected into the supernatant or at the polyurethane surface after 24 hours of contact. After the coating of the device and its sterilization, the antimicrobial activity of the (PAR/HA) remained unchanged. Same results were obtained after an accelerated aging. A final set of experiments was launched after inflation and deflation of the balloon performed into a test tube to mimic the contact with the intranasal cavity during the device introduction. Once again, the antimicrobial activity remained the same.
Conclusion and discussion: These results show the capacity of (PAR/HA) films to ensure antimicrobial activity when coated on the polyurethane balloon of CAVI-T device. (PAR/HA) coating appears to be a simple and powerful system, compatible with an utilization in polyurethane based scaffolds and medical devices. Next step of this study will be the evaluation of the coating for its ability of promoting healing in the specific case of intranasal bleeding. Immunomodulatory and hemostatic properties of the film will be assessed. Thus, the coating design could be tuned to fit polyurethane based scaffolds and devices specific needs.
References: 1 Özçelik et al., Adv. Healthc. Mater. 4, 2026-2036 (2015). 2 Mutschler at al., Chem. Mater. 28, 8700-8709 (2016). 3 Mutschler et al., Chem. Matter. 29, 3195-3201 (2017). 4 Patent WO2017191110A1 - EP3241570A1
20941826586
Introduction
Glioblastoma (GB) is the most frequent and lethal primary brain tumor. GB has currently no cure and the standard care regimens only provide patients a median survival of 12-15 months after diagnosis. Indeed, considering the unfeasibility of performing complete surgical resection and the low efficacy of chemoradiotherapy in eliminating the remaining GB cells, it is not possible to circumvent tumour recurrence. Therefore, there is an urgent need to develop an efficient treatment for GB. This work proposes a hydrogel designed for the direct injection into the resection cavity, allowing starting the treatment immediately after surgery. Being locally administered, it can overcome possible problems related to e.g. systemic side effects and reduced brain penetration. Moreover, the viscoelastic behaviour of the matrix allows the desirable interaction and attachment of the surrounding cancer cells, without the detrimental effects of the tumor microenvironment. Thus, it simultaneously promotes the sustained release of drugs to efficiently damage GB cells while avoiding stimulatory extracellular matrix effects on tumour cells.
Methodology
The hydrogel is based on hyaluronic acid (HA) functionalized with the fibronectin inhibitor peptide Arg-Gly-Asp-Ser (RGDS) and physically crosslinked with 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) large unilamellar liposomes (LUVs) encapsulating doxorubicin (DOX). The peptide was synthesized in an automated peptide synthesizer, followed by its characterisation by HPLC-MS. Then, it was used to functionalize HA through carbodiimide chemistry. HA functionalization was confirmed by NMR and ATR-FTIR spectroscopy. LUVs were prepared by the thin-film hydration method followed by extrusion. LUVs’ characterisation included the determination of size distribution, surface charge, morphology, phase transition, stability and drug concentration. Hydrogels were characterized in terms of viscoelastic and thermal properties and structure. Drug release profile from hydrogels was evaluated by HPLC. The ability of the matrix metalloproteinase-2 (MMP-2) produced by GB cells to break the peptide-HA binding was also assessed. The human primary GB cell line GBML42 and astrocytes were used to assess the therapeutic value and safety of the developed formulation.
Results
LUVs with DOX presented a homogeneous size of ≈121.7 nm and a slightly negative zeta potential (≈-2.43 mV). Moreover, DOX was encapsulated in relevant concentrations (≈68.2 µM in 1 mM LUVs) considering the DOX IC50 results (≈3.82 µM at 24 h of treatment). The hydrogel presented rheological properties similar to the healthy brain and was able to sustain the release of DOX. In vitro assays demonstrated the efficacy of unmodified HA hydrogels with liposomes encapsulating DOX to damage GB cells. Conversely, RGDS-functionalized HA hydrogels presented cytotoxicity even without DOX incorporation. Indeed, MMP-2 disrupted the peptide-HA bond. Thus, the internalization of free RGDS can lead to GB cells apoptosis. Importantly, RGDS-functionalized HA hydrogels incorporating liposomes with DOX efficiently damaged GB cells without affecting the metabolism and viability of astrocytes, proving their safety.
Conclusions
RGDS increased the cytotoxicity of the system, proving how it can act synergistically with the incorporated drug for GB treatment. Thus, this work shows the potential of this formulation to be used as a safe and effective local treatment for GB.
Acknowledgments: H2020 (668983-FORECAST), FCT (PTDC/BTMSAL/28882/2017–Cells4_IDs) and NORTE2020 (NORTE-01-0145-FEDER-000021).
83767225355
"Introduction: The Blood-Brain Barrier (BBB) is a dynamic interface which regulates the movement of solutes. The physical barrier consists of endothelial cells (ECs) with extrinsic barrier properties induced by interactions with the neurovascular unit (NVU). Neither static nor dynamic in vitro BBB models fully capture in vivo-like conditions, and while coculturing EC monolayers with other NVU cell types has improved barrier properties, the complexity of these culture conditions detracts from their usefulness for high-throughput drug discovery, testing, and disease modelling [1]. The model proposed here utilises material-driven fibronectin (Fn) fibrillogensis by poly(ethyl acrylate), which is biologically compatible with many cell types including ECs, to present a EC monolayer with growth factors (GFs) in synergy with integrin binding sites on Fn [2]. Radically, we hypothesise that an effective in vitro model can be created using GFs and EC monolayer grown on a PEA-Fn coated electrospun membrane scaffold. PLLA electrospun fibres additionally provide a physiological-like scaffold in comparison to commercial polycarbonate or polyethylene-terephthalate semi-porous membranes.
Experimental Methods: Electrospun membranes are produced from poly L-lactic acid (PLLA) 8% solution in hexafluoro-2-propanol and coated with plasma-polymerized PEA (pPEA). Membranes were characterised using scanning electron microscopy, transmission electron microscopy, and atomic force microscopy. hCMEC/D3, were grown on Fn-coated membranes with Fn coating with the addition of GFs at small concentrations (100 ng/ml) on the membrane. Cell/barrier characterisation includes TEER, FITC-dextran permeability and tight junction (TJ) immunofluorescence. Inserts to hold membranes with inbuilt TEER electrodes for continuous resistance measurement in cell culture were designed using AutoDesk Fusion360 and printed on a Prusa 3D printer in PLLA.
Results: hCMEC/D3 cells have low expression of TJs on membranes and their overall TEER values are considered very low. However, we were able to show high level of ZO-1 staining in specific pPEA-Fn-GF conditions, including low permeability to 10/40kDa dextran. Barrier characteristics were further improved by combining different GFs, such as FGF-2 and BDNF, and ECM proteins, such as collagen IV and laminin, and exploration of arranged electrospun fibres in differing geometries. In the investigation of key barrier-inducing GFs, novel GFs which induce higher levels of permeability than VEGF have been discovered.
Conclusion: We demonstrate that this versatile and tuneable design can induce barrier characteristics in immortalised hCMEC/D3 cells. With continued investigation into the changes the cells undergo and optimisation for increased BBB-characteristics, this is primed to be a powerful BBB model. There is room for further optimisation, particularly for the electrospun scaffold and growth factor choice and their combinations, although the model proves promising even at this early stage. This system offers a promising platform, with prospects for the study of BBB physiology and pathology, as well as for high-throughput BBB drug permeability testing.
41883636764
"Introduction
Corneal regenerative medicine in recent times has taken a focus on the recapitulation of the limbal epithelial stem cell (LESC) niche. Located peripheral to the central cornea, this pool of stem cells is vital for the preservation of sight throughout adult life. The limbus as an anatomical feature has striking topographical characteristics which are readily observed using clinical ophthalmological equipment. Despite this accessibility, it has remained a challenge to recapitulate its true structure in vitro in a manner which is truly biomimetic both in terms of shape and tissue physical properties. Current methods include structure mimicry techniques, such as lithography [1] and tissue mechanics replication which can involve biological (or biological-like) substrate engineering [2]. In an approach to unify surface shape and mechanical properties, soft polymeric wrinkling [3] was utilized to form a topographical surface for LESC culture and fate control.
Methodology
To produce the substrates, chips of polydimethylsiloxane were exposed to a dual oxidation treatment using strong acid and low temperature oxygen plasma. Subsequent to this, the surfaces were coated with 10% GelMa containing 0.25% LAP through UV crosslinking. Afterwards, substrates were re-hydrated to produce the wrinkled-like surfaces. Human LESC’s were isolated from human corneas (NHSBT) using a 2mg/ml Collagenase IV digest of dissected limbal region sections at 37⁰C overnight. The grown monolayer cells were detached and seeded on the sterilised substrates for up to 7 days culture before fixation. Cell response was characterized using immunofluorescence of key limbal markers: ABCG2, p63, Vimentin, and of key corneal markers: CK3, CK12, Nestin. Imaging was taken using a confocal microscopy. The depth-resolved morphology of the elastomer was examined using Optical Coherence Tomography (OCT).
Results
The substrates have demonstrated regular aligned wrinkles with the dimension (70.86±25.39µm) close to limbal crypt width. OCT images showed consistent crypt depth around 17.14±4.64 µm across the whole substrates. Limbal cells responded to the topographic features well by the clear ordering and alignment with elongation of cells in plane along wrinkle propagation physically. The phenotype change toward the substrate was demonstrated through the changes in marker expression, in particular the marked increase of CK3 and Nestin expression in comparison to monolayer controls in conjunction to a decrease in ABCG2 expression. It is worthy to note that the time in culture on the substrate affected marker expression, stem cell markers were preserved at day 3 but showed transition to corneal commitment at day 7.
Conclusion
In this study, we present a novel fabrication technique to produce biocompatible smart material with physically instructive nature of the topography which is able to replicate the shape of key limbal anatomical features whilst provide mechanobiological signals to exert cell fate control, driving commitment of LESC’s towards epithelial cells.
References
Kang, K B. et al., Nat. Sci. Rep. 9,1, 1-8, 2019.
Tan, Y. et at., Macro-Mol. Bio. 100441, 2021.
Dimmock, R L et al., Rec. Prog. In Mat. 2,1,2020."
62825443626
Introduction
Severe bone injuries can result in incapacities and thus affect a person's quality of life. Mesenchymal stem cells (MSCs) can be an alternative for bone healing by growing them on scaffolds that provide mechanical signals for differentiation. Such scaffolds can give the appropriate ques to the cells in order to induce their differentiation into mature osteoblasts and later on to be transplanted into the body. Until now lots of attention has been given to create appropriate nano and micro patterns that can work as signal inducers limiting the research in only 2.5 dimensions. On the other hand, our work introduces true, well- defined 3D environments to the research of MSCs differentiation.
Methodology
In our approach, we fabricated hierarchical auxetic mechanical metamaterials and ultra-light ultra-stiff scaffolds via two photon polymerization and used them as scaffolds to investigate the differentiation of MSCs into osteoblasts. Those scaffolds consist of unit cells comparable to the diameter of MSCs which is approximately 50 to 100μm, so only a couple of cells can fit inside thus ensuring the optimal mechanical environment for each cell. In the case of auxetic scaffolds, the unit cells are able to bend without breaking such that the cells can adapt their environment to their needs, whereas the kelvin foam is stiff non elastic scaffold that shows no deformation in response to the forces exceeded by the cells. We investigated the localization of YAP protein, a key protein transcription factor that acts as a mechanotransduction mediator and compared it to common osteogenic markers in both protein and gene levels by using confocal microscopy and qPCR .
Results
Interestingly, YAP protein is translocated to the nucleus even after 21 days of culture and RUNX2 gene shows a 10-fold increase in auxetic scaffold in comparison with the control only after 7 days. Long term cultures up to 28 days shows high mineralization of the extracellular matrix after Alizarin red staining. Moreover, SEM pictures revealed different cell morphology in those different scaffolds because of the different geometries used. Auxetics pushes the cells into more elongated phenotypes whereas kelvin foam in more broad cells bodies.
Conclusion
Auxetic scaffolds are ideal for osteogenic differentiation as they can maintain and promote the osteogenesis efficiently even after 28 days of culture. Our work paves the way for the use of more complicated metamaterials into the tissue engineering field.
Acknowledgement
This work was funded by In2Sight: Horizon 2020 GA: 964481
52354563999
The low regeneration potential of the central nervous system (CNS) represents a challenge for the development of new therapeutic strategies. Mesenchymal stem cells (MSCs) have been proposed as a possible therapeutic tool for CNS disorders, namely due to the beneficial actions of their secretome. Indeed, the latter possesses a broad range of neuroregulatory factors that promote an increase in neurogenesis, inhibition of apoptosis/glial scar, immunomodulation, angiogenesis, neuronal and glial cell survival, as well as relevant neuroprotective actions into different pathophysiological contexts. Considering their protective action in lesioned sites, MSCs, and their secretome, might also improve the integration of local progenitor cells in neuroregeneration processes. In this sense their use could represent an important vehicle for the establishment of future CNS regenerative therapies. In the present talk the role of MSCs, and their secretome, on phenomena such as in vitro and in vivo neuronal/glial survival will be addressed. Additionally, their possible applications, for Spinal Cord Injury and nerve repair be presented. For several years we have been dissecting the role of the secretome of adipose tissue derived stem cells (ASCs), as well as its individual vesicular and proteic individual fractions, in in vitro and in vivo models of axonal growth, inflammation and spinal cord injury, respectively. In vitro experiments revealed that the unfractionated secretome had a significant effect growth, when compared to its protein or vesicular fractions, on axonal growth and neuroinflammatory profile of microglial cells. Following on this data we then evaluated the impact of ASCs secretome on the histological and functional recovery of transection and compression based models of SCI in mice. Results of these experiments revealed that ASCs secretome induced a significant improvement of the locomotor performance of SCI mice, when compared to untreated animals, as assessed by the Basso Mouse Scale test (BMS). This was particular evident in the animals that were injected systemically (IV through the tail veins) with ASCs secretome, when compared to a local delivery. Additionally, the histological analysis has indicated that this motor improvement is closely related with a consistent reduction of the lesion volume, as well as a decreased activation of inflammatory cells (microglia) activation after treatment, as well as an robust increase on the regeneration of new axons. Finally we have also developed approaches for the encpasulation of the secretome in biodegradble systems to facilitate and potentiate their local application.
31451701305
TBA
Introduction
Multi-Photon Polymerization (MPL) is a Direct Laser Writing (DLW) technique that combines ultrafast (femtosecond, fs) laser pulses and Computer Aided Designs (CADs) for the fabrication of high precision scaffolds that find application in fields such as tissue engineering [1, 2]. We used such scaffolds for mono- and co- cultures of murine N2a neuronal and SW10 glial cells in order to investigate how topography affects the cell behavior on the 3D environment for various timepoints.
Methods
A novel bridge-shaped 3D designed of dimensions of 400μm x 400μm x 60μm was fabricated using a femtosecond fiber laser operating at 780 nm (pulse duration: 120 fs, repetition rate 80 MHz). The material used for polymerization was a hybrid material consisting of organic and inorganic components (3-Trimethoxysilyl propyl methacrylate, MAPTMS/ Methacrylic acid, MAA and Zirconium propoxide, ZPO). 4,4’- Bis (diethylamino) benzophenon, Bis, and Sudan Black B were both used as photoinitiators (PIs). The fabricated 3D structures were used as scaffolds for the mono- and co-cultures of N2A neuronal and SW10 glial cells for timepoints starting from 7 days. Cultures were monitored both by Scanning Electron Microscopy (SEM) and Confocal Microscopy for both morphological and intra-cellular investigation.
Results
Cell cultures were conducted on both 3D scaffolds and glass coverslips for various timepoints. Cell growth and survival between the different conditions/ culture periods were investigated to determine the optimal culturing conditions. Comparison of the cultures exhibited a strong preference of directionality for cell elongation and axon growth dictated by the topography of the scaffolds compared to the control glass coverslips. Our findings not only show that our scaffolds can sustain both mono and co- cultures of N2a and SW10 cells, but also that by carefully designing a suitable topography, cell behavior can be influenced towards a desired way.
Discussion/Conclusions
Our findings show the effect of topographical properties on cell growth and behavior and the ability to influence the aforementioned behavior in a beneficial way by designing 3D scaffolds with specific geometries based on the application. We highlight the potential of the development of an in vitro model for the study of neurodegenerative diseases which may find further application in tissue regeneration.
Acknowledgments/ Funding
In2Sight: Horizon 2020 GA: 964481
References
1. Sun, H.B. and Kawata, S., Eds., ed Berlin, Heidelberg: Springer Berlin Heidelberg, 170: 169-273 (2004).
2. Nguyen, A. K. and Narayan, R. J., Materials Today, 20, (6):314-322 (2017).
73296328364
Peripheral nerve injuries (PNI) affect millions of patients worldwide and cause motor and sensory dysfunction leading to reduced quality of life and increased healthcare costs. The primary treatment option for repairing large PNIs is to use patient’s own nerve graft – an autograft, which is limited by availability and donor site morbidity. In this study, we aim to prepare an off-the-shelf advanced nerve guidance conduit (NGC) with capacity to regenerate critical-sized PNI as effectively, but overcoming the associated limitations of utilising autografts.
Advanced NGCs were composed of two phases – an outer tubular shell composed of collagen type I (Coll) and internal matrix composed of Coll, chondroitin-6-sulphate (CS) that was enriched by adding series of extracellular matrix derived molecules, namely fibronectin, laminin 1 and laminin 2 (Coll-CS-ECM). NGCs with Coll-CS and Coll-CS-ECM were tested for their neurotrophic and immunomodulatory potential in vitro using rat dorsal root ganglia (DRGs) explant culture. Following this, NGCs with Coll-CS (n=16) and Coll-CS-ECM (n=16) and autografts (n=16) were implanted in a large (15 mm) critical-sized rat sciatic nerve defect model.
In vitro analysis showed that in comparison to Coll-CS conduits, Coll-CS-ECM significantly decreased DRGs’ secretion of inflammatory markers such as interferon gamma-induced protein 10, monocyte chemoattractant protein 1 and macrophage inflammatory protein 1a and significantly increased DRGs’ production of nerve growth factor, vascular endothelial growth factor and interleukin-6.
In vivo analysis showed that sensory and motor function recovery improved significantly over time in all animals. Notably, the response of the two NGCs to electrical stimulation was similar to the autograft group and no differences were seen in recordings of compound nerve action potential and compound muscle action potential either. Consistent with these results, no significant differences in muscle weight loss were observed between either NGC and autograft group. Importantly, the total area of neurofilament positive staining and the number of myelinated axons within both NGCs was similar to autografts. However, in agreement with our in vitro results, Coll-CS-ECM significantly increased vascularisation inside the conduit when compared to Coll-CS and autograft. This demonstrates the ability of the ECM molecules to direct early regeneration across a large nerve defect.
Collectively, our results demonstrated that enrichment of a NGC with ECM derived molecules such as fibronectin, laminin 1 and laminin 2 resulted in a biomaterial capable of modulating immune response and increasing secretion of pro-repair molecules that ultimately resulted in bridging large PNIs to a level equivalent to an autograft indicating its potential as a new clinical therapy for repairing large nerve defects.
94238163729
Introduction
Peripheral nerve tissue engineering aims to create biomaterials that can replace and possibly even therapeutically surpass the current gold standard nerve autograft. Tissue-engineered constructs can be designed to deliver a combination of benefits to the regenerating nerve, such as supportive cells, alignment, extracellular matrix, soluble factors, and biomechanical integration. An emerging therapeutic opportunity in nerve tissue engineering is the use of electrical stimulation (ES) to modify and enhance therapeutic cell function.
ES has been shown to positively affect four key cell types; neurons, endothelial cells, macrophages, and Schwann cells, involved in peripheral nerve repair1. Briefly, neurons experience faster neurite outgrowth and increased protein adsorption, endothelial cells upregulate angiogenic factors, macrophages may experience a phenotypic shift towards pro-repair phenotype and Schwann cells increase neurotrophic growth factor and exosome secretion. To leverage these phenotypic benefits associated with ES, a conductive tissue engineered scaffold may be used to provide stimulation that improves the regenerative environment, or directly stimulates regenerating axons within the construct. This work attempts to explore how tissue engineering strategies can make use of this therapeutic stimulus to improve nerve regeneration.
Methodology
A novel conductive tissue engineered construct was developed, comprised of conductive organic semiconducting polypyrrole (PPy) nanoparticles distributed within a cellular or acellular collagen matrix, which is then aligned using gel aspiration ejection (GAE) to generate an engineered neural tissue. The GAE technique has been utilized previously for peripheral nerve tissue engineering of cellular collagen gels2 and has therefore been further developed to provide a rapid method to achieve conductive collagen scaffolds in under 1 hour. A fully hydrated hydrogel is aspirated into a cannula, which simultaneously removes the bulk of the interstitial water within the construct and aligns the fibrous collagen with the construct.
Results
The resultant construct is stabilized through this process and due to the conductive PPy nanoparticles distributed throughout the aligned collagen matrix. The material exhibited conductive properties before and after processing with GAE. The conductive engineered tissue was tested in vitro to assess neural cell compatibility and ability of ES to modulate cell phenotype and regeneration.
Conclusions
ES has provided promsing results to short nerve gap injuries. This approach provides a promising new method for investigating whether ES can be used to enhance nerve tissue engineering, and importantly address clinical need within 'critical length' nerve injury gaps.
References
41883656488
"Introduction. Extracellular vesicles (EVs) are involved in a plethora of physiological and pathophysiological contexts, and their potential regenerative applications have attracted special interest. Ease of autologous isolation, low immunogenicity and lack of reproductive potential are only some of the enticing characteristics that turn the spotlight increasingly towards EV-based therapy. However, too many unknowns regarding the biology of EVs remain. Within the scope of our research, we focus on peripheral nerve regeneration, where we challenge the gold standard of autologous reconstruction with alternative therapeutic approaches, including EVs. Schwann cells (SCs) have been ascribed an essential role in nerve repair. Their response to nerve damage includes debris clearance, attraction of macrophages, and providing structural and trophic support for the regrowing axon. In this study, adipose tissue derived stem cells (ASCs) serve as a source for EVs. Here, we follow the journey of ASC-EVs to the recipient SCs and decipher how they are able to transmit their multifaceted signals and actuate downstream processes, including proliferation.
Methods. EVs were isolated by differential ultracentrifugation and characterized according to the MISEV guidelines. Imaging flow cytometry (IFC) allowed immunophenotyping with a single-vesicle resolution, while nanoparticle tracking analysis (NTA) was used to determine concentration and size distribution. The entire SC – ASC-EV interaction was observed with live-cell imaging (LCI), from initial contact to subsequent internalization and perinuclear translocalization, which was confirmed with 3D image reconstructions of high-resolution confocal micrographs. Scanning electron microscopy enabled us to elucidate the initial contact in detail, while transmission electron microscopy granted us a closer look at the vesicle transit through the cellular membrane. We further broke down the membrane transit on a molecular level by pairing well-established pharmacological inhibitors of major endocytotic mechanisms with state-of-the-art IFC. The cellular response to ASC-EV treatment was quantified via EdU incorporation during DNA synthesis.
Results. Upon initial contact with SCs, ASC-EVs were moved along the membrane until they were internalized and subsequently transported towards the cell’s nucleus, where they were accumulated. The inhibition of specific endocytosis pathways revealed that in SCs, the internalization of ASC-EVs is mainly mediated by clathrin, though alternative modes of membrane transit are likely involved, as no complete block of ASC-EV-internalization could be achieved. Upon internalization of ASC-EVs, we observed an increase in SC proliferation in a time- and dose-dependent manner, up to 2.5-fold compared to untreated SCs within 72h.
Conclusions. We established that ASC-EVs can enhance proliferation in SCs, crucial for peripheral nerve regeneration. This response is activated upon internalization of ASC-EVs. We identified the major mode of internalization, however, alternative modes of internalization likely involved. The potential therapeutic application of EVs necessitates understanding the underlying processes, especially the interaction with target cells. Our investigations provide a deeper understanding of the cellular signal transduction during peripheral nerve regeneration upon stimulation with ASC-EVs and adds to the knowledge needed to harness the full potential of EVs for therapeutic purposes."
41883636477
Mesenchymal stem cells are a promising source for externally grown tissue replacements and patient-specific immunomodulatory treatments. This promise has not yet been fulfilled in part due to production scaling issues and the need to maintain the correct phenotype after re-implantation. One aspect of extracorporeal growth that may be manipulated to optimise cell growth and differentiation is metabolism. The metabolism of MSCs changes during and in response to differentiation and immunomodulatory changes. MSC metabolism may be linked to functional differences but how this occurs and influences MSC function remains unclear. Understanding how MSC metabolism relates to cell function is however important as metabolite availability and environmental circumstances in the body may affect the success of implantation. Genome-scale constraint based metabolic modelling can be used as a tool to fill gaps in knowledge of MSC metabolism, acting as a framework to integrate and understand various data types (e.g., genomic, transcriptomic and metabolomic). These approaches have long been used to optimise the growth and productivity of bacterial production systems and are being increasingly used to provide insights into human health research. Production of tissue for implantation using MSCs requires both optimised production of cell mass and the understanding of the patient and phenotype specific metabolic situation. This presentation will discuss the current knowledge of MSC metabolism and how it may be optimised along with the current and future uses of genome scale constraint based metabolic modeling to further this aim.
41935603124
"Introduction
In recent years, the clinical application of injectable bone pastes has been increasing because of their benefits including ease to adaptation into irregular-structured defects and injectability suitable to minimally invasive surgery. These advantages are widely advocated to reduce patient complications and health care costs. Here, we successfully developed injectable bone pastes integrating biphasic calcium phosphate (BCP, HA:β-TCP=1:4) nanoparticles with poly(ɛ-lysine) dendron activated by phosphoserine at generation 3 (G3-K PS). Furthermore, the incorporation of strontium (Sr) element into the BCP nanocrystals was also considered to minimize bone resorption.
Methodology
The preparation of materials including G3-K PS semi-dendrimers, BCP, SrBCP (with 15 mol% Ca2+ replaced by Sr2+), BCPG3 (BCP in G3-K PS carrier) and SrBCPG3 (SrBCP in G3-K PS carrier) was adapted from previously protocols1. G3-K PS semi-dendrimer in ethanol solution was added at 1% (w/w) to BCP or SrBCP slurry under 200 rpm and 37 °C stirring until gelation occurred. In vitro cell tests to assess the osteogenic potential of the synthesized biomaterials were performed using bone marrow stromal cells (MSCs) isolated from ovariectomized rats and the expression of specific gene markers (Runx2, ALP, Cxcl9, RANKL) at 1, 3 and 7 days of cell culture were analyzed by RT-PCR. To confirm the role of Cxcl9 and Sr ions in material induced osteogenesis, additional Cxcl9 protein (250 ng mL-1) was introduced into the BCPG3 and SrBCP groups in culture medium, and Sr ions (3 mM) were introduced into the BCP and BCPG3 groups in cell culture medium. In vivo studies were performed using ovariectomized female Sprague Dawley rats. Several bone metabolic markers including P1NP, CTX-I and Cxcl9 were evaluated using ELISA assay at week 12. Immunohistochemistry analysis, micro-CT and scanning electron microscopy investigations were performed to evaluate the new bone tissue formation and osteointegration.
Results
Injectable bone paste material integrating BCP nanoparticles with G3-K PS carrier was successfully synthesized with or without the doping of Sr element into the BCP nanocrystals. Both in vitro and in vivo findings showed that the integration of G3-K PS would downregulate Cxcl9 gene and protein expressions to achieve an enhanced bone regeneration effect, with respect to a higher BMD and BV/TV. Immunohistological staining demonstrated lower expression of Cxcl9 in BCPG3 and SrBCPG3 groups, meanwhile Runx2 and RANKL positive expressions were more in BCPG3 group than others. The results were in accordance with in vitro gene and protein expression and in vivo serum biomarker analysis. This study demonstrated for the first time that G3-K PS carrier could down-regulate Cxcl9 expression and no additional benefit to osteoporotic bone regenerating ability of BCPG3 material was found with Sr incorporation.
Conclusion
The results indicated that the BCPG3 bone paste can become a high-performance bone filler in the treatment of osteoporotic bone defects.
References
1. Raucci, M. G. et al., Tissue Eng Part A 20, 474-85 (2014).
Acknowledgements: The authors would like to thank the Progetto MIUR PRIN2017 – ACTION Grant N. 2017SZ5WZB and H2020-MSCA-RISE-2016, SECOND.R.I., Grant Agreement No 734391 for financial support"
94238145306
"Introduction
Bone is a highly dynamic tissue that undergoes continuous remodeling through lifetime. During this process, two cell types, osteoblasts and osteoclasts, are responsible for bone formation and bone resorption respectively. Mechanical stimuli applied on bone tissue can shift the balance between these two cell populations [1]. Different studies have investigated the impact of the mechanical stimuli on cell proliferation and differentiation [2, 3]. Pre-osteoblastic cells are well established to evaluate in vitro osteogenic responses [4], and they can differentiate into mature osteoblasts under appropriate culture conditions, allowing for the validation of bone tissue formation. In this study, the application of uniaxial compression on pre-osteoblastic cells seeded onto PLLA/PCL/PHBV scaffolds was examined by assessment of cell viability and differentiation markers to determine the effect of the mechanical stimulation on cellular responses.
Methodology
MC3T3-E1 pre-osteoblastic cells (7x104 cells/scaffold) were seeded onto blend scaffolds (5 mm x 5 mm x 1 mm) consisting of PLLA/PCL/PHBV (90/5/5) and cultured under both dynamic and static conditions for 21 days. The scaffolds were subjected to mechanical stimulation for 40 min every day. Three different frequencies of uniaxial compression, 0.5, 1, and 1.5 Hz, with a strain equal to 8% of the scaffold side (400 μm of displacement), were employed to assess their effect on pre-osteoblasts’ differentiation. Cell proliferation and morphology were monitored via a cell viability assay and scanning electron microscopy (SEM). Measurement of alkaline phosphatase (ALP) activity and calcium secretion were conducted to determine the effect of the mechanical stimulation on the osteogenic responses of pre-osteoblasts, and were compared to the static condition.
Results
All dynamic conditions depicted lower cell number than the static equivalent, however comparable at all experimental time periods. These finding are in line with the SEM images showing that the cells adhered strongly to the scaffolds from the early experimental time points.
The ALP activity in all dynamic conditions demonstrated significantly higher values (p<0.0001 for 0.5 Hz, and p<0.001 for 1 and 1.5 Hz) than those of the static one on Day 7. Similarly, the calcium secretion by pre-osteoblasts demonstrated the highest values in all dynamic conditions on Day 7, with the condition at 0.5 Hz indicating the highest level, followed by those at 1 and 1.5 Hz. At Day 14, the calcium concentration decreased, while at Day 21 all conditions displayed similar levels to Day 14.
Conclusions
This study demonstrated that the applied mechanical stimuli affected the cell viability and osteogenic differentiation of the pre-osteoblastic cells leading to their differentiation into mature osteoblasts, revealing that the most efficient applied stimuli condition was at 0.5 Hz.
This project has received funding from the European Union’s Horizon 2020 research and innovation program under grant agreement No 814410.
References
1. Lang, T. et al., J Bone Miner Res, 19(6), 1006-12 (2004)
2. Zhou, Z. et al., Materials Science and Engineering: C, 93, 975-986 (2018)
3. Lee, J.A. et al., J Biomed Mater Res A, 103(10), 3188-200 (2015)
4. Danilevicius, P. et al., Applied Surface Science, 336, 2-10 (2015)
"
73296331205
"The establishment of vascularisation is one of the current challenges, especially in critically sized bone defects [1,2]. Therefore, the aim of the present work is to develop a biomimetic hydrogel scaffold mimicking the bone osteoid in which proper vasculature is induced in order to maximally stimulate osteogenic regeneration. Thiol-ene step-growth photocrosslinking was herein selected to enable superior control, homogeneous network formation and lower radical concentrations compared to traditional chain-growth systems [3]. The networks were benchmarked against traditional chain-growth systems in a physico-chemical and an in vitro osteo- and angiogenic evaluation.
For osteogenic purposes, a novel norbornene-modified aminated gelatin was developed with a degree of substitution of 135% compared to the amount of amines present in native gelatin type B (by addition of only 1.2 equivalents of norbornene carboxylic acid). It is, to the best of our knowledge, the highest substitution reported for norbornene-functionalised gelatins [4]. Thiol-ene crosslinking with thiolated gelatin as cell-interactive crosslinker resulted in networks with full norbornene conversion and a gel fraction of 99%. The increase in storage modulus compared to non-aminated thiol-ene and gelatin-methacryloyl systems (through reaction with the same amount of equivalents) was attributed to an increase in chemical crosslinking (1.8-fold and 4.6-fold respectively) and a decrease of swelling (3.2-fold and 2.9-fold). The biodegradation properties of this hydrogel were preserved and direct contact cell viability data indicated an extended morphology and excellent biocompatibility (96% viability after 7 days). Osteogenic differentiation seeding tests indicated an increase in alkaline phosphatase (ALP) production after 7 days (2.8-fold and 1.5-fold) and a rise in calcium deposition after 21 days (5.2-fold and 1.7-fold). In addition, the incorporation of amorphous calcium phosphate into the thiol-ene hydrogel further contributed to the osteogenicity (1.1-fold increase in ALP-production and 1.2-fold increase in stem cell-based calcium deposits).
Thiol-ene systems were also benchmarked against chain-growth systems with regard to angiogenic stimulation. The homogeneous networks showed similar attachment, yet a more pronounced vascular signalling capacity in terms of fibroblast growth factor-1 (1.7-fold increase after 1 day) and epidermal growth factor (1.2-fold increase after 1 day) secretion compared to the more heterogeneous chain-growth networks which signalled mainly through the production of placental growth factor (2-fold increase after 1 day). Moreover, the thiol-ene networks showed an increased seeded endothelial cell viability (1.2-fold after 7 days) and metabolic activity (1.3-fold after 7 days). The incorporation of human placenta substrate to the thiol-ene networks greatly stimulated the sprout junction density, the total tube length and the number of branches within the developed vascular network in these hydrogels.
The results show that highly controlled networks could be created with tailored topology and viscoelastic behaviour which can stimulate angio- and osteogenesis. Ongoing work focusses on the processing of these hydrogels as bio-inks through extrusion-based 3D-printing and evaluation of the 3D-printed constructs towards vascularised osteogenesis.
[1] Parmentier L, et al. Materials, 2020;
[2] Nguyen LH, et al. Tissue Eng Part B Rev, 2012;
[3] Hoyle C, Bowman C. Angew Chem Int Ed, 2010;
[4] Van Hoorick, J, et al., Macromol Rapid Commun, 2018."
83767247288
"INTRODUCTION
Understanding the interaction between cells and materials is fundamental to tissue engineering. The scientific community's focus on biomaterial-cell interactions has largely fallen on cellular phenotypes, proteins, and nucleic acids, while molecular mechanisms within cells are often overlooked. However, it is expected that cellular metabolism will be significantly influenced by biomaterials. In this regard, direct measurement of metabolites can elucidate perturbation in cellular metabolism, helping to explain and predict changes in cellular phenotypes. In this work, we performed in vitro based metabolomics study to identify changes in cellular metabolism upon interaction with calcium phosphate based biomaterials.
METHODS
Calcium phosphates, including hydroxyapatite (HAP), β-tricalcium phosphate (β-TCP), and bicalcium phosphate (BCP), were applied as the model materials to investigate the cellular response. Sintered materials were characterized by XRD to determine the crystalline structure. NIH/3T3 cells were directly seeded on the materials and harvested on days 1, 3, 5, and 7, following methanol-chloroform mixture extraction protocol to collect metabolites and lipids. Targeted quantitative metabolite analysis was carried out utilizing several HPLC methods in combination with mass spectrometric detection. Obtained metabolomics data were further analysed using MetaboAnalyst 5.0.
RESULTS
The results showed that different calcium phosphate components induced different changes in cellular metabolism. Pathway analysis comparing metabolites profiles between cells grown on HAP, β-TCP, and BCP from different time points and control cell culture showed that metabolism and biosynthesis of several amino acids, acylcarnitines, and lipids were changed. For example, we observed changes in glutathione and arginine metabolism which plays important roles in antioxidant defence and immune response. The observed changes in amino acid metabolism were inter-interpretable with previously reported gene expression and protein activity data for selected materials.
CONCLUSIONS
In our study, we confirmed the calcium phosphate based biomaterials influence on cell metabolism. Distinct changes in metabolite profiles for cells seeded on calcium phosphate ceramics compared to controls were observed. Most of the detected metabolites exhibited time-dependent changes indicating a cellular adaption mechanism to biomaterial-induced perturbation. These results provide evidence about the biomaterials chemical composition influence on cellular metabolism and its link to gene expression level. This proves that metabolomics is a useful tool for studying metabolomics-material interactions to improve the current understanding of the mechanism governing cell behaviours."
31412759499
"Tissue engineering proposes an innovative therapeutic approach to support and induce regenerative processes in damaged tissues. Scaffolds should provide a suitable environment for proliferation, differentiation, and maturation of cells and formation of new tissue and blood vessels. Furthermore, scaffolds should be gradually resorbed at a rate commensurate with bone formation. Calcium phosphates (CaPs) are commonly used in bone tissue engineering due to their chemical composition similarity to the main component of the inorganic part of bone. Among CaPs, β tricalcium phosphate (β-TCP) exhibits a suitable resorption rate for tissue engineering applications [1]. However, highly porous ceramics demonstrate high brittleness and poor surgical handling. Polymeric coatings tend to improve the durability of bioceramic scaffolds and may serve as carriers of biologically active substances [2]. Poly(3-hydroxybutyrate) (P(3HB)) is a biocompatible and biodegradable biopolymer, which degradation products are harmless to the surrounding tissues. For these reasons, we used P(3HB) as a coating on β-TCP scaffolds. The physicochemical and biological properties of the obtained materials have been examined.
β-TCP scaffolds were fabricated by a foam replication method using three types of polyurethane matrices with different pore sizes (S-small, M-medium, L-large). The bioceramic scaffolds were soaked in the P(3HB) solution, dried, and subjected to further studies. Obtained materials were assessed by X-ray diffraction, scanning electron microscopy, hydrostatic weighing, compression tests, and chemical stability in vitro. Degradation products of P(3HB) were analysed via UHPLC-MS. Furthermore, hMSC adhesion, growth, and differentiation were assessed.
The ceramic scaffolds were uniformly covered with the biopolymer, which was evidenced by SEM observations. The P(3HB) coating did not significantly influence the total porosity of the materials obtained ( Ptotal~70 vol%). Composites possessed higher comprehensive strength (up to 4.5 ± 0.5 MPa) compared to uncoated β-TCP. The degradation of P(3HB) during incubation in water was confirmed by UHPLC-MS as (R)-3-hydroxybutyric acid and its oligomers were identified in the extracts. In vitro studies revealed that hMSCs adhere, grow, and proliferate on both uncoated and coated scaffolds (viability over 85% at 7 and 21 days). The number of cells increased in day 21 if compared to day 7 on all materials. The pore size affected the depth of penetration of the cells. The cells efficiently penetrate the materials (even 650-700 µm into the scaffold with large pores).
P(3HB) can serve as a coating on ceramic-polymer composites improving mechanical properties and the durability of the scaffolds. In addition, the released (R)-3-hydroxyacids may nourish the surrounding tissues. Preliminary in vitro studies using hMSC revealed no cytotoxicity of β-TCP as well as β-TCP/P(3HB) scaffolds. Developed materials can act as scaffolds for bone tissue regeneration. Further in vivo studies are necessary.
ACKNOWLEDGEMENTS: Research funded by the National Centre for Research and Development, Poland, grant Techmatstrateg no. TECHMATSTRATEG2/407507/1/NCBR/2019
REFERENCES: [1] Putri, et al., Journal of Biomedical Materials Research Part A, 108(3) (2020), 625-632. [2] Skibiński, et al., Ceramics International 47(3) (2021), 3876-3883."
83767259297
"Surgical repair of large skull bone defects is often performed using patient-specific metallic implants.(1) These implants, however, have poor osseointegration ability, and their life-long fixation is dependent on osteosynthesis screws.(2) It is often suggested that a composite model bone graft substitute could be used to overcome these problems, combining desired properties from individual material classes.(3) We recently developed new materials that combine the mechanical properties of a titanium alloy (Ti6Al4V) with the bioactivity of a calcium phosphate ceramic (CaP) using additive manufacturing, resulting in porous Ti6Al4V 3D implants without or containing 5 or 10 wt% beta-tricalcium phosphate (TCP).(4) The osteogenic differentiation and tissue formation capacity of bone marrow-derived human mesenchymal stromal cells (hMSCs) on these implants were examined in vitro, and the implants were subsequently tested in vivo in a large animal calvarial defect model.
Standardized cylindrical implants (diameter 5 mm, height 2 mm) were seeded with hMSCs and cultured in basic or mineralization medium. On days 14 and 28, the constructs were analyzed for cell metabolic activity, DNA content, tissue formation, alkaline phosphatase (ALP) activity, osteopontin and osteocalcin secretion, and osteogenic gene expression. Next, critical-size defects were made in the frontal bone of skeletally mature minipigs. Test or control scaffolds (diameter 15 mm, height 5 mm) were used for reconstruction, or the defect was left untreated. The animals were sacrificed after 12 weeks, and the implants were retrieved and analyzed for new bone formation and bone ingrowth.
hMSCs cultured on the scaffolds in vitro remained metabolically active and showed a similar proliferation profile on all scaffold types. hMSCs produced ALP, osteopontin and osteocalcin. Additionally, we observed tissue formation throughout the porous scaffolds in all conditions after 14 and 28 days. RUNX2 and ALP expression showed an inverted relationship with increasing TCP content, whereas osteocalcin and osteopontin were more expressed with increasing TCP content. A detailed analysis of the in vivo results of new bone formation and bone ingrowth into the porous structure is ongoing and results will be summarized in the presentation. Overall, the implants showed a good intra-operative handleability and no complications regarding the implant site were observed.
We have shown successful cell survival and tissue formation on our newly developed Ti6Al4V-TCP scaffold types in vitro. Bone formation and bone ingrowth into the 3D porous implant structure in the physiologically complex minipig model will enhance our understanding of the effect of the individual components and structural characteristics on the biological response.
52354527524
Cell morphology plays an important role in controlling cell functions. Application of micropatterned surfaces in cell biology provides reproducible cell morphology and relative stable adhesion and cytoskeleton pattern for investigation of stem cell functions. We have used photo-reactive polymers and UV lithography to prepare micropatterns to control cell size, shape, adhesion area, aspect ratio and chirality to investigate their influences on stem cell differentiation and gene transfection. In this study, the independent influence of adhesion and spreading area on differentiation of human bone marrow-derived mesenchymal stem cells (MSCs) was investigated by using micropatterned surfaces to precisely control cell adhesion and spreading areas.
The micropatterns were prepared by micropatterning non-adhesive PVA on cell adhesive TCPS surface. Ten micropattern structures were designed and prepared to control cell adhesion area and cell spreading area separately. The micropatterns were composed of many TCPS microdots having a diameter of 2 μm in a round circle having a diameter of 70, 60 and 50 μm. The TCPS microdots and round circles were surrounded by PVA. The micropatterns were designed to control the cells to have the same spreading area but different adhesion area, or to have the same adhesion area but different spreading area. MSCs were cultured on the micropatterns. The formation of FAs and the cytoskeletal organization in the cells were investigated to evaluate cell adhesion and spreading state. The mechanical properties of micropatterned cells and the transduction of cytoskeletal force into nucleus were characterized to reveal the mechanism of the influence. The osteogenic and adipogenic differentiation of MSCs on the micropatterned surfaces were evaluated.
When cell spreading area was the same, cells with small adhesion area formed FAs at cell edge. Their cytoskeletal structure was mainly composed of radically assembled DSFs. The lack of myosin binding to DSFs resulted in low cytoskeletal tension. And the YAP/TAZ mainly distributed in cytoplasm. Therefore, cells with small adhesion area preferred to differentiate into adipocytes. Increasing in cell adhesion area reinforced the cell/material adhesion strength. Cells formed integrated actin network including VSFs, DSFs and TAs. Association of myosin with VSFs and TAs generated high cytoskeletal tension. The cytoskeletal tension stimulated accumulation of YAP/TAZ into nucleus. Cells with large adhesion area showed high potential to become osteoblasts. When cell adhesion area was the same, changing spreading area did not significantly affected stem cell fate determination. Cells with the same adhesion area showed similar potential of osteogenic or adipogenic differentiation. The results indicated that the adhesion area rather than spreading area played more important roles in manipulating stem cell functions. Large adhesion area facilitated the osteogenic differentiation, while small adhesion area promoted the adipogenic differentiation.
20967804808
Introduction
There is a dire short of donor corneas for cornea transplantation, leaving millions of visually impaired patients without treatment1. 3D bioprinting holds tremendous potential for fabrication of cornea mimicking structures. One of the key technological challenges in 3D bioprinting is the establishment of bioink compositions that allow both ideal printability as well as biocompatibility. To address these needs, we developed a hyaluronic acid (HA)-based bioink for 3D bioprinting of cornea tissue engineering (TE).
Methodology
HA-based bioink was prepared using hydrazone crosslinking chemistry2. Crosslinking components were combined with rheological modifiers to obtain a printable bioink. The shear thinning property and viscosity of the bioink as well as the mechanical stability of the printed structures were determined with a rheometer. Extrusion-based 3D bioprinting was used. The shape fidelity and self-healing properties of the bioink were explored. Human stem cells, such as human adipose stem cells (hASCs) and hASC-derived cells were chosen for printing cornea stroma equivalents. The printed constructs were evaluated for their cell viability, proliferation and microstructure with LIVE/DEAD® and PrestoBlue™ viability assays, immunofluorescence (IF) and hematoxylin and eosin stainings. Key protein expression was determined with IF and quantitative PCR. Moreover, 3D printed stromal equivalents were implanted into ex vivo porcine corneal organ cultures to explore integration to host tissue. Finally, human pluripotent stem cell derived neurons (hPSC-neurons) were 3D bioprinted to the periphery of the cornea stroma equivalents, and the integration of neuronal extensions to the printed structures was explored.
Results
The developed HA-based bioink showed excellent shear thinning property, viscosity as well as printability. Quality prints with high-resolution and good shape fidelity were achieved. Moreover, HA-DA bioink discs showed self-healing after 24 hours of healing. Importantly, HA-based bioink demonstrated excellent biocompatibility with all explored human stem cells and human stem cell derived cells. Cells in printed structures showed good tissue formation seen with positive expression for connexin 43 and formation of cellular networks. Corneal stroma equivalents with appropriate cell organization and positive expression of lumican were successfully manufactured. Moreover, 3D bioprinted cornea stromal equivalents demonstrated excellent integration to host tissue in ex vivo organotypic cultures after 21 days. While inspecting the innervation of cornea stromal equivalents in 3D bioprinted cornea tissue model, the printed structures with HA-based bioink allowed the ingrowth of long neuronal extensions. Target cells in the cornea stromal equivalents accelerated the neuronal extension growth compared to printed structures without cells.
Conclusions
We have developed a HA-based bioink using click chemistry that fills the demands of next-generation bioinks with excellent printability, stability, biocompatibility as well as tissue formation. Here, we demonstrated that the developed bioink is feasible for 3D bioprinting of cornea stroma equivalents. Moreover, we manufactured the first 3D bioprinted cornea tissue model with innervation. The developed bioink and the printed human stem cell derived cornea stromal equivalents hold great potential for future cornea TE applications.
References:
1. Gain, P. et al., JAMA Ophthalmol. 134(2):167-73 (2016).
2. Koivusalo, L. et al. Biomaterials. 225, 119516 (2019).
20941860968
Following spinal cord injury, a complex scar forms around a lesion cavity, preventing axonal regrowth. Despite ongoing development of stem cell treatments for spinal cord injury, effective repair of the cord remains a challenge in part due to the lack of a supportive environment and cell death. Therapeutics that physically bridge the cavity with a neurotrophic environment while simultaneously delivering stem cells to restore lost tissue may have potential. Building off success in our lab in peripheral nerve repair[1], we aimed to identify neurotrophic extracellular matrix (ECM) proteins to develop novel, biomimetic functionalized hyaluronic acid scaffold implants and to determine their trophic capacity for spinal cord applications using multiple cell models[2]. By optimizing scaffold stiffness and matrix composition for stem cell delivery, it was hypothesized that the pro-regenerative signaling properties of trophic induced pluripotent stem cell (iPSC) derived astrocyte progenitors could be enhanced by scaffold physiochemical properties to promote cord repair.
To identify neurotrophic ECM proteins, astrocytes and various neuronal cells were cultured on a range of ECM combinations to identify a novel neurotrophic substrate. Following incorporation of the neurotrophic substrate mix into freeze-dried 3D hyaluronic acid scaffolds of varying stiffness, scaffold physicochemical properties were characterized. Astrocytes, neurons, dorsal root ganglia (DRG) and iPSC-derived astrocyte progenitors were cultured in scaffolds up to 21 days and the effect of scaffold stiffness and matrix composition was assessed. Additionally, the impact of scaffold properties on the therapeutic effectiveness of iPSC-derived progenitors was assessed using various models.
Screening of central nervous system ECM components revealed that a combination of collagen-IV (Coll-IV) and fibronectin (FN) synergistically enhanced neuronal and astrocyte outgrowth compared to control substrate poly L-lysine. Subsequently, hyaluronic acid scaffolds functionalized with Coll-IV/FN were manufactured using different concentrations of hyaluronic acid to produce scaffolds of varied stiffnesses ranging from soft to stiff (0.8-3kPa). Astrocytes cultured in soft, Coll-IV/FN functionalized scaffolds, increased secretion of IL-10 and exhibited morphologies typical of resting phenotypes. Furthermore, soft CIV/FN scaffolds significantly enhanced neurite outgrowth from DRG explants (a model of axonal growth) compared to stiffer scaffolds. Soft, but not stiff Coll-IV/FN scaffolds also promoted iPSC progenitor infiltration, differentiation and glutamate uptake (a measure of functional capacity) while encouraging iPSC-derived spheroid growth. Furthermore, conditioned media taken from soft, CIV/FN iPSC-loaded but not stiffer scaffolds significantly enhanced neurite outgrowth 2.8 fold. Finally, mouse spinal cord and DRG explants cultured on soft, Coll-IV/FN iPSC scaffolds promoted astrocyte migration and long axonal extensions between DRG and iPSC neurospheres within scaffolds.
These data indicate that by appropriately tuning the physicochemical properties of scaffolds to mimic that of the uninjured spinal cord, significantly enhances astrocyte responses while promoting neurite extension. Furthermore, biomimetic scaffolds promote the paracrine activity of patient-derived progenitor cells, enhancing their therapeutic capacity. Overall, the impact of biomaterial properties on the therapeutic effectiveness of stem cells has significant implications for spinal cord repair applications.
This work is funded by the IRFU-Charitable Trust, Anatomical Society and SFI-AMBER centre.
[1]Hibbitts (et al.), Matrix Biology, (2022)
[2]Woods & O’Connor (et al.), Adv Healthcare Mat., (2021)
31412720648
Introduction: The past few years saw an increasing trend in liver disease prevalence [1]. Orthotopic transplantation is the current gold standard treatment. However, its practice is affected by strong complications represented by the paucity of available organs and the necessity of long-term immunosuppressive therapies. Tissue engineering strategies represent suitable alternatives by combining scaffolds matrices and patients’ autologous cells. In this setting, CRISPR/Cas9 genome edited HLA knockout human induced pluripotent stem cells (hiPSCs) represent a promising cell candidate for a universal strategy, conjugating high cell availability and wide tissue-specific differentiation capacity [2]. The present work aims to rebuild a functional liver substitute seeking for the definition of a promising strategy to evade T cell surveillance upon in vivo transplantation.
Methodology: hiPSCs underwent CRISPR/Cas9 genome-editing to prevent expression of HLA class I and II. HLA Class I/II-/- hiPSCs were differentiated according to standard protocols to obtain definitive endoderm (DE), hepatic endoderm (HE) and immature hepatocytes (IH) cells in a 2D monolayer [3]. hiPSCs-IH were transferred to Matrigel® 3D culture in hepatic organoid expansion medium to generate self-replicating hepatic organoids [3]. To obtain decellularized scaffolds, mouse livers were cannulated via portal vein and decellularized via already established detergent-enzymatic treatment [4]. 17 M cells were obtained from enzyme mediated organoid disaggregation and were further seeded in the decellularized scaffold via the portal vein. Repopulated livers were cultured in a custom-designed bioreactor in a dynamic condition provided by peristaltic pump (flowrate=0.5 ml/min). Constructs were cultured for 7 days in hepatic organoid expansion medium and further supplemented with Oncostatin-M and Dexamethasone to induce terminal hepatocyte differentiation for the following 7 days [5]. Histological and immunohistochemistry analysis were performed to study scaffold repopulation and expression of hepatic maturation and biliary polarity markers. qRT-PCR analysis was performed to analyse the expression of mature hepatic markers including cytochrome 3A4,1A2 (CYP3A4, CYP1A2), hepatocyte nuclear factor 4 alpha (HNF4α).
Results: Complete HLA double knockout was confirmed by genome-sequencing analysis. H&E staining showed successful scaffold repopulation supported by the bioreactor. Immunofluorescence analysis revealed the expression of mature hepatocytes markers (HNF4α, Alpha1-anti-trypsin, human albumin) together with biliary polarity markers such as zonula occludens 1 (ZO1) and multi-drug resistance protein 2 (MRP2), proving a nearly complete mature state. HLA class I and II expression were not detected in immunofluorescence staining, supporting sequencing analysis. qRT-PCR results showed enhanced expression of HNF4α, CYP3A4 and CYP1A2 in the bioreactor culture compared to the static in vitro control, highlighting the effect of the scaffold environment and the dynamic culture on hepatic maturation.
Conclusion: Universal therapeutic strategies are mostly unavailable for the majority of end-stage diseases, including liver diseases. The present work demonstrated the development of a universal functional liver graft by combining organotypic acellular scaffolds and universal hepatocytes obtained from HLA class I/II-/- hiPSCs. The decellularized 3D liver microenvironment efficiently supported hiPSCs-derived hepatocytes engraftment and proliferation. This experimental evidence proves that universal hiPSCs represent a valid candidate to be employed in “ready-to-use” tissue engineering and regenerative therapies, with the promise to overcome immune rejection upon graft transplantation.
41883617739
Recent progress in 3D bioprinting technologies has shown promising results for the multi-material design of interface tissue engineering. However, the achievement of structural integrity between tissues with different mechanical strengths is a great challenge in the bio-fabrication of tissue interfaces, such as bone-cartilage interfaces1. In this study, we used multimaterial 3D bioprinting approach for the deposition of hydrogel structures based on an aspiration-on-demand capillary method. The obtained construct showed a high degree of structural integrity at the interface between adjacent ink segments mimicking the bone-cartilage tissue interface. To do so, specific amounts of different cell-laden hydrogels were extruded in the same capillary and deposited at the desired geometrical pattern2. The ink segments, comprised of Alginate (Alg)/Gelatin (Gel), and Carboxymethylcellulose (CMC)/ Gelatin for soft and hard tissue, respectively. The inks were prepared as the following: Alg and CMC were dissolved in PBS separately by forming amide bonds by carbodiimide-mediated precipitation of material's carboxyl groups with Tyramine's amino groups utilizing EDC/NHS reaction. Therefore, a specified amount of tyramine and NHS were applied to the CMC and Alg mixture followed by adding EDC was added to the NHS at an equal molar ratio to make Alg-Ph and CMC-Ph. Two polymer mixtures were stirred for 24 h, dialyzed against deionized water, and were lyophilized for 3 days. Gelatin was added at 15% w/v in PBS and incubated for 1 day at 37 °C to acquire Gel-Alg-Ph and Gel-CMC-Ph. The hydrogels were crosslinked at the presence of ruthenium (Ru) mixture and sodium persulfate (SPS) photo-initiating system under a visible light source at the concentration of 0.1/1 Ru/SPS (mM/mM). Mesenchymal stem cells (MSCs) were grown and added to the two ink mixtures in various cell counts for the targeted tissue. Mechanical properties of the casted samples were assessed with different parameters such as various Alg-Ph, CMC-Ph, Ru/SPS concentrations, and the visible light exposure time. Differentiation of MSC cells expected after 21 days, histology, immunohistochemistry, and a live/dead test for the bioprinted structure were carried out. [ASF1] The outcomes of the in vitro biochemical studies were corroborated by the result of the histological staining. This research, which investigated the multilayered and hierarchical architecture of osteochondral tissue using 3D biofabricated material compositions, could lead to an improved regeneration of osteochondral lesions in the future.
1. Diloksumpan, P. et al. Biofabrication 12, (2020).
2. Nadernezhad, A. et al. Sci. Rep. 6, 1–12 (2016).
41883643605
"Introduction
Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, fatal form of diffuse interstitial lung disease which is associated with substantial mortality and a median survival of 3 years from the time of diagnosis. Acute exacerbations (AE) of IPF have been defined as acute, clinically significant respiratory deterioration of unidentifiable cause. Available data suggest that 46% of deaths in IPF are preceded by AE, and the median survival of patients with IPF who experience AE is approximately 3.5 months. Mesenchymal stem cells (MSC) derived from term amniotic fluid (TAF) are neonatal and can be propagated to extremely high amounts for use in cell therapy. TAF-MSC are derived from different tissues of the fetus. Therefore, sorting strategies using tissue-specific markers can prepare a sorted population of cells from each tissue-type. We hypothesize that lung-specific MSC can be used to reduce the overall severity of AE-IPF and lower the risk of mortality due to the pharmacological effects (anti-inflammatory, immunomodulatory, regenerative, proangiogenic, and antifibrotic) these cells may have.
Methodology
RNASeq data from TAF-MSC clones was used to identify tissue-specific markers present on these MSC. Several prospective lung markers were tested by flow cytometry of cultured TAF-MSC. One of these markers was used for cell-sorting using Tyto MACSQuant cell sorter and the positive cell population was expanded to passage 4 (TAF-lung-MSC). Rats were instilled intratracheally with bleomycin to induce fibrosis at day 0. At Day 4 TAF-lung-MSC or vehicle was administered by an IV injection. On day 28 lungs were prepared for histology, stained and scored.
Results
After collection, processing and expansion of TAF cells in a defined media system, different subtypes of MSC were identified. Of these, one type had unique RNA expression and cell surface phenotype expression profiles. This signature was partially similar to MSC collected from lung tissue. After identifying lung-specific clones, candidate marker genes were identified. The validity of the surface marker genes was established using flow cytometry with antibodies directed against the indicated lung surface markers. One of the prospective markers was used for lung-MSC sorting. The positive fraction was further propagated until passage 4 when it was used to treat bleomycin induced pulmonary fibrosis in a rat model. TAF-lung-MSC showed anti-fibrotic effect with significantly less fibrosis in the lungs of rats at 24 days after infusion of MSC than in the bleomycin-treated control group (p<0.05) as assessed both by histopathological evaluation (percent parenchyma affected) and fibrosis scoring using the Modified Ashcroft scale. Further, at the termination of the study on Day 28, TGF-β plasma levels were higher in bleomycin-treated control rats than in rats treated with TAF-lung-MSC. No adverse reactions from cell injections were reported. No remaining TAF-lung-MSC could be detected after Day 24, supporting that MSC were cleared from the rats after exerting their effect.
Conclusion
Several markers were found to be good lung-specific markers for sorting of TAF-MSC using the Tyto MACSQuant cell sorter instrument. Sorted cells were further propagated and TAF-lung-MSC were effective in reducing lung fibrosis in a rat bleomycin induced pulmonary fibrosis model."
62825419269
"Introduction: Spinal cord injury (SCI) is a condition that hampers the communication between the brain and the body, resulting in several comorbidities that decrease the patient’s life quality. The limited regeneration after SCI is mainly attributed to the injury complexity composed of several interconnected mechanisms. Although reestablishment of lost nerve tracts is essential for functional recovery, it is also important to restore the destroyed blood vessels. Adipose-derived stem cells (ASCs) have been addressed as therapeutic agents do their ability to modulate several repair processes, among neuronal and vascular growth, both at a paracrine and non-paracrine level. However, until now, ASCs did not elicit a total satisfactory repair. To enhance the regenerative features of ASCs, several approaches are being explored. Inflammatory pre-conditioning has shown to augment the anti-inflammatory properties of these cells, but with few reports showing the effects at a neuronal and vascular level.
Therefore, we aimed to understand the impact of pro-inflammatory (LPS+IFN-γ) and pro-regenerative molecules (IL-10) on the neuroregenerative and angiogenic potential of ASCs at three levels: indirect contact, secretome and direct contact.
Methodologies: The neuroregenerative potential was evaluated by analysis of the neurites produced by rat dorsal root ganglia (DRG) explants. The angiogenic potential was assessed by plating human umbilical vein endothelial cells (HUVECs) on a matrigel matrix, with further morphological analysis of the formed vessels. Each assay was adapted to the type of cell communication studied. Gene analysis for several vascularization and axonal growth-related molecules was performed after ASCs stimulation.
Results: ASCs induced neurite growth in all DRG assays. In the secretome and direct contact assay, the control did not induce any growth. No differences between inflammatory stimulations were found regarding neurite area, length and arborization pattern, in any DRG assay. LPS+IFN-γ enhanced the vascular potential of ASCs secretome, forming more vessel-like structures with higher average length and interconnectivity. This effect was lost in the indirect co-culture, with no differences between stimulations at any level. IL-10 did not alter ASCs behavior. Inflammatory stimulation led to alterations in ASCs gene expression, but without a clear shifted phenotype.
Conclusion: Overall, this work showed that ASCs and their secretome can modulate neurite and vascular processes essential for successful CNS regenerative applications. Furthermore, pro-inflammatory stimulation enhanced secretome angiogenic properties, holding great potential to enhance ASCs therapeutic properties.
Funding: This work has been funded by grants from Prémios Santa Casa Neurociências ‐ Prize Melo e Castro for Spinal Cord Injury Research (MC-04/17); PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF) FEDER, through the Competitiveness Internationalization Operational Programme (POCI), and by National funds, through the Foundation for Science and Technology (FCT), under the scope of the projects TUBITAK/0007/2014; PTDC/DTP-FTO/5109/2014; POCI-01-0145-FEDER-029206; POCI-01-0145-FEDER-031392; PTDC/MED-NEU/31417/2017; NORTE-01-0145-FEDER-029968; project UIDB/50026/2020 and UIDP/50026/2020 ICVS Scientific Microscopy Platform, member of the national infrastructure PPBI - Portuguese Platform of Bioimaging (PPBI-POCI-01-0145-FEDER-022122); and by the projects NORTE-01-0145-FEDER-000013 and NORTE-01-0145-FEDER-000023, supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the ERDF."
73296328146
"INTRODUCTION Therapeutic application of mesenchymal stromal cells (MSCs) has been suggested as a promising regenerative treatment for osteoarthritis (OA) by virtue of their paracrine-mediated chondroprotective and immunomodulatory activity. Nonetheless, collection of MSCs is an invasive procedure and the therapeutic efficacy of autologous MSCs are subject to large variability due to the varying potency of individual MSCs.1 In the current study we therefore set out to explore human induced pluripotent stem cell (hiPSC) derived MSCs (hiMSCs) as sustainable cell sources for OA stem-cell treatment by identifying and characterizing the therapeutic OA-secretome of hiMSC that contribute to a beneficial chondrocyte state.
METHODOLOGY An established protocol was applied to generate hiMSCs with high similarity to hBMSCs.2 To mimic the inflammatory environment, generated hiMSCs and hBMSCs were exposed to different cytokines (IL6 or a combination of TNFα and IFNγ). RNA sequencing was performed to determine the secretome of licensed hiMSCs (N=6). Data were analyzed in R using DESeq2 package while considering significant differentially expressed genes (DEGs) in comparison to unlicensed controls when FDR<0.05. Luminex was applied to quantify cytokine levels in cell culture media. Effects of licensed hiMSCs on human primary articular chondrocytes (hPACs; N=3, RAAK study) were determined by RT-qPCR following 3 days of co-culture in transwells.
RESULTS Cell licensing with IL6 resulted in modest changes in gene expression effect sizes and 5 FDR-significant genes. Of note among these genes was SOCS3 encoding suppressor of cytokine signaling 3 (FC=2.0, FDR=2.2x10-2). In contrast, TNFα+IFNγ showed substantial differences as compared to unlicensed cells. Particularly, with respect to well-known immunomodulatory genes such as IDO1 (over 1000-fold induction), MCP1, and HLA-DRA (both around 100-fold induction). Likewise, we demonstrated significant increase of secreted cytokines in the culture media of TNFα+IFNγ exposed hiMSCs. The changes in licensed secretome of hiMSCs were highly comparable to those in hBMSCs, independent of the licensing factor applied, suggesting that the therapeutic mode of action is the same.
Effects of hiMSCs licensed with either IL6 or TNFα+IFNγ were explored in co-cultures with hPACs. The inverse effects of both licensing methods on chondrocytes with respect to expression of COL2A1 and OA markers MMP13, ADAMTS5, CD55 and IL11 suggested that IL6-licensed hiMSCs exerted direct pro-chondrogenic activity.
CONCLUSION Our results demonstrate that hiPSC-derived MSCs should be considered promising candidates as stable source for application in cell therapy. Particularly, IL6 licensed hiMSCs showed direct chondroprotective effects as reflected by decrease in OA markers. Transcriptome wide analyses revealed SOCS3 as a candidate effector. Further studies will address whether, in vivo, this may result in additional beneficial effects by virtue of its immune-suppressive activity.
In line with previously determined similarities between characteristics of our hiMSCs and hBMSCs, the current study demonstrates potential of iPSC-derived MSCs in response to inflammatory environment. Findings pave the way to further explore their application in the clinic as off-the-shelve cell source to treat osteoarthritis.
References
1. Barry et al, J Orthop Res 37, 1229-1235 (2019)
2. Rodriguez-Ruiz et al, Cell Tissue Res 386, 309-320 (2021)"
52354519528
TBA
"Introduction
Proteoglycan-4 (PRG4) is a mucinous glycoprotein with critical roles in the bio-lubrication of articular cartilage and joint health1. Intra-articular injection of PRG4 protects against cartilage deterioration in in vivo models of osteoarthritis, an effect that is largely attributed to restored bio-lubrication. Thus far, tissue-engineered, collagen-glycosaminoglycan (coll-GAG) scaffolds2 developed by our lab have shown significant promise in treating focal yet critical-sized osteochondral defects within several in vivo animal models and in humans Such scaffolds alone, however, lack the requisite frictional qualities of articular cartilage. The regenerative capacity of collagen-based biomaterials can be improved through the incorporation of therapeutic molecules4 and we have previously demonstrated that the frictional properties of coll-GAG scaffolds are enhanced by soaking scaffolds in recombinant human (rh)PRG4-solution in vitro3. The goal of this study was, therefore, to investigate an alternative approach to PRG4-injections by developing a scaffold-based recombinant human (rh)PRG4 delivery system for superior bio-lubrication of cartilage biomaterials.
Methods
1) The effect of rhPRG4 (Lµbris Biopharma3) treatment on mesenchymal stem cell (MSC) proliferation (DNA, Picogreen) and sulfated-GAG (sGAG, Blyscan Assay) production was examined in 2D-culture (wells). 2) Subsequently, rhPRG4 was incorporated via bulk-addition to a collagen I-hyaluronan slurry (blended mixture) and lyophilized, previously optimized for controlled molecule release4. The coll-rhPRG4 scaffold properties were assessed including pore architecture (JB4, toluidine-stain), rhPRG4 release (ELISA) and molecular weight (western blot). 3) The scaffolds’ mechanical properties including the bio-lubrication (coefficient of friction3, COF) and compressive stiffness were characterized. 4) Finally, the biological response of MSCs in 3D-culture (scaffolds) after 14, 21, and 28 days was assessed.
Results
1) In 2D, rhPRG4-treatment significantly increased MSC proliferation (+61%) and sGAG production (+4.3x). 2) rhPRG4 was successfully incorporated within scaffolds as demonstrated by western blots indicating intact rhPRG4 of molecular weight ~460kDa, rhPRG4 released gradually from the scaffold up to day 14 (67% release by day 7). The novel biomaterial was highly porous with uniformly interconnected pores (121.9 to 160µm). 3) The coll-rhPRG4 scaffolds were lubricious, with a 42% reduction of COF (Coll-GAG-rhPRG4: 0.068 ±0.01 vs. Coll-GAG: 0.118±0.02, p<0.001), however incorporation of PRG4 reduced the stiffness of the scaffolds. 4) Coll-GAG-rhPRG4 scaffolds produced 40 to 80% more sGAG than controls across multiple timepoints (Days 14, 21, and 28).
Conclusion
An innovative scaffold based rhPRG4 release system was successfully designed for cartilage repair applications. Additionally, beyond rhPRG4’s ability to support bio-lubrication, a previously undescribed biological effect was revealed, rhPRG4 increased cell proliferation and sGAG formation in vitro. Therefore, PRG4 containing biomaterials may be suitable for other applications (e.g., contact lens materials). To conclude, scaffold-based delivery of PRG4 may circumvent the need for multiple intra-articular injections whilst also providing cues to promote cartilage regeneration.
References
1. Jay et al, Matrix Biology, 2014; 2. Levingstone et al, Biomaterials, 2016; 3. Matheson et al, Journal of the Mechanical Behavior of Biomedical Materials, 2021; 4. Almeida et al, Acta Biomaterialoa, 2014.
Acknowledgements
ReCaP: European Research Council Advanced Grant (Grant: 788753), National Science Foundation- Science Foundation Ireland (NSF-SFI) (Grant: NSF_ 17_US_3437), ADMIRE Marie Sklodowska-Curie-Action-Cofund (EU Horizon 2020: 945168, SFI: 12/RC/2278_2)"
31412741608
"Introduction
Annually, millions of people die because of liver failure, while the waiting duration for a donor liver is around 12 months.[1] Herein, we target hybrid 3D-printed scaffolds to serve liver tissue engineering(LTE) applications.
As starting materials gelatin in combination with a polysaccharide was used to develop printable hydrogels. As polysaccharides dextran (Dex) and chondroitin sulphate (CS) were selected as mimics for the liver extracellular matrix (ECM) to explore their effect on the cell response. Methacrylated gelatin (GelMA) served as benchmark. The hydrogel materials were characterized on 2D-as well as on 3D-level.
HepG2 cells were used to assess the in vitro biocompatibility of the developed hydrogels.
Materials and methods
Thiolated gelatin (GelSH)[2] was crosslinked with the norbornene-functionalized polysaccharides DexNB and CSNB. Gelatin was methacrylated using methacrylic anhydride (GelMA) as reference material.
The different materials were characterized on both 2D- and 3D-level to assess the physico-chemical properties and the biocompatility. 3D-hydrogel scaffolds of the materials were developed by indirect 3D-printing[3].
Results and discussion
On a 2D-level, DexNB-GelSH and CSNB-GelSH were superior over GelMA as their crosslinking kinetics were significantly faster and they mimicked natural liver tissue (NLT) to a greater extent with respect to swelling and mechanical properties. The swelling ratio of GelMA, DexNB-GelSH and CSNB-GelSH were respectively 9.1±0.5 and 9.6±0.5 and 8.7±0.2 which is in line with the swelling of NLT (i.e.10)[3].
Atomic Force Microscopy (AFM) measurements revealed superior microscale mechanical properties of the DexNB-GelSH hydrogel sheets compared to the other materials. DexNB-GelSH exhibited a stiffness of (196±24)kPa, CSNB-GelSH of (106±2)kPa and GelMA of (291±11)kPa. Healthy NLT exhibits an average surface stiffness of (183±48)kPa. The higher the stiffness, the more the material mimics the ECM of unhealthy cirrhotic liver ((411± 63)kPa)[4].
On a 3D-level, DexNB-GelSH scaffolds exhibited a compressive modulus of (4.8±1.6)kPa which is in excellent agreement with that of NLT (i.e. 1–5kPa)[5] as compared to GelMA which resulted in a modulus of (8.5±1.9)kPa and CSNB-GelSH (12.6±1.9)kPa.
So far, the biocompatibility of CSNB-GelSH and GelMA were compared. However, the in vitro biocompatibility of both materials was comparable based on the MTS assay. The live-dead staining showed that the cells grew more into clusters on the DexNB-GelSH scaffolds compared to the more spread morphology which the cells exhibited on the GelMA material.
Conclusions and future perspectives
DexNB-GelSH and CSNB-GelSH scaffolds are promising hybrid materials to support LTE as they exhibit similar physico-chemical properties compared to NLT (cfr. microscale stiffness, compressive modulus, swelling ratio and chemical compostion), while cell viability and proliferation of the hepatocytes were preserved.
In future research, different cell types such as primary hepatocytes and organoids will be included in the biological evaluation. Furthermore decellularized liver ECM will be incorporated into the hydrogel material in order to improve the cell interactivity and proliferation.
1)Emek, E. et al. Transplant. Proc. 51 (7), 2413-2415(2019)
2)Van Vlierberghe, S. et al. Eur. Polym. J. 47 (5), 1039-1047(2011)
3)Mattei, G. et al. Acta Biomater. 10, 875–882(2014)
4)Zhao, G. et al. J. Surg. Oncol. 102, 482-489(2010)
5)Mattei, G. ACTA Biomater. 10(2), 875-882(2014)"
73296310004
"Introduction
The fabrication of three-dimensional (3D) scaffolds able to promote a spatiotemporal guidance of cell infiltration, vascularization and innervation are of great interest in tissue engineering and regenerative medicine (TERM) applications. To this end, peptide sequences displaying fast and slow proteolytic rates towards urokinase plasminogen activator uPA, namely GTAR and DRIR, the QK vascular endothelial growth factor mimetic peptide and the IKVAV laminin-devired peptide for neuronal adhesion and proliferation have been described in literature as promising candidates [1,2]. Hydrogels based on Elastin-like recombinamers (ELRs) also have shown applicability in this regard, as their tailorable recombinant nature allow the genetic encoding of proteolytic sequences into their backbone and their peptide sequence enable the covalent tethering of bioactive residues such as the QK peptide or IKVAV.
Methodology
The ELRs prepared in this work were decorated with the GTAR or DRIR proteolytic sequences, the RGD cell-adhesion domain, the QK pro-angiogenic peptide or the IKVAV pro-innervation peptide. Furthermore, click catalyst-free crosslinkable domains were attached to the ELRs to produce the intended hydrogels. In vitro studies allow to determine the effect of IKVAV peptide over C6 glial cells adhesion, whereas the porous structure of the prepared hydrogels was evaluated by microscopic techniques. To assess the ability of ELRs to promote angiogenesis and neurogenesis, we fabricated a 3D construct containing two different cylindrical ELR hydrogels. In detail, the first cylinder contains the QK peptide with the GTAR fast-proteolytic sequence, whereas the second cylinder contains the IKVAV peptide with the GTAR fast-proteolytic sequence, in order to evaluate both bioactivities. In contrast, the outer part lack or display the DRIR slow-proteolytic sequence. Cell infiltration, vascularization and innervation were analyzed by histology and immunohistochemistry (IHC) upon subcutaneous implantation in Swiss CDR-1 mice with time.
Results
Microscope analysis showed the porous structure of the fabricated hydrogels. In vitro studies confirmed the effect of the IKVAV peptide over the cell-adhesion of C6 glial cells. Furthermore, in vivo studies of 3D ELR models revealed a marked increase in cell colonization in the interior tubes containing fast-proteolytic sequences, when compared to the outer part lacking or bearing slow-proteolytic sequences. Histology and IHC results showed the effect of the QK peptide triggering angiogenesis, and the effect of the IKVAV peptide triggering innervation in the pre-design orientation
Conclusions
The combination of proteolytic-sensitive sequences, the QK pro-angiogenic peptide and the IKVAV pro-innervation peptide into 3D ELR hydrogels confirmed the ability to spatiotemporally control angiogenesis and innervation in vivo. Specifically, the cylinder displaying the QK peptide promote a faster endothelialization, whereas the cylinder displaying the IKVAV peptide promote a faster innervation, following the pre-designed orientation. These results set the basis for the development of ELR-based scaffolds for TERM applications where the spatiotemporal control of vascularization and innervation play an important role.
References
1. González-Pérez, F. et al., Acta Biomater. 130 149–160 (2021).
2. Farrukh, A. et al., ACS Appl. Mater. Interfaces 10, 41129−41137 (2018)."
94238101804
"Introduction: Osteoinductive bone morphogenetic proteins (BMPs) possess the ability to induce bone formation and therefore have been the basis of osteoinductive devices designed for bone regeneration. Osteogrow C is a novel autologous bone graft substitute comprised of recombinant human Bone Morphogenetic Protein 6 (rhBMP6) within autologous blood coagulum (ABC) with synthetic calcium phosphate (CaP) ceramic particles. CaP particles serve as a compression resistant matrix and are available in a broad range of shapes and sizes. The aim of this study was to investigate the time course of ectopic bone formation in rats following subcutaneous implantation of rhBMP6/ABC with CaP particles in a size range from 2360 to 4000 µm.
Methodology: Osteogrow C osteoinductive device was prepared as follow: rhBMP6 (20 µg per implant) was added to autologous blood (500 µL), mixed with synthetic ceramic particles (size range: 2360-4000 µm; chemical composition: TCP/HA 80%/20%; porosity: 86%; average pore size: 246 µm) and left to coagulate at room temperature. Subcutaneous pockets were created in the axillary region of Sprague Dawley rats (male, 6-8 weeks, 250-300 g), and following blood coagulation, osteoinductive devices were implanted in pockets. Animals were killed on days 7, 14, 21, and 35 following implantation. Extracted implants were analyzed on histological and microCT sections to investigate the time course of ectopic bone formation.
Results: MicroCT analyses revealed that Osteogrow C implants induced extensive bone formation two weeks after implantation at rat ectopic site. Histological analyses have shown that seven days after implantation large areas of endochondral ossification were present only at the peripheral parts of the implants while on day 14 endochondrally formed bone was present throughout the implant between ceramic particles. On day 21 following implantation BMP-induced osteogenesis has reached its final stage and ectopic bone was present at the ceramic surfaces, in the pores, and between the particles. At the end of the observation period (day 35) the structural properties of newly formed bone were similar as on day 21, however, the thickness of trabeculae between the particles was decreased while the number of adipocytes was increased and they became the predominant cell population in the bone marrow.
Conclusions: In the present study we have elucidated dynamics of ectopic bone formation following implantation of rhBMP6 in ABC with ceramics (Osteogrow C). Osteogrow C implants with large (2360-4000 µm) ceramic particles induced bone in rat ectopic site proving excellent osteoinductive properties of tested implants. Therefore, Osteogrow C is a promising novel therapeutic solution for bone regeneration."
41883630105
"Introduction: Human umbilical cord blood stored in blood banks cannot always be used for hematopoietic stem cell transplantation as up to 80% of stem cells are lower than the cut-off required for such application, being potentially available for non-transfusion applications such as the source of platelet growth factor [1]. In this study, we fabricated a fibrin-based drug delivery system to provide a local and sustained release of cord blood platelet lysate (CBPL) at the wound site. Also, we assessed the temporal evolution of skin lesions using photoacoustic imaging (PAI) and near-infrared spectroscopy (NIRS), non-invasive technologies translatable to a clinical setting. [2,3]
Methods: Fibrin scaffold loaded with CBPL was fabricated by a peculiar spray process (IT Patent application pending N. 102021000025664). Excision wounds were created on the dorsum of genetic diabetic mice (db/db) using a biopsy punch (8 mm diameter). Wounds were treated with fibrin scaffolds with or without CBPL. Oxygen saturation (%SO2) was monitored in the wound area and surrounding tissue through PAI and NIRS imaging on days 0, 7, and 14 after lesion induction. At each time point, whole blood was collected for flow cytometry, and on day 14 wound tissue was retrieved for histological analyses.
Results: Diabetic mice treated with CBPL scaffold showed a significantly higher closure of about 82% of the initial lesion size, 14 days after the intervention. Animals treated with the unloaded scaffolds showed closure of only 62% of the initial lesion size. Histological analysis demonstrated an improved reepithelization and collagen deposition in granulation tissue in mice treated with CBLP scaffold in comparison to unloaded fibrin scaffold. The flow cytometric quantification of circulating fraction variations (compared to baseline) of endothelial progenitor cells (EPCs, CD45-/CD34+/KDR+) showed an increase in the CBPL fibrin scaffold treated mice when compared with the fibrin scaffold alone. Moreover, a statistically significant correlation (P = 0.03, R = 0.754) has been observed on day 14 after induction of skin lesion between wound repair areas and the variations of EPCs fraction in the CBPL fibrin scaffold treated mice. In all the lesions, the %SO2 signal from both PAI and NIRS showed a typical trend characterized by an increase one week after the wound induction (day 7) followed by a decrease toward the initial intact skin %SO2 values (day14).
Conclusions: This study allows developing a new approach based on imaging and circulating biomarkers to describe the inflammatory state and healing dynamics during the regenerative process. The present findings pave the way for clinical translation of the developed experimental patches and combined molecular imaging.
[1] Eppley BL et al. Plast Reconstr Surg. 114(6), 1502-8 (2004)
[2] Steinberg, et al., Photoacoustics. 14, 77–98 (2019)
[3] Landsman A. Wounds. 32(10), 265-271 (2020)
Acknowledgments: This work was supported by Fondazione Pisa grant number No. 2016.0163, Project acronym: PREVISION. The project has been supported by the Multi-Modal Molecular Imaging Italian node - Euro Bioimaging (MMMI- EuBi)."
73296363497
Kidney organoid derived from pluripotent stem cells (PSCs) have gradually become a platform to understand kidney morphogenesis, development and diseased states. Despite the abundant differentiation protocols to obtain relevant renal cell types and organoids, most of these only reach a developmental immature stage (1). We hypothesize that the limited maturation is due to absence of relevant ECM molecules that interact with signalling of the developing kidney. With this, we synthesized heparan sulphate analogue hydrogels to observe if organoid maturation will proceed upon encapsulation. Precursor molecules were successfully functionalised with both 5.2% norbornene, and 1.7 SO3 groups per disaccharide of backbone. Adsorption studies on these hydrogels show strong affinity towards growth factors relevant to the reciprocal induction signalling in the developing kidney. This demonstrates the materials’ mimicry on ECM modulated signalling of heparan sulphate. In addition, further maturation of encapsulated organoids was observed by means of immunocytochemical staining with the presence of principal and intercalated cell populations, along with reduced interstitial cells. Overall, organoid maturation may be realised with these class of materials with a more physiological model for drug discovery, disease modelling, and understanding further the developmental biology of kidney.
Reference
1 - Geuens, T., et al, Biomaterials, 275, 120976.
62825448726
"Introduction: Growth factors (GFs) are a key component of tissue engineering, but their exogenous administration has proven costly and ineffective. Extracellular matrix-inspired biomaterial approaches have sought to sequester these molecules, regulating their activity and presentation to cell receptors.1 Our previous work has shown that molecularly imprinted nanoparticles (MINPs) can play this role in standard 2D and 3D cell cultures, combining high recognition specificity, stability, and cost-effectiveness.2 Taking this concept a step forward, here we tested MINPs against transforming growth factor (TGF)-β3, a regulator of tenogenesis, in hydrogel systems with bioinspired ordered microstructures. Our hypothesis is that combined control over biophysical and biochemical cues will synergistically contribute to more robust tenogenic commitment of stem cells.
Methodology: A TGF-β3 N-terminal epitope was used as template molecule for solid phase imprinting by polymerization of acryloyl-containing monomers. MINP affinity and selectivity were assessed by surface plasmon resonance (SPR), Western blot and dot blot. Aligned polycaprolactone meshes were first produced by electrospinning, followed by cryosectioning into 50-µm microfibers. To enable their remote orientation within hydrogels, superparamagnetic iron oxide nanoparticles were synthesized by thermal decomposition and incorporated in the electrospinning solution. Finally, tenogenic constructs were prepared by encapsulating human adipose tissue-derived stem cells (hASCs), along with microfibers and MINPs, in transglutaminase-crosslinked gelatin hydrogels. Microfibers were unidirectionally aligned by applying a uniform magnetic field during gelation.
Results: SPR demonstrated a remarkable affinity of MINPs for the template (KD = 18±13 nM), in the range of some monoclonal antibodies. In comparison, the interaction between TGF-β3 epitope and MINPs imprinted against biotin was negligible, demonstrating the impact of imprinting on the molecular recognition potential of nanoparticles. hASCs remained viable for at least 14 days in hydrogel systems, showing a preferential orientation along the microfiber alignment axis. Preliminary qPCR findings indicate a positive correlation between MINP concentration and tendon-associated gene expression markers (SCX, TNMD) in aligned constructs, which is not observed in gels with randomly oriented microfibers. Furthermore, osteogenesis-associated ALP expression was downregulated as MINP concentration increased, corroborating the hypothesis of phenotypic steering toward tenogenesis. Protein synthesis is currently being analyzed by immunoassays to further bolster these results.
Conclusions: Our findings demonstrate the potential of molecular imprinting as a cost-effective complementary strategy in tissue engineering approaches. The endogenous GF sequestering ability of MINPs allows an efficient replacement of expensive recombinant GFs. Moreover, we also show that its combination with microstructural cues synergistically drives stem cell differentiation toward tenogenesis in engineered constructs. Thus, this strategy not only holds potential to significantly improve tendon healing after injury, but its principles can also be applied to engineer different tissues.
References:
1. Teixeira, S.P.B. et al. Adv. Funct. Mater. 30, 1909011 (2020).
2. Teixeira, S.P.B. et al. Adv. Funct. Mater. 31, 2003934 (2021).
Acknowledgements: EU HORIZON 2020 for projects ACHILLES (H2020-WIDESPREAD-05-2017-Twinning-810850) and MagTendon (ERC-2017-CoG-772817); FCT/MCTES for scholarships PD/BD/143039/2018 (S.P.B.T.) under PhD-PATH (PD/00169/2013) and PD/BD/129403/2017 (S.M.B.) under PhD-TERM&SC (PD/59/2013), for project SmarTendon (PTDC/NAN-MAT/30595/2017), and individual contracts 2020.03410.CEECIND (R.M.A.D.) and CEECIND/01375/2017 (M.G.F.); Xunta de Galicia for postdoctoral grant ED481B2019/025 (A.P.).
Authors declare no conflicts of interest."
41883603688
The bone is a complex and dynamic tissue, in which the equilibrium between bone deposition and resorption can be perturbed by various pathological conditions, including bone metastases. Against them, no effective therapy has been developed yet, and available treatments are primarily palliative, aiming at restoring bone homeostasis. To improve the process of anti-metastatic drug discovery, new pre-clinical models are required, since available in vivo and in vitro models are limited by species specific differences in tumor mechanisms and by an oversimplification of the bone environment, respectively. Furthermore, potential side effects can be neglected with available models, resulting into unexpected toxicity of candidate drugs in clinical trials. In this scenario, advanced 3D in vitro models could become relevant assets for research and pharma industry for the discovery of new drugs against bone tumors, overcoming limitations of current models. To this end, our work aims at developing complex 3D in vitro models of bone tissue, taking into account its heterogeneous composition, to be exploited for the test of anti-metastatic drugs. To this end, we firstly developed microfluidic devices and millimeter-scaled vascularized bone models based on osteoblasts, osteoclasts, vascular cells and mesenchymal stromal cells embedded in a 3D hydrogel loaded with hydroxyapatite nanoparticles. We demonstrated that the simultaneous presence of all cell types and of the mineral component increased the bone turnover, as compared to simpler culture conditions. Then we added immune cells and breast cancer metastatic cells, showing that tumor cells were able to colonize the bone microenvironment, particularly in the perivascular niche. Furthermore, we were able to investigate the behavior of immune cells, when in co-culture with vascular and cancer cells within a bone-like environment. To test the effects of anti-tumor drugs in our system, we added rapamycin and doxorubicin, two FDA-approved anti-tumor drugs, known to have side effects on the vascular compartment, demonstrating how tumor cell resistance to the drugs was increased by the presence of a bone microenvironment. Furthermore, we were able to show the antiangiogenic effects of the drugs, by monitoring the damage to the microvascular network in the model. Finally, we tested our 3D bone model also with cells deriving from Ewing sarcoma, a pediatric bone tumor, showing that they could proliferate in our mineralized bone model. Sarcoma cell viability was affected by the knockdown of relevant genes and by the addition of oxidative stress inducers, in accordance with results shown in mouse experiments. Overall, we showed that the screening of anti-metastatic drugs in an in vitro model recapitulating the complexity of bone environment allowed to better estimate the effects of potential drugs both on their intended target and on other components of the microenvironment as compared to simpler models.
31451703655
"Development of novel bone biomaterials inevitably has a phase of in vitro testing for cytocompatibility and functional osteogenesis. This relies on defined conditions under which osteoblasts, or their precursors, are stimulated to drive a mature, matrix depositing osteoblast phenotype. Based on the in vitro results obtained, a selection is made to be further tested using in vivo studies. While this is the classically adopted route, the correlation between in vitro and in vivo results is not as strong as would be expected (1). This may not be surprising when considering the methods typically being used are decades old. This suggests there is room to optimize in vitro tests to obtain more translationally relevant data. Often overlooked is the desired route to new bone formation. Direct osteogenesis is favored when cells experience 2D environments, while embedding cells within a hydrogel leads to a 3D environment, that may be more favorable to indirect bone formation, or endochondral ossification. Which raises the question: should the culture media used in vitro be tailored to the exact bone forming mechanism desired? It has been suggested that 10 mM β-glycerophosphate typically used in osteogenesis medium can lead to spontaneous ectopic calcification, an artifact often observed when using cell free material. Classical dexamethasone containing osteogenic media also has the potential to drive adipogenesis. However, many studies typically only investigate markers associated with an osteogenic phenotype, potentially overlooking conflicting signals driving differentiation into other lineages. Taken together, this suggest that improvements are possible.
Therefore, studies testing novel biomaterials are increasing in complexity with the aim to develop more representative models and more accurately represent in vivo conditions (2).
Bioreactors can be used to improve flow and nutrient exchange, and coculture of cells can highlight interactions that may occur naturally in vivo but are lacking in monoculture studies. Increasingly, single cell sequencing is being adopted to study the differentiation of cells over time, identifying new markers and even distinct osteogenic differentiation pathways. This is key as the current range of markers may not be broad enough to make accurate predictions. Whole bone explant cultures can be combined with materials to investigate aspects such as osseointegration. As the body of knowledge increases, it is time to rethink how materials are tested
Combining bioreactors, coculture systems and improved markers and assays offers the opportunity to improve the accuracy of the in vitro results and reduce subsequent animal use as a result. While this may take some time, the outcomes will be rewarding.
"
41935603609
Introduction: Bone marrow is one of the most preferable sites for metastasis, but the complicated in vivo metastatic niches make it challenging to study cancer cell colonization. We took a novel approach to establish an in vitro complex bone marrow environment on a dynamic 3D culture system, Bone marrow (BM)-on-chip. With a specific focus on prostate cancer, we designed, fabricated, and performed an in vitro on-chip cell culture to compare the tumorigenic potential of PTEN-negative PC3 prostate cancer cells with or without intact hemidesmosomes (HD).
Methodology: The BM-on-chip is designed in a standardized 96-well microplate format with a lumen structure, yielding a high throughput platform that allows co-culture and real-time observation of the colonization process. The 3D microenvironment of the bone marrow niche is created by sequentially loading tdTomato-expressing MC3T3 osteoblasts and GFP-positive PC3 cells into collagen I-coated microchannels at days 0 and 7 respectively. The cells are supplemented by the bi-directional flow of medium through the microchannels. Using Leica SP8 Falcon confocal microscope in a live-cell chamber, the assessment of osteoblast and PC3 cells co-culture was done first on day 14, followed by the addition of 0.5 nM or 1 nM DTX into the culture medium. The next imaging was on day 21 to analyze the effect of DTX on cell survival. The areas of MC3T3 cells were compared by analyzing the image data using IMARIS x64 9.2.1 software.
Results: HD-deficient PC3 cells were generated by knocking out the expression of α6-integrin subunit (α6-KO) using CRISPR/Cas9-mediated gene editing. We found that PC3 cells attached on top of fibrillar-shaped osteoblast and formed relatively small foci while α6-KO cells formed much larger cell clusters that were tightly integrated into the osteoblastic structures. Interestingly, co-culture of PC3 cells with osteoblast caused the reduction of MC3T3 osteoblasts whereas this effect was not observed in co-cultures containing α6-KO cells. Next, the cells were treated with docetaxel (DTX) which is a drug commonly used in prostate cancer treatment. Comparative analysis revealed dose-dependent reduction of PC3 cells area and volume after 7 days of incubation with DTX. In contrast, PC3 α6-KO cells appeared relatively resistant to DTX treatment, possibly due to their tight integration into MC3T3 osteoblasts. These observations are in line with our mouse model and 2D cells analysis results showing increased metastasis and DTX-resistance of HD-deficient α6-KO PC3 cells.
Conclusion: Our data shows that the BM-on-chip model can be successfully used for functional analysis of osteoblasts-prostate cancer cells co-culture. It reveals that PC3 α6-KO cells readily colonize osteoblast niches where they show robust resistance to DTX treatment when compared with control PC3 cells.
73296334839
Intro: Our broader understanding of the immune system's role in determining the success of intrinsic repair mechanisms has led to increased research focus on immunomodulatory therapies1. These therapies could be particularly effective in abnormal fracture healing, where bone tissue engineering has thus far failed to provide safe and reliable clinical therapeutics. While current developments rely heavily on endpoint assessment of bone formation in pre-clinical studies, the effect on key preceding processes, including hematoma formation, and the immune response to fracture, are seldomly taken into account2.
Aim: The aim of this study was to engineer an innovative in vitro model that facilitates studying the link between the immune response and mineralization during fracture healing.
Methods: Collagen-hydroxyapatite (CHA) scaffolds were incubated with human whole blood, mimicking the fracture hematoma formed when the scaffold is implanted in vivo. The effect of blood-biomaterial interactions on the microarchitecture of the scaffold, as well as the production of inflammatory mediators (IL-1β, IL-4, IL-6, IL-8, IL-10, MCP1 and VEGF), was assessed. The response of human bone progenitor cells (HBCs) to the blood-biomaterial interactions was evaluated in terms of cell infiltration, proliferation, and mineralization in the scaffold.
Results & discussion: The interaction between blood and CHA scaffolds led to the infiltration of erythrocytes, monocytes, and platelets, and the formation of a fibrin network. The porous microarchitecture of the scaffold was maintained in the presence of blood, while its support for HBC infiltration was limited by the presence of blood. The scaffold stimulated a limited production of pro-inflammatory factors (IL-6 and IL-8) by blood cells. However, the blood-biomaterial interactions significantly upregulated the production of IL-6 and IL-8 by HBCs by a factor of 10. The production of pro-inflammatory factors was temporally regulated, peaking between day 1 and 5, and tapered off by day 28. While blood-biomaterial interactions had no impact on the proliferation of HBCs over 28 days, blood impacted their capacity to mineralize, with a 28% reduction in calcium quantification, and a 50% reduction in intracellular alkaline phosphatase activity. Taken together, these data indicate that a transient hematoma-like pro-inflammatory matrix can be recapitulated in vitro, which can then be used to assess the effect of blood-biomaterial interactions on downstream processes of fracture healing, including mineralization. On-going experiments are assessing the capacity of blood-biomaterial interactions to alter the drug release kinetics of rhBMP-7, a commonly used inducer of bone formation, while the effect of rhBMP-7 on fibrin network formation and its ability to steer the immune response will be evaluated. Ultimately, the capacity of blood-biomaterial interactions to modulate the osteoinductive effects of rhBMP-7 will be determined.
Conclusion: In this study, a unique platform to bridge the knowledge gap between the immune response and mineralization during fracture healing was engineered, while also taking into account blood-biomaterial interactions, representing a significant advancement over current in vitro models of the fracture hematoma.
Acknowledgements: The authors would like to thank the Orthoregeneration Network & Orthopedic Research Society (Kick-Starter Grant) for providing financial support to this project.
62825433305
Bone is a complex composite material, so any scaffold or model designed to reconstruct its integrity or model its behaviour must have a high degree of complexity and fulfil several requirements, including high biocompatibility, suitable surface and mechanical properties, adequate architecture, tailored degradability. Obtaining a bone-mimetic composition is also crucial, because hydroxyapatite (HA), the mineral phase of bone, dictates the behaviour of both healthy and tumour cells.
Here, for bone regeneration and modelling purposes, biomimetic nanostructured coatings are designed, manufactured by Ionized Jet Deposition (IJD) and deposited onto 3D printed metallic and polymeric scaffolds.
Coatings are obtained by ablation of biogenic hydroxyapatite, derived from bovine bone. Polymeric scaffolds are obtained by Fused Deposition Modelling of PLA filaments, while metallic scaffolds are obtained by selective laser sintering starting from Ti-6Al-4V powder.
Surface morphology (FEG-SEM, AFM), composition (GI-XRD, FT-IR, EDS) and stability profile in culture medium (immersion in alpha-MEM at pH 7.4 and FEG-SEM at 24h, 7 days and 14 days) are assessed, for the coatings. Then, coverage of the 3D printed coatings is optimized and the relevance of shadowing effects is evaluated.
Finally, the interactions between the coatings and cells (MSCs for bone regeneration and SAOS-2 for bone tumour modelling) is assessed, by studying their adhesion to the scaffolds, morphology, early proliferation and differentiation (for the MSCs).
Coatings have a submicrometric thickness, that can be selected by tuning deposition time, nanostructured surface morphology and biomimetic composition. Indeed, the nanostructured coatings are constituted by multi-doped carbonated hydroxyapatite (Na 0.28 ± 0.08, Mg 0.16 ± 0.01 wt%) and are constituted of nanoscale aggregates (diameter ~ 50 nm) grouping in clusters up to 2 microns diameter. A nominal thickness of 450 nm is selected. Upon exposure in medium, they progressively dissolve, but maintain stability for over 14 days. Upon deposition on the 3D printed scaffolds, nanostructured coatings grow on all the surface of the fibres without altering their shape or porosity and coat their entire surface with no shadowing effects.
In addition, they promote cells colonization of the whole scaffolds, different from controls, where cells tend to concentrate on the outer layers.
For bone regeneration, coatings dictate MSCs morphology and sustain early proliferation and differentiation towards an osteogenic lineage. For bone models, they permit optimal viability at early and late timepoints.
As a consequence, the developed coatings appear promising for applications in regenerative medicine and bone modelling.
ACKNOWLEDGEMENTS: Funding from 5 per mille provided to IRCCS - Istituto Ortopedico Rizzoli, and from the Euronanomed III project NANOVERTEBRA is acknowledged.
20941835526
A limitation, when it comes to 3D printed biomimetic structures with micrometer and sub-micrometer precision are computer aided design (CAD) programs. Existing CAD software is usually based on “manual” step-by-step design principles intended and suitable for subtractive and formative manufacturing methods rather than organic designs for additive manufacturing. The resulting structures can hence deviate strongly from their natural tissue counterparts and small design changes of complex objects usually result in time consuming workflows [1–4]. Alternatively, tissue imaging dataset derived designs have been used[3,5] to recapitulate native geometries accurately, but lack systematic variation and adjustment of individual design parameters[1].
Algorithmic design based on parametric and algorithmic modelling provides an alternative and allows to efficiently explore and optimize geometries based on a set of logical operations and user defined rules.[6–8] Algorithmic design algorithms can yield hierarchical branching patterns resembling those found in nature[4,8] and enable scalable design automation (e.g. scan to print).
To experimentally realize perfusable biomimetic microtissue, we designed an alveoli network using algorithmic design principles. This lead to a set of hollow alveoli surrounded by a capillary network (A). Both alveoli and capillaries can be contacted via distinct in- and outlets for cell seeding, medium perfusion and tidal ventilation. This algorithmic design approach allows for deliberate design permutations such as alveoli size, degree of vascularization and wall thickness (B). Using dip-in mode TPS printing,[9] the algorithmic design was printed both with acrylate-based resin and imaged with scanning electron microscopy (C, left), as well as with gelatin-based resin and imaged with two-photon fluorescence microscopy (C, right).
73296324244
Cancer, as a cause of death, is only surpassed by cardiovascular diseases. Thus, it is critical to achieve progress in its treatment and prevention. Given the complexity and heterogeneity of cancer, various therapeutic targets are being investigated, including components of the tumor milieu. The tumor microenvironment (TME) consists of several types of cells (vascular cells, tumor-associated fibroblasts, immune cells, mesenchymal stem cells, and adipocytes) embedded in extracellular matrix soaked by interstitial fluid rich in soluble factors secreted by cells [1]. Increasing evidence indicates that tumor progression depends on the interaction between the tumor and its microenvironment and that the effectiveness of anti-cancer therapies is modulated by changes in the TME [1-3]. Therefore, extensive research efforts are devoted to investigating the spatial organization of the native TME and to build in vitro models of the TME using three-dimensional (3D) bioprinting [4] and tissue-on-a-chip techniques [5].
In this work, we report the 3D bioprinting of avascular structures that recapitulate several features of the TME [6]. In our model, the tumor is represented by a hydrogel droplet uniformly loaded with breast cancer cells, whereas the microenvironment is modelled by rings of hydrogel loaded with peritumoral cells: tumor associated fibroblasts and peripheral blood mononuclear cells. The tumor cells used in our experiments came from a commercial cell line (SK-BR-3), while the peritumoral cells were obtained from breast cancer female patients in different carcinoma stages. The cells were embedded in CELLINK Universal Bioink at concentrations of 1 million cells per milliliter, and the tumor models were fabricated using extrusion bioprinters (INKREDIBLE and BIO X, CELLINK, Sweeden). For the optimization and precise control of the printing process, we developed in-house Python scripts able to generate the G-code instructions for the two bioprinters based on the geometries of the digital models. Our workflow was designed to permit the subsequent bioprinting of desired constructs on multiwell plates of different dimensions. After two weeks of in vitro culture, histological cryosections of the tumor models showed that the hydrogel used in this study was appropriate for sustaining cell growth and proliferation. When tumor models were implanted subcutaneously, in the dorsal region of CD1 Nu/Nu immunosuppressed mice, within 28 weeks in vivo they became vascularized and grew about 5 times in diameter. In conclusion, our work presents a reliable methodology for building models of the TME using extrusion bioprinting. Such models can be used for fundamental research or, if built from patient-derived cells, for testing the effectiveness of anti-cancer therapies, thereby contributing to personalized treatment plans.
References:
[1] Turley, S.J., Cremasco, V., Astarita, J.L., Nat. Rev. Immunol. 15, 669–682 (2015)
[2] Shieh, A.C., Ann. Biomed. Eng. 39, 1379–1389 (2011)
[3] Privat-Maldonado, A., Bengtson, C., Razzokov, J., Smits, E., Bogaerts, A., Cancers 11, 1920 (2019)
[4] Li, J., Parra-Cantu, C., Wang, Z., Zhang, Y.S., Trends Cancer 6, 745–756 (2020)
[5] Shang, M., Soon, R.H., Lim, C.T., Khoo, B.L., Han, J., Lab Chip 19, 369–386 (2019)
[6] Bojin, F., et al., Micromachines 12, 535 (2021)
73296318819
Bioinspired engineered microenvironments provide cells with a holistic “instructive niche” that offers the adequate entourage for cellular control both in space and time. Biomaterials provides essentially surface signals. We hypothesise that volume characteristics of biomaterials have a negligible influence on cell-behaviour and new tissue formation as compared with interfacial mechanical, topographic or biochemical properties. We explore this problem using minimalistic approaches, emplying scaffoldess strategies or by exposing cells to biomaterials that are designed to maximise the area to the volume. Examples from our group are presented, including: (i) a new concept of cell fiberoids, where fibres made of cells are engineered without any biomaterials; (ii) cell-sheets and cell-stamps obtained by magnetic force based-tissue engineering; (iii) cell encapsulation in liquified capsules with thin biomaterials shells for the autonomous development of microtissues; or (iv) ultrathin cell carriers with controlled geometry, and cell caps obtained by surface cell engineering. Such elements can be used as building blocks to be assemble into large constructs to produce macroscopic tissues using bottom-up tissue engineering methodologies.
Vasculature plays an essential role in skin physiology and its architecture and function are altered in aged and diseased skin. Quite remarkably, papillary and reticular dermis show very distinct extracellular matrix (ECM) and vascularization. Furthermore, fibroblasts freshly isolated from their native microenvironment have different gene expression patterns, morphology and proliferation rate. Whereas 2D culture or embedding in hydrogels has been used to characterize both fibroblast subpopulations, the lack of relevant models has hindered investigation of their contribution to vascularization. We thus cultured human papillary and reticular fibroblasts as cell sheets over two to three weeks and used RNA-seq differential expression analysis to identify genes involved in microenvironment generation. Bioinformatics analysis revealed that cell sheet culture maintains specific expression of matrisome gene signatures resulting in papillary and reticular ECMs that differ in composition and structure. The transcriptomic also revealed layer-specific expression of angiogenesis-related genes. The impact of secreted and ECM-bound factors was then assessed using two independent 3D angiogenesis assays. The first assay consisted in stimulating endothelial cells embedded in a fibrin gel with conditioned media from each fibroblast subpopulation, while the second assay was based on cell-sheet co-culture of fibroblasts and endothelial cells, thus allowing direct interactions of endothelial cells with the microenvironment generated by fibroblasts. Vascularization was analysed in 3D using in-house developed software (3D-skel; Atlas et al, 2021) with both assays. These analyses revealed that papillary fibroblasts secrete highly angiogenic factors and produce a microenvironment characterised by ECM remodelling capacity and formation of dense small vessels, whereas reticular fibroblasts produced more structural components of the ECM associated with less but larger vessels. These features mimick the characteristics of both the ECM and vasculature of native dermis subcompartments. In addition to showing that skin fibroblast populations differentially regulate angiogenesis via both secreted and ECM factors, our work emphasizes the importance of papillary and reticular fibroblasts for tissue engineering and modelling dermis microenvironment and its vascularization.
Atlas Y, Gorin C, Novais A, Marchand MF, Chatzopoulou E, Lesieur J, Bascetin R, Binet-Moussy C, Sadoine J, Lesage M, Opsal-Vital S, Péault B, Monnot C, Poliard A, Girard P, Germain S, Chaussain C, Muller L. Microvascular maturation by mesenchymal stem cells in vitro improves blood perfusion in implanted tissue constructs. (2021). Biomaterials. 268: 520194
52354554306
Introduction: Atopic dermatitis (AD) is a chronic inflammatory and common skin disorder that is frequently associated with other atopic diseases such as allergic rhinitis and asthma. The immunopathogenesis of AD is a complex process, as both innate and adaptive immune systems are involved in the development of eczema in those patients. Keratinocytes, mast cells, dendritic cells, and T cells, among others, are involved in skin inflammation, however, in AD, there is a tendency towards T-helper 2 (Th2) responses. There is not a complete cure for AD, and most of the current treatments focus on symptom relief. Therefore, new treatments to improve the long-term control of AD are necessary. Murine models have been developed to mimic aspects of AD pathophysiology; however, they usually show markedly different responses to drug treatments that may lead to high failure rate of drug development.
Methodology: Purified peripheral blood human monocytes were differentiated into monocyte-derived dendritic cells (Mo-DC) using GM-CSF and IL-4. Purified naïve CD4+ T cells were CD3/CD28 activated, stimulated with IL-2 and polarised into Th2 cells using IL-4 and anti-IFNγ. Cells were characterised by qPCR and flow cytometry for key cell markers. To develop in vitro human skin models, type I collagen matrix populated with human dermal fibroblasts were seed on the apical surface with N/TERT immortalised human skin keratinocytes and Mo-DC, and cultured at an air-to-liquid interface before analysis. Th2 cells were incorporated into the dermal component.
Results: Mo-DC displayed successful differentiation by expression of cell-specific markers including CD1a, CD11c and CD207, and also high levels of proinflammatory markers such as IL-18 in response to allergens. Th2 cells showed increased CD119, D154, CD4 and CCR4, and secreted increased levels of IL-4, IL-5, IL-6, IL-13 and thymic stromal lymphopoietin (TSLP). Tissue-engineered skin models based on dermal fibroblasts and keratinocytes showed a keratinised, stratified squamous epidermis on top of a well-populated fibroblast containing dermis that histologically mimicked human skin. T cells were successfully incorporated into the dermis and Mo-DC into the epidermis, as determined by histological analysis.
Conclusion: Multiple immune cells can be differentiated from peripheral blood human monocytes and cultured in a 3D environment together with other human skin cells. The next aim is to show immune cell functionality within a 3D model in response to human allergens.
62825462739
Introduction:
Stromal vascular fraction (SVF) cells, isolated from adipose tissue are an abundant easily accessible stromal cell source for bone tissue engineering. These characteristics make them a good alternative to bone marrow derived mesenchymal stem cells (BM-MSCs). Previous studies from our group provided a proof-of-concept that Adipose-derived Stromal Cells (ASCs), resulting from the expansion of SVF-cells, can generate bone tissue through endochondral ossification (ECO) by forming hypertrophic cartilage tissue (HCT) in vitro which in turn remodels into bone tissue when implanted in vivo. However, little is known about the underling mechanisms of ASCs chondrogenesis and their in vivo remodeling process.
In this work, we hypothesized that (i) freshly isolated SVF-cells are better suited for generating mature HCTs than expanded ASCs (P0-ASCs) and (ii) that more mature HCTs are more prone to remodel into bone tissue in vivo.
To that aim, we assessed the effect of SVF-cells monolayer expansion and its consequences on the proteomic signature and chondrogenic potential. In addition, we sought out to determine which HCT-properties can predictively determine the in vivo bone forming capacities of ASCs.
Methodology:
SVF-cells or P0-ASCs (0.5x106) were first seeded onto collagen sponges (V=40 mm3) and then maintained in chondrogenic cultures for 3, 4, 5 or 6 weeks. The cartilage maturation of these adipose-derived HCTs (A-HCTs) was evaluated qualitatively by histological staining and quantitively using Elisa and compared to the ones generated by BM-MSCs (BM-HCTs). Next, these HCTs were implanted ectopically in nude mice for 12 weeks to evaluate their bone forming capacities. In addition, the proteomic profiles of SVF-cells and P0-ASCs were compared by mass spectromer.
Results:
SVF-cells from all tested donors formed mature HCT within 3 weeks whereas P0-ASCs needed at least 4 to 5 weeks (depending on the donor). Longer in vitro differentiation increased the degree of maturation of HCTs which was characterized by a denser cartilagenous matrix and more mineralization. Interestingly, the HCTs degree of maturation obtained in vitro was indicative of their bone forming capacity in vivo, with an optimal bone remodeling obtained not for the highest degree of maturation but for an average degree of maturation. In fact, excessive in vitro maturation resulted in mineralized tissue rather than bone in vivo. In contrast, insufficient maturation resulted in major scaffold resorption in vivo. A-HCTs showed more mineralization, higher content of IL-10 and TNF-alpha relative to BM-HCTs. However, A-HCTs formed mature bone organ in vivo similarly to BM-HCT when suitably matured in vitro. When looking at their proteomic profile, P0-ASCs presented a different profile to the ones of the SVF-cells; most notably regarding energy related pathways such as glycolysis, TCA-cycle and lipid metabolism.
Conclusion
Our data showed that SVF-cells possess a superior chondrogenic potential compared to P0-ASCs. In addition, we were able to not only control the degree of maturation of the HCTs but also modulate their in vivo fates.
Perspective:
We will assess whether a more physiological culture environment would better preserve the specific metabolic status of SVF-cells and thereby better retain their differentiation potential when expanded in vitro.
62825458959
Our newly developed self-feeding hydrogels with enzyme-empowered degradation capacity have demonstrated high biological performance in-vitro and in-vivo as a novel self-maintained and biocompatible 3D scaffold1. Photo-crosslinkable platelet lysates (PL)-based hydrogels have exhibited to support distinct human-derived cell cultures owing to their high content of bioactive molecules, such as cytokines and growth factors2. To take advantage of all features of both PL and self-feeding hydrogels, here we combined UV responsive laminaran-methacrylate (MeLam) and PL-methacrylate (PLMA) derivatives plus glucoamylase (GA) to fabricate a multicomponent hybrid hydrogel (GLMPL). This hydrogel emerged as an unique scaffold due to the combination of sustained delivery of glucose produced via enzymatic degradation of laminaran and granting cell adhesin by presence of PL. Besides, this biomaterial was also applied to fabricate high-throughput freestanding microgels with controlled geometrically shapes. Impressively, such multicomponent hybrid hydrogel was successfully implemented as a glucose supplier bioink to fabricate complex and well-defined cell-laden structures using a support matrix.
MeLam and PLMA were synthesized following the previous reports2,3. In order to obtain a gradual production of glucose over time, GA enzyme was incorporated into MeLam/PLMA mixtures before UV exposure. We applied superhydrophobic surfaces (SH) patterned with wettable shaped domains (SL)4, where suspensions of cells and GLMPL hydrogel precursor could be dispersed and microgels with different geometries were produced after UV irradiation. To demonstrate the outstanding bifunctionality of such bioink, a mixture of GLMPL hydrogel precursor and cells suspension were deposited into agarose support matrix by extrusion 3D-printer. Thereafter the printed structures were exposed to UV to form gels. In vitro studies were performed on these biomaterials by encapsulating Human adipose-derived stem cells (hASCs) cultured in glucose free Dulbecco's Modified Eagle Medium. As such, any difference in cells response could then be attributed directly to the presence of enzyme and consequently glucose accessibility for the encapsulated cells.
CellTiter-Glo assay has shown hASCs metabolic activity significantly increased in GLMPL hydrogels over 21 days. Moreover, pronounced cell proliferation was confirmed through DNA quantification. Live-Dead assay confirmed encapsulated hASCs stretched inside the GLMPL hydrogel. DAPI/phalloidin staining have approved that cells elongated in GLMPL hydrogels and formed interconnected networks with neighbouring cells. Live-dead assay showed that up to 7 days of culture, most of the hASCs remained viable and elongated inside the microgels and 3D printed structures. It is noteworthy to mention that freestanding microgels and bioprinted scaffolds showed well-preserved architecture during the culture.
In conclusion, these results, combined that most current bioscaffolds suffer from lack of nutrient diffusion and adhere motifs, clearly suggest the potential of this multifunctional hybrid hydrogel in future developments of 3D structures in a wide range of biotechnological applications as an autonomous cell supporting system.
Authors acknowledge the project CICECO-Aveiro Institute of Materials UIDB/50011/2020 & UIDP/50011/2020, SFRH/BD/143883/2019 and CEECIND/02713/2017.
References
1. Zargarzadeh, M., et al., Materials Horizons (2022).
2. Santos, S.C., Custódio, C.A. & Mano, J.F, Advanced Healthcare Materials 7, 1800849 (2018).
3. Custódio, C.A., Reis, R.L. & Mano, J.F. , Biomacromolecules 17, 1602-1609 (2016).
4. Neto, A.I., et al., Advanced Materials 28, 7613-7619 (2016).
83767222599
Background:
Urethral reconstruction is performed in patients with urethral strictures or for correction of congenital disorders. In most instances, foreskin or buccal mucosa flaps are used in these surgeries. However, complications may occur due to limited availability of tissue. In the future, tissue engineering (TE) might offer alternative solutions as it enables a detailed design to closely mimic native tissue, and provides opportunities for creating a scaffold for urethral reconstruction. Here we describe the cellular responses to fluidic flow of cell types that have the potential to be used in urethral TE.
Methods:
Human umbilical vein endothelial cells (HUVECs), and bladder and urethra derived epithelial cells (primary cells, isolated from male pigs by PdG) were exposed for 72 hours to fluidic, causing shear stress of τmax= 10.0 dyn/cm2 and τmax= 20.0 dyn/cm2 in a ibidi flow system. Cell elongation, cell alignment and actin fiber alignment were analyzed.
Results:
Bladder and urethra derived epithelial cells elongate and align when exposed to fluidic flow induced shear stress similar to endothelial HUVEC cells. Despite their different origin [1], we could see no differences between bladder and urethral epithelial cells in the flow experiments.
Conclusion:
Both bladder and urethral epithelial cells similarly adapt to fluidic flow. So in this respect, both cell types could potentially be used in TE for urethral reconstruction. This is important information, because the harvesting of bladder epithelium is much easier than of urethra epithelium: bladder epithelial cells can be isolated from urine or bladder washout or may be obtained by biopsy, in contrast to urethra epithelial cells. Next steps in our approach would be creating intermitted flow to mimic voiding patterns in patients
[1] de Graaf P, van der Linde EM, Rosier PFWM, et al. Systematic Review to Compare Urothelium Differentiation with Urethral Epithelium Differentiation in Fetal Development, as a Basis for Tissue Engineering of the Male Urethra. Tissue Eng Part B Rev. 2017;23(3):257-267.
73296312607
Introduction
Tendon injuries are common in equine athletes. Tissue healing occurs via biomechanically inferior scar tissue deposition, often resulting in re-injury. This is mediated, in part, by excessive proinflammatory cytokine release in the acute stages following injury. Stimulating equine tenocytes with interleukin 1 beta (IL-1β) alters tendon-associated and extracellular matrix remodelling gene expression in 2D culture and impairs 3D collagen gel contraction1. Similar findings were reported in humans tenocytes2, with activation of the NF-κB pathway suggested to facilitate these effects1,2. However, it is unknown how IL-1β modulates global gene expression in equine tenocytes in 3D culture and whether pharmacological NF-κB inhibition can attenuate the deleterious effects of IL-1β stimulation.
Methodology
Five biological replicates of adult tenocytes isolated from the equine superficial digital flexor tendon were cultured in a 3D collagen gel for 14 days under control or IL-1β-stimulated (1 nM) conditions, with media changes every 3-4 days. Daily gel contraction was examined before cells were harvested for RNA at day 14. Transcriptomic analysis was performed on a NovaSeq6000 platform, utilising Salmon-based alignment3 and differential expression analysis with DESeq2 (padj<0.05 and LogFC>1)4. Validation of several up-and-down regulated genes was undertaken with in-house qPCR. Gene Ontology and pathway analysis was performed with Panther Slim5 and Enrichr6, respectively. The interleukin 1 receptor antagonist (IL1Ra) protein1 and the NF-κB inhibitors JSH-237, IMD03548, and PF-066508339 were administered to tenocytes with-or-without IL-1β (1 nM); we are currently determining the rate of rescue for 3D collagen gel contraction, NF-κB P65 protein cytosol-nuclear shuttling after 60 min stimulation in 2D, gene expression after 72 hr in 2D, and IL-6 protein secretion with ELISA after 72 hr in 2D and during 14 days in 3D culture.
Results
Of the 18435 mapped genes, 2517 were differentially expressed following IL-1β exposure; 954 genes were upregulated and 1563 were downregulated. Gene ontology and pathway enrichment revealed that IL-1β enhanced NF-κB - but not JNK, P38, ERK, or STAT – signalling. Furthermore, IL-1β inhibited extracellular matrix organisation. IL1Ra fully rescued collagen gel contraction when co-administered with IL-1β, but only partially rescued tendon-associated and matrix remodelling gene expression at day 14. JSH-23 attenuated P65 nuclear translocation, reduced IL-6 secretion in 2D, and rescued matrix remodelling - but not tendon-associated - gene expression in 2D. Conversely, collagen gel contraction was not impacted by JSH-23 in the presence of IL-1β. Additional NF-κB inhibitor work is ongoing.
Conclusion
Exogenous IL-1β promotes NF-κB transcriptional signalling in equine tenocytes in 3D culture. These findings were verified with enhanced IL-6 secretion, a known target of P65. JSH-23 rescued IL-6 secretion and matrix remodelling gene expression after 72 hr in 2D, but had no influence on collagen gel contraction during 14 days in 3D culture. As IL1Ra fully rescued collagen gel contraction, this suggests a more global attenuation of NF-κB signalling is required to restore tenocyte function as other NF-κB proteins (i.e., c-REL, P50) may target tendon contraction genes. This will be addressed with the ongoing IMD0354 and PF-06650833 experiments.
62825403004
Introduction: Fecal incontinence has a high impact on patient quality of life. Available treatments based on surgical and non-surgical approaches range from change in diet, to bowel training or sacral nerve stimulation, none of which represent a long-term solution. Novel therapies are emerging that aim to regenerate the sphincter muscle and, therefore, restore continence. These approaches usually consist of administering a suspension of previously expanded cells to the damaged tissue. This strategy often results in a reduced cell viability due to the harsh step of cell harvesting from the culture platform in which cells were expanded, as well as the unnatural way the unattached adherent cells are delivered as a suspension.
Methodology: Here, we propose a new strategy for the treatment of fecal incontinence, by means of a two-step process. First, skeletal muscle cells (SkMCs) are expanded under static and planar culture conditions until relevant cell numbers are reached. The expanded SkMCs are then combined in bioreactors with implantable, biocompatible and biodegradable polymeric microcarriers, prepared by thermally induced phase separation (TIPS). Different bioreactor culture scenarios were tested: (1) SkMCs from different commercially available sources vs from primary muscle samples from different patients, (2) vertical wheel bioreactor (VWBR) vs spinner flask, (3) xeno(geneic)-free vs non-xeno-free conditions, (4) culture time, (5) agitation scheme. These parameters were optimized to maximize cell adhesion efficiency. Cell viability (calcein and DAPI staining) and distribution throughout the microcarriers, presence of the CD56 myogenic marker (flow cytometry), cell differentiation potential (desmin staining to assess myotube formation) and cell migration from the microcarriers were also assessed.
Results: The optimized adhesion process allowed us to obtain a 70-80% efficient SkMC adhesion onto the TIPS microcarriers. This was achieved by applying an intermittent agitation scheme to patient-derived SkMCs adhered to the microcarriers in VWBR, under xeno-free conditions, after 24h. SkMCs maintained their myogenic features (expression of CD56 marker) after the expansion phase in planar systems under static conditions, as well as after adhesion and culture in the microcarriers. SkMCs were able to migrate from the microcarriers and differentiate into multinucleated myotubes, as well as maintaining high cell viability throughout the process.
Conclusion(s): By optimizing the choice of bioreactor, as well as its operating conditions, we were able to obtain a high percentage of viable SkMCs adhered to TIPS microcarriers, with relevant muscle regeneration potential. Additionally, by performing the entire cell adhesion process under xeno-free conditions, we avoid the use of fetal bovine serum, which addresses regulatory issues. The xeno-free conditions established for the cell-microcarrier combination, associated to the single-use feature of the bioreactor selected, make this process more amenable to GMP compliance. The use of implantable microcarriers should also greatly increase the likelihood of success of the proposed cell-based therapy, as it avoids the drawbacks associated with harvesting of SkMCs and their subsequent delivery in an unattached state to the damaged tissue.
The AMELIE project has received funding from the European Union's Horizon 2020 research and innovation programme under grant agreement No 874807.
94238169579
Introduction:
Regenerative Medicine is a relatively new field found at the intersection of science, engineering and medicine. Researchers in this field are traditionally biologists, chemists, materials scientists, data scientists, engineers or physicians who have acquired skills and knowledge beyond their basic training and stepped into the rapidly evolving field of TERM. Many of them, however, still find themselves approaching their research questions from the perspective of their own traditional discipline finding it difficult to adopt the much needed multidisciplinary approaches. Having recognized the need for a new researcher profile that would receive basic training in multiple disciplines, at Maastricht University we have taken the initiative of designing and developing an undergraduate program focusing on Regenerative Medicine and Technology. Within this program we aim to educate a new generation of researchers that will be able to swiftly adapt to any area of TERM, and contribute to the design and development of medical therapies, products and devices for research and clinical use.
Methodology:
A multidisciplinary Curriculum Committee and a broader Consultative Committee were assembled with the goal of developing an outline of a possible curriculum for the envisaged program using the principles of backward chaining. A total of nine Developing Groups have been installed to develop and constructively align the first two years of education keeping the CCCS (Constructive, Contextual, Collaborative, Self-Directed) learning principles in mind.
Results:
Applying the principles of problem- and research-based learning our program intends to offer a solid science and engineering foundation which would be strongly integrated with the relevant aspects of medicine. To this end, the desired competencies have been defined and in turn translated to year intended learning outcomes and a three-year curriculum. The first year offers a strong foundation in science, engineering and regenerative medicine. The second year is intended to focus on application of the acquired foundation in TERM. An example of this is a real-world student research project defined by clinicians within the MUMC+ university hospital. In addition, special attention will be paid to the development of translational science skills necessary to bring new therapies and products onto the market. We also envisage a close collaboration with the industry to bring in real-life success and fail stories. The third year would offer a minor and a semester-long research graduation project in TERM. The program aims to integrate the perspectives and input of the TERMIS-EU community and holds the ambition to catalyze the creation of an international network in education.
Conclusions:
The design and development of a new undergraduate program with the focus on TERM has been initiated at Maastricht University. The first ideas on the competencies and final qualifications have been translated into a curriculum outline draft. The education will be developed in further detail keeping the principles of constructive alignment and the CCCS learning principles in mind. The subsequent steps to be taken are the preparation of a macro-efficiency file and, in case of a positive evaluation, also an initial accreditation procedure. The program’s envisioned start date would then be September 2023.
62825438124
TBA
Human mesenchymal stem/stromal cells (MSCs) are a therapeutically relevant, heterogenous cell entity with immunomodulatory and pro-regenerative potentials. Apparently, MSCs mediate a huge proportion of their therapeutic effects via extracellular vesicles (EVs). Connected to several advantages in using cell-free products for the therapeutic setting, MSC-EVs emerged as promising novel therapeutic agent for various diseases, including graft-versus-host disease (GvHD), ischemic stroke, COVID-19 and sepsis.
It is our current mission to optimize the MSC-EV production strategy in a scaled, GMP compliant manner, and to set up an appropriate quality control platform to translate MSC-EVs into the clinics. One of the challenging aspects in this context is inherited from the MSC field, i.e. contradictory reports on the efficacy of MSC therapies. Apparently, not all MSC products mediate therapeutic effects when applied into patients. Similarly, we observe functional differences among independent MSC-EV preparations; even when same MSC stocks were used as starting material. Thus, to avoid draw backs as they occurred in the MSC field by failing to show efficacy in a phase III clinical trial for GvHD treatment, it is one of our most important missions to address the heterogeneity aspect by establishing appropriate read outs and set up strategies for allowing scaled and reproducible manufacturing of potent MSC-EV products.
94355106237
In the last years, the role played by extracellular vesicles (EVs) in inter-cellular and inter-organ communication through the delivery of signal molecules has been revealed and, nowadays, it is considered to be of utmost importance. EVs are secreted by almost all cell types and have an important role in several research fields ranging from oncology to immunology and diagnostics to regenerative medicine. However, the therapeutical employ has not yet been established, although their enormous biological potential is well known. The major limitation to EVs clinical use is the inability to localize the in vivo benefits into strategically defined sites of interest to avoid side effects. Numerous recent discoveries have shown how injectable hydrogels can be used for biomedical applications in regenerative medicine. Specifically, hydrogel-based drug delivery systems are found to be more efficacious than the conventional systemic administration.
Based on these premises, we proposed a new strategy to harness stressed endothelial cell-derived EVs and their angiogenic cargo in injured tissues. EVs, derived from human endothelial cells were collected and used as bioadditives for Gelatin Methacrylate (GelMA) formulation and functionalization. Our results showed that 3D bioprinted structures loaded with EVs support the formation of a functional neovascular network in-situ, constituted of perfused microvessels recapitulating the print pattern. In addition, we assessed the EVs-GelMA effects on cardiac remodeling after acute myocardial infarction (AMI). For this purpose, the EVs-GelMA was injected and polymerized into the ischemic ventricular cardiac wall after the surgical induction of AMI. Results showed improved cardiac performance and a reduction of the ischemic area with partial revascularization of the cardiac wall in the treated animals. Taken together, these findings support the development of new advanced regenerative applications for the revascularization of ischemic organs and peripheral tissues.
41883634604
Introduction: Human cord blood (CB) represents a rich source of several stem cell (SCs) types including hematopoietic stem and progenitor cells (HSPCs). Thus, clinical application of CB cells has become an alternative for the bone marrow transplantation. However, successful application of CB-HSPCs in adult patients requires the development of effective strategies improving their ex vivo expansion, homing and regenerative potential. One of the promising approaches for enhancement of SCs functionality includes their treatment with extracellular vesicles (EVs), which were shown to harbor and transfer bioactive content. Thus, in our study, for the first time we have evaluated an impact of human induced pluripotent SCs (hiPSCs)-derived EVs (hiPSC-EVs) on selected functions of CB-HSPCs, important for their hematopoietic potential in vitro and in vivo.
Methodology: hiPSC-EVs were harvested from media collected from feeder-, serum- and xeno- free cultures of hiPSCs by sequential ultracentrifugation. Next, CD45dimLin-CD34+ cell fraction enriched in HSPCs was isolated from CB by magnetic- (MACS) and fluorescence-activated cell sorting (FACS) and further expanded in dedicated serum- free media. Subsequently, we evaluated the influence of hiPSC-EVs on several biological and functional properties of CB-HSPCs in vitro and in vivo.
Results: Our results revealed that hiPSC-EVs may transfer their bioactive content and improve functional properties of CB-HSPCs including metabolic activity, hematopoietic and clonogenic potential, as well as survival, chemotactic response to stromal cell-derived factor 1 (SDF-1) and adhesion to the model components of hematopoietic niche in vitro. Importantly, hiPSC-EVs enhanced homing and engraftment of CB-HSPCs in vivo. Additionally, we have demonstrated that the treatment with hiPSC-EVs may activate signalling pathways in CB-HSPCs on both gene expression and the protein level.
Conclusion: In conclusion, our findings suggest that the “priming” with hiPSC-EVs may improve several functions of CB-HSPCs important for their homing and hematopoietic activity following the transplantation. These results support the new concept envisioning hiPSC-EVs as next-generation tools that may enhance future applications of CB in hematology.
31412704386
"INTRODUCTION: Bronchopulmonary Dysplasia (BPD) is a life-threatening disorder affecting premature newborns, for which no definite cure is available1,2. Lung fibrosis is one of the main problems that affect young patients. The aim of this work was to investigate the mechanism of action of human extracellular vesicles (EVs) both in vitro and in an animal model of hyperoxia-induced BPD. Specifically, we evaluated the effects of EVs on the development of fibrosis and on functionality of lung epithelial cells.
METHODOLOGY: GMP-grade EVs were produced by human Wharton-Jelly derived MSCs (Exo Biologics, Belgium), isolated by tangential flow filtration and characterized according to MISEV2018. Rat pups were divided in 3 groups: normoxia + PBS vehicle (control group), hyperoxia with PBS (untreated), hyperoxia with MSC EVs in PBS (treated). Both PBS and EVs were injected intratracheally (IT) on day 3, 7 and 10 and pups were sacrified on day 14. The expression of the genes TGFβ1 and aSMA was analysed in lungs. To evaluate epithelial secretory function, the expression of glycosaminoglycans (Alcian blue staining) and of surfactant protein C (SFTPC) was analyzed by histology and immunofluorescence. Collagen deposition was assessed by Sirius Red staining. Macrophages from rat bone marrow were treated with TGFβ, cultured and analyzed for aSMA and CD90 expression by flow cytometry.
RESULTS: Pups under hyperoxia exhibited an increase in collagen deposition in the lungs. This parameter was reduced by treatment with MSC EVs. The area of lung tissue expressing glycosaminoglycans was significantly increased in MSC EV-treated rat pups in respect to untreated animals. In addition, cells expressing SFTPC were significantly increased in MSC EVs treated pups with respect to the untreated group. In vitro, MSC EVs suppressed the induction of aSMA expression in macrophages.
CONCLUSIONS: Intratracheal administration of clinical-grade MSC-EVs counteracts the development of fibrosis and improve pulmonary epithelial function in a neonatal model of hyperoxia-induced lung injury. These results can contribute to understand the mechanism of action of these nanoparticles in preventing the development of BPD.
1- Porzionato, A. et al., Am J Physiol Lung Cell Mol Physiol 316: L6–L19, (2019)
2- Hansmann, G. et al., Pediatric Research 89:446 – 455, (2021)"
41883644586
In regenerative medicine, extracellular vesicles (EVs) have been increasingly studied as alternative acellular therapies overcoming the limitations of cell-based strategies. Derived from mineralising osteoblasts, EVs demonstrated their osteogenic potency suggesting their potential as a novel bone regenerative therapy. However, the clinical translation of EVs remains limited by issues associated with the scalability, reproducibility and purity of these naturally-derived nanoparticles. In this study, the characterisation of mineralising-osteoblast-derived EVs (MO-EVs) was performed to inspire the development of osteogenic synthetic EVs.
EVs were collected from cultures of mineralising osteoblasts over a 2-week period and the EV-isolation was performed by ultracentrifugation. Subsequently, the size, ζ-potential, morphology and particle concentration of these nanovesicles was characterised and the presence of tetraspanin markers (CD9, CD63 and CD81) was confirmed using nano-flow cytometry. Furthermore, the pro-osteogenic capacity of MO-EVs was assessed in vitro via quantifying alkaline phosphatase (ALP) activity and calcium deposition. From the composition analysis of MO-EVs, bio-inspired proteoliposomes harboring ALP and/or annexin VI were formulated via the thin-film hydration method followed by extrusion. Both the activity of the proteins post-insertion and their resulting incorporation efficiency in proteoliposomes were then determined. Additionally, cell-derived nanovesicles (CDNs) were produced by the serial extrusion of mineralising osteoblasts and the resulting synthetic EVs were similarly characterised.
The isolation of MO-EVs was validated as positivity for all tetraspanin markers was reported for these sub-100 nm vesicles. Notably, their osteogenic potency was confirmed in vitro on osteoblasts as MO-EVs increased significantly ALP activity, calcium deposition and collagen production after a 2-week treatment. MO-EVs were found enriched in several annexin proteins which guided the formulation of bio-inspired proteoliposomes. The insertion of both ALP and annexin VI was successful with >30% incorporation efficiency obtained for all formulations. Importantly, EV-inspired liposomes harboring annexin VI or ALP were found to be functional with the validation of the mediation of Ca2+-influx by annexin VI inside proteoliposomes and the confirmation of ALP enzymatic activity. Moreover, CDNs were also successfully produced as a nanoparticle population with an EV-size was obtained after serial extrusion. Both EV-inspired proteoliposomes and CDNs’ osteogenic potencies were then compared to MO-EVs after 14 days in osteogenic conditions.
Taken together, these results shows the potential of the development of synthetic EVs as biomimetic nanocarriers to accelerate the clinical translation of EV-based therapies for bone regeneration.
20941834084
Spinal cord Injury (SCI) is a life changing event with a high number of new cases reported every year. The most common cause of SCI comes from traumatic events, such as traffic accidents, falls, violence and sports activities, while the non-traumatic events (tumours, neurodegenerative and infectious diseases) are less prevalent. The injury incurred in the spinal cord tissue triggers several pathophysiological events which cause damage and supress axonal growth in the spinal cord tissue. These events consist of activation of apoptotic pathways, the release of inflammatory cytokines and the formation of a glial scar that primarily contains further damage, but also releases biomolecules that inhibit axons outgrowth. It affects nerve fibres passing through the lesion site causing motor dysfunction and altered sensation, causing incapacitating conditions to SCI patients. In this work we suggest a new multidisciplinary approach, combining the value of Adipose Tissue Derived Stem Cells (ASCs) secretome and Electrical epidural stimulation (EES). ASCs secretome was reported to promote regaining of function after SCI in a mouse model, likely promoted by the factors present in it which include anti-apoptotic and angiogenic factors, neuroprotectants and immunomodulators which may prime the unfavourable environment created upon SCI to a more neuroprotective/regenerative one. These benefits combined with the EES, which promote spinal cord plasticity, central pattern generator activation and stepping initiation is expected to improve functional gains after SCI. In an organotypic model of ex vivo spinal cord, we have observed that the secretome from ASCs is a potent modulator of axonal growth and inflammatory cells migration. We observed an increase in neurite outgrowth as well as migration of iIba-1+ cells to the outside of the explant (P<0.05). This confirms the paracrine effect of the secretome in the spinal cord environment with a potential translatable effect in vivo. On this wise, we decided to combine the ASCs secretome with EES in a rat model of SCI (severe contusion) in vivo. We observed a synergetic effect of both treatments on the locomotor score (BBB scale), body weight support, maximum speed, number of steps and dragging time during the stepping cycle, with the combinatory approach demonstrating superior performance than control rats in all parameters analysed. We also detected an interesting result in the Randall Sellito test (pain response), which may suggest an increase in intraspinal plasticity after the treatment. Altogether, this provides evidence of the therapeutic potential of ASCs secretome after SCI, supported by indications on the positive effects exerted on neuroinflammation, axonal outgrowth and regeneration in vitro. This potential is also highlighted when combined with EES in vivo, with functional gains observed in the locomotor performance when compared to control.
52354522124
"Traditionally, tissue engineering strategies employ a “top-down” approach, where cells are randomly seeded in polymeric scaffolds or hydrogels. As a result, engineered tissues are often at best homogenous in composition, lacking the morphological or structural features of native tissues. Alternative “bottom-up” approaches, that leverage the self-organizing capacity of stem cells, have shown promise for engineering human tissues [1]. During early limb development stem cells aggregate and condensate before differentiating along a chondrogenic lineage. In fact, pellet culture, where mesenchymal stem cells (MSCs) are forced to aggregate using centrifugation, has been the standard culture system for initiation of stem cell chondrogenesis in vitro as it allows cell-cell interactions that are analogous to those that occur during pre-cartilage condensation during early joint development [2]. Therefore cellular aggregates, microtissues or organoids might represent promising biological building blocks for the engineering of functional tissues.
This talk will describe how phenotypically distinct microtissues generated from stem/stromal cells can be integrated to engineer a biphasic osteochondral implant containing a biomimetic layer of engineered articular cartilage. The osseous region of this osteochondral graft was engineered using islands of hypertrophic cartilage microtissues capable of executing an endochondral programme, while the chondral region of the graft was formed by the self-organisation of early-cartilage microtissues into a unified and structurally organised tissue mimetic of native AC. Furthermore this talk will examine whether implantation of such an engineered plug into critically-sized caprine osteochondral defects can result in effective biological joint resurfacing and prevent the deleterious cascade of events that typically follow an untreated osteochondral injury.
REFERENCES
[1] Nichol, JW. et al. Soft Matter 5, 1312 (2009).
[2] Johnstone, B et al. Exp. Cell Res. 238, 265–272 (1998)."
52419506939
Organoids (self-assembled 3D tissue structures from a cluster of cells) can be used for patient-specific drug testing or for the generation of larger tissue constructs, which can in turn be used as implants to restore and/or replace damaged tissue. For cartilage, human chondrocytes, both healthy and diseased (derived from patients with osteoarthritis (OA)), show the potential to self-assemble into organoid structures. However, these organoids do not show the distinct collagen architecture that is needed to withstand the loading conditions in joints. Recently, the convergence of self-assembled equine articular cartilage-derived progenitor cells (eACPCs), which were stimulated with BMP-9 to increase the rate of matrix formation, with micrometer-scale reinforcing PCL fibres (made with melt electrowriting (MEW)) shows the potential to create abundant cartilage-like tissue while providing a mechanical support structure that is capable of withstanding in vivo loading conditions. We envision that the convergence of reinforcing technologies, such as MEW, with the human OA-derived organoids allows us to tackle the challenges of the availability of cell sources and limited mechanical properties of implants and with that steers towards patient-specific implants.
31451702964
"Despite immense interests, growing organoids resembling the human musculoskeletal system (including bone, cartilage, muscle, tendon, ligament) in a petri dish remains a major challenge in front of the tissue engineering and regenerative medicine (TERM) community. Bone is a vital organ that contains billions of bone cells as well as a sophisticated internal architecture across several length scales. Recapitulating the structural and functional complexity in bone requires the development of high resolution biofabrication techniques that faithfully recreate tissue architecture down to the micrometer-scale accuracy. One promising approach is to combine computer models derived from biomedical imaging data with light-based tissue manufacturing.
Here, we present an image-based subtractive biomanufacturing process to create microengineered 3D bone cell models in biocompatible hydrogels. To this end, new computer models that mimic the topology of lacuno-canalicular network (LCN) in bone are developed by sequential immunostaining and confocal microscopic imaging of osteocytes in bone specimen. These models are converted into stereolithography (.STL) files through image processing. Using two-photon subtractive lithography, we demonstrate the fabrication of LCN-mimicking microstructures inside a photodegradable polyethylene glycol hydrogel at high spatial resolution. The structural fidelity is highly dependent on the laser processing parameters such as laser power, writing speed and photosensitivity of the hydrogel matrices. The inclusion of a soluble two-photon photoinitiator can greatly decrease the laser threshold. Lastly, preliminary success on biomimetic subtractive 3D microprinting in the presence of living bone cells and guided 3D cell growth will be presented towards a living bone organoid."
52354555506
Bone organoids are an emerging novel platform to study human bone biology and bone formation. Biofabrication techniques such as extrusion bioprinting have been used to produce mineralized in vitro bone models. However, cell-cell interactions and mineralization rates are influenced by the initial cell printing density [1]. Here, we investigated the effect of cell density on mineral formation, organoid stiffness, and cell morphology in human 3D bioprinted cell-laden hydrogels under dynamic culture conditions.
Osteoblasts isolated from a femur bone chip of a 15-year-old male were encapsulated in 0.8% (w/v) alginate, 4.1% (w/v) gelatin, 0.1% (w/v) graphene oxide hydrogels [1]. Cells were extrusion bioprinted at 5x106 or 10x106 cells/mL of hydrogel and cultured in compression bioreactors for 10 weeks. Organoids were subjected to uniaxial cyclic compressive loading with 1% strain at 5Hz for 5 minutes 5 times per week. Live/dead assays were performed to determine cell viability after bioprinting (day 1) and after two weeks (day 15) of mechanical loading. F-actin nuclear staining was performed after 30 and 70 days of loading to investigate cell spreading morphology. Time lapsed micro-computed tomography (micro-CT) scans were taken weekly to monitor mineral volume and density. After 10 weeks of loading, organoids were fixed and cryosectioned for histology, immunohistochemistry, and scanning electron microscopy (SEM) to evaluate cellular phenotypes and mineralized matrix formation.
Patient-derived organoids exhibited high cell viabilities after bioprinting (>90%) and after two weeks of daily mechanical loading (>85%). F-actin staining after 30 and 70 days, revealed increased cell spreading and dendrite number in higher cell density organoids. While time lapsed micro-CT images revealed similar endpoint mineral volumes, significant differences were found when comparing mineralization rates and mineral densities between the two cell density groups. Higher cell density organoids exhibited the highest mineral formation rates in the earlier timepoints (28-35 days) while lower cell density organoids reached peak mineral formation after 49-56 days. Notably, a significantly higher average mineral density of 230.8 ± 15 mg HA/cm3 was found in higher cell density organoids compared to 176.9 ± 21.42 mg HA/cm3 in low cell density organoids after 70 days of culture. In line, higher cell density organoids exhibited increased stiffness as compared to lower cell density organoids. A 10-fold increase in stiffness was observed in higher cell density organoids at endpoint compared to day 15 measurements. Meanwhile, lower cell density organoids only showed a 2-fold increase in stiffness during this time. Histology, immunohistochemistry, and SEM imaging revealed distinct cell morphologies in the organoids, including osteoblastic and osteocytic characteristics.
Here, we have established a methodology that better supports the formation of mineralized patient-derived bone organoids resembling native bone tissue. Bioprinting at higher cell densities increased organoid stiffness and mineral density. These clinically relevant bone organoids combine primary patient cells with physiological loading conditions to study mineral formation of healthy bone. In future studies, this platform will be employed to investigate pathological bone and evaluate potential therapies.
References:
[1] J. Zhang et al., Acta Biomaterialia, 117, 307-322 (2020).
94238117924
Intramembranous ossification is the most common pathway used in tissue engineering (TE) of bone. However, therapeutic effects in case of large bone defects are suboptimal due to hindered vascularization. On the other hand, TE grafts generated via the endochondral ossification pathway, where the bone formation occurs through cartilage intermediaries, become vascularized and form bone upon implantation. In order to build bone, the chondrocytes have to become hypertrophic. The goal of this study was to develop fiber-reinforced bone decellularized extracellular matrix (bdECM)-based hydrogels for enhanced hypertrophic differentiation of chondrogenicly primed human bone marrow MSC (hMSC). </div>
<div>The hydrogels were composed of either 10 mg/ml type I collagen or bdECM mixed with alginate (30 mg/ml). As the reinforcement, 3D fibrous scaffolds were fabricated using PCL and PCL-based composites containing 25 wt% of β-TCP and FDM technology. HMSC were encapsulated in the pre-gel at density of 20x106/ml, infused into the 3D scaffolds (50 μl of cell suspension/ construct) and cultured in chondrogenic medium for 28 days. To measure cell viability, live/dead staining and MTS assay were performed. Secretion of cartilaginous matrix was evaluated by staining for safranin O, collagen type I, II and X. Moreover, quantification of GAGs was performed using 1,9-dimethylmethylene blue assay and collagen using total collagen assay. Additionally, alkaline phosphatase (ALP) activity was measured using colorimetric assay with para-nitrophenyl phosphate as a substrate. </div> <div>The cell metabolic activity did not decrease upon culture, although the overall viability measured by live/dead staining decreased, suggesting cell proliferation. A synergistic effect of the dECM and β-TCP on hypertrophic differentiation of hMSC was measured my means increased ALP activity and visualized by more intense staining against type X collagen. The aforementioned finding suggests that bone dECM hydrogels reinforced with β-TCP/PCL scaffolds are suitable materials for chondrogenic priming of MSC and bone TE via osteochondral pathway. 52354583529Introduction
Autologous bone grafting is currently the gold standard treatment for bone defects. However, it is still associated with multiple drawbacks such as limited availability and donor site morbidity. Various approaches have been explored to overcome this, of which endochondral bone regeneration (EBR) has emerged as a promising approach. EBR aims to mimic the process where bone is remodeled from a cartilage template, which naturally takes place in a fracture callus. Clinical translation of EBR would benefit from creating an off-the-shelf product consisting of donor-derived (allogeneic) and devitalized tissues. We have previously shown that such allogeneic devitalized cartilage tissues display accelerated full-bridging of a femoral defect in immunocompetent rats [1]. However, there is still a need to fully elucidate the interplay between the immune response and new bone formation. To explore this further, the early and late immune response elicited by vital and devitalized cartilage derived from autologous or allogeneic cells were compared in a subcutaneous rat model.
Methods
Rat multipotent mesenchymal stromal cells (rMSCs) were isolated from the bone marrow of Brown Norway (BN; autologous/syngeneic) and Dark Agouti (DA:allogeneic) rats. rMSCs were encapsulated within collagen hydrogel spheroids and chondrogenically differentiated for 28 days, followed by devitalization [1]. Two chondrogenic spheroids per group were implanted into subcutaneous pockets in BN rats (n=7) for 3,7,14,28 and 84 days. New bone formation and immune response were analyzed via micro-CT, histology (H&E, histomorphometry) and immunohistological staining (CD68,iNOS,CD206,CD3,myeloperoxidase).
Results and Discussion
Histological analyses revealed that the vital autologous spheroids were remodeled into bone including marrow cavities within 28 days. At this time, remnants of non-remodeled cartilage were still observed for the vital allogeneic and both devitalized groups. After 84 days, 5/7 samples from the vital autologous group could not be retrieved possibly due to resorption. Histomorphometry analyses from day 28 and 84 revealed no significant differences in bone formation or cartilage between groups (excluding vital autologous due to reduced sample size). The onset of bone formation appears to positively correlate with the presence of osteoclasts on day 14 in the vital autologous and both devitalized groups. Presence of osteoclasts in the vital autologous and both devitalized groups on day 84 indicate that active remodeling is still taking place. Initial analyses of the immune response revealed no significant differences between groups in terms of presence of macrophages (CD68, CD206 or iNOS) or T lymphocytes in the tissue surrounding the implants on day 3, 7 or 14. Further analysis of later timepoints and different immune cells are still on-going.
Conclusion
No differences in bone formation was observed between all groups. However, the vital autologous group demonstrated the fastest bone formation, most of which were resorbed by 84 days. Early analysis indicate that the onset of bone formation coincides with the presence of osteoclasts from as early as 14 days. Additional analyses are currently ongoing to elucidate the interplay of immune cells and bone formation at later time points.
References
1. Longoni, A. et al (2021).Acceleration of Bone Regeneration Induced by a Soft-Callus Mimetic Material.Adv Sci.mDOI:10.1002/advs.202103284
52354526488
Introduction: Bone remodeling is the combined process of bone resorption by osteoclasts and bone formation by osteoblasts. This process is regulated by mechanosensing osteocytes. It is the most fundamental physiological process that defines living bone. An imbalance in this process can cause metabolic bone diseases such as osteoporosis. Currently, no complete in vitro bone remodeling model is available. Such models have the potential to increase our knowledge on the physiological and pathological processes underlying bone remodeling and could potentially improve drug development processes. Bone-on-a-chip technology has the great potential to advance bone research, allowing for the study of low cell numbers in high temporal and/or spatial resolution. In this study, microfluidic chip technology is used to create a bone remodeling model. Currently, the study focusses on three-dimensional (3D) bone formation inside the microfluidic chip by osteogenic differentiation of human bone marrow derived mesenchymal stromal cells (MSCs) into osteoblasts. Next, the aim is to achieve bone-remodeling-on-a-chip by facilitating interaction between osteoblasts, osteocytes and osteoclasts.
Methods: A bone-on-a-chip microfluidic device that facilitates 3D in vitro bone-like tissue formation was developed. A polydimethylsiloxane (PDMS) microfluidic device was fabricated by means of photo- and soft-lithography. The device contained rectangular-shaped cell culture channels that were coated with fibronectin and seeded with MSCs. The MSCs were dynamically cultured for a period of 21 days by applying medium flow, resulting in shear stresses of around 2.3 mPa acting on the cells. Osteogenic medium was used to differentiate the MSCs along the osteogenic lineage.
Results: Time-lapse brightfield imaging revealed self-assembly into 3D constructs within the channel. At the end of the 21-day culture period, deposition of calcium (Alizarin Red staining) and collagen (Picrosirius Red staining) in the extracellular matrix produced by the cells was visible. Confocal microscopy revealed the formation of 3D bone-like struts through self-assembly. Immunohistochemical staining confirmed the formation of collagen type 1 and revealed the expression of osteopontin and DMP-1, confirming the differentiation of the MSCs into the osteogenic lineage.
Conclusion: Overall, the results revealed mineralized bone-like struts. With this, the developed bone-on-a-chip microfluidic device showed the first step towards a 3D in vitro bone remodeling model, exhibiting 3D bone-like tissue formation. In future research, osteoclasts will be added to the model to facilitate the bone resorption process.
31412708155
"Introduction
Minimally invasive surgery for the restoration of bone tissues lost due to diseases and trauma is preferred to reduce patient complications and health care costs. The current challenge is to design material at the site of surgery with specific behaviors for mimicking the natural structures and delivering appropriate signals to cells promoting tissue repair/regeneration. Selection of a suitable injectable is often based on material characteristics (including mechanical properties, drug release kinetics and degradation) that serve for the specific treatment function. Micro or nano-structured materials in the form of gels, nanoparticles and nano-composites have gained increasing interest in regenerative medicine because they are able to mimic the physical features of natural extracellular matrix (ECM) at the sub-micro and nano-scale levels. Different strategies are implemented for engineering bioactive and osteoinductive injectable materials to optimize their interfaces with cells and bone tissue environments.
Here, it discussed injectable bone materials integrating biphasic calcium phosphate nanoparticles prepared by sol-gel synthesis with different polymers, nano-materials and antimicrobial compounds for specific bone infections conditions.
Methodology
Innovative natural polymer-based double network hydrogels (DNs) were developed by a two-step network-formation procedure to obtain photocrosslinkable methacrylated hyaluronic acid (HAMA) and maleated hyaluronic acid (MAHA). Different chemical modification of hyaluronic acid (HA) followed by the development of nanocomposites hydrogels were directly prepared in situ by sol-gel synthesis [1]. Furthermore, injectable hybrid material based on graphene oxide nanosheets and hydroxyapatite prepared by sol-gel approach is described. The presence of GO increases the bioactive and osteogenic material properties.
One more approach is based on the use of antimicrobial injectable materials [2]. Several systems based on Ionic Liquids (IL) at different alkyl-chain length incorporated in Hydroxyapatite through the sol-gel process were developed to obtain an injectable material with simultaneous opposite responses toward osteoblasts and microbial proliferation.
In vitro cell tests to assess the osteogenic potential of the synthesized biomaterials were performed using human mesenchymal stem cells (hMSC) and the expression of specific osteogenic markers (ALP, OCN) was analyzed.
Results
Nanocomposite materials based on chemically modified HAs and in situ sol-gel CaP, were successfully developed and characterized in terms of physico-chemical, morphological, mechanical and biological properties. Injectable bone materials integrating BCP nanoparticles with HA based materials, GO and IL were successfully synthesized. For HA-GO system, it was found that the spindle-like hydroxyapatite nanoparticles were intercalated between GO nanosheets. The oxygen-containing functional groups of GO sheets play an important role in anchoring calcium ions, as demonstrated by FTIR and TEM investigations, thus improving the bioactive and osteogenic properties. The systems based on CaP-ILs showed a higher osteogenic activity and antimicrobial performance by increasing the IL alkyl chain. These systems are able to induce osteogenic differentiation and also inhibit biofilm formation.
Conclusion
The results indicated that all the proposed injectable materials can be considered a high-performance bone filler in the treatment of bone defects."
41935603297
Glycosaminoglycans (GAGs) within extracellular matrices (ECM) control the presentation of soluble, cell-instructive signals. The incorporation of GAGs into engineered polymer networks can therefore provide a powerful and versatile means of directing cell fate. We have established a rational design strategy for ECM-inspired hydrogels based on multi-armed poly(ethylene glycol), GAGs of different sulfation patterns, and functional peptides to systematically explore the related options. Micro-processing schemes (cyrogelation, solvent-assisted micro-molding, microfluidic microgel fabrication, multicomponent inkjet bioprinting) allow for the fabrication of multiphasic and multifunctional GAG-based gel materials with spatiotemporally adjusted signaling characteristics. Applications of the materials platform include 3D culture models of stem cell and tumor microenvironments as well as scaffolds for exploring new therapeutic approaches to chronic wounds and neurodegenerative diseases.
References
(1) Freudenberg, U.; Liang, Y.K.; Kiick, K.; Werner, C.: Glycosaminoglycan-based biohybrid hydrogels: a sweet and smart choice for multifunctional biomaterials. Advanced Materials 40 (2016) 8861-8891, DOI:10.1002/adma.201601908
(2) Gvaramia, D.; Müller, E.; Müller, K; Attalah, P.; Tsurkan, M.V.; Freudenberg, U.; Werner, C.: Combined influence of biophysical and biochemical cues on maintenance and proliferation of hematopoietic stem cells. Biomaterials 138 (2017) 108-117, DOI:10.1016/j.biomaterials.2017.05.023
(3) Müller, E.; Pompe, T.; Freudenberg, U.; Werner, C.: Solvent-assisted micromolding of biohybrid hydrogels to maintain human hematopoietic stem and progenitor cells ex vivo. Advanced Materials 29 (2017) Article number: 1703489, DOI:10.1002/adma.201703489
(4) Husman, D., Welzel, P. B., Vogler, S., Bray, L. J., Träber, N., Friedrichs, J., Körber, V., Tsurkan, M. V., Freudenberg, U., Thiele, J., Werner, C.: Multiphasic microgel-in-gel materials to recapitulate cellular mesoenvironments in vitro, Biomater Sci (2019), DOI:10.1039/c9bm01009b
(5) Kühn, S., Sievers, J., Stoppa, A., Träber, N., Zimmermann, R., Welzel, P., Werner, C.: Cell-instructive multiphasic gel-in-gel materials, Advanced Functional Materials (2020), DOI:10.1002/adfm.201908857
(6) Magno, V., Meinhardt, A., Werner, C.: Polymer hydrogels to guide organotypic and organoid cultures, Advanced Functional Materials (2020), DOI: 10.1002/adfm.202000097
"Introduction. One challenge of developing biomaterials for tissue engineering is the capability to precisely engineer desired properties, while keeping robustness and versatility in the system. Cell-encapsulating hydrogels are used as extracellular matrix mimics for basic study of cell function, high-throughput drug screening, and therapeutic delivery. In their molecular design, the crosslinking chemistry plays a vital role in regulating important properties, such as gelation rate, mechanical strength, and bioactivity [1]. Despite the many covalent crosslinking strategies reported so far, they are often not economic, user-friendly, or tunable enough to facilitate the adaptability of the encapsulating system to a variety of biomedical scenarios. To overcome this challenge, and inspired by the biochemistry of fireflies, we present a bioinspired covalent chemistry for the fabrication of precisely tunable, inexpensive, and versatile polyethylene glycol (PEG) hydrogels for 3D cell culture [2]. It is based on the condensation reaction between cyanobenzothiazole and cysteine groups, known as “luciferin click ligation”.
Methodology. 4-arm, 20-kDa PEG macromers bearing cyanobenzothiazole or cysteine functional groups were synthesized. Hydrogels were prepared under physiological conditions (37°C, HEPES buffer, pH 7-8). Crosslinking process, biofunctionalization with enzymatically cleavable and cell-adhesive peptides, and encapsulation of human mesenchymal stem cells (hMSCs) took place one-pot. The resulting hydrogels were cultured for 1-3 days. Cell viability, cell behavior and cell-materials interactions were evaluated by live/dead assay, F-actin cytoskeletal and morphological characteristics of cell analyses, respectively. Cell proliferation ability was assessed by Ki67+ nuclei staining. Mechanical strength and gelation kinetics of hydrogels were characterized by shear rheology.
Results. PEG hydrogels showed efficient and pH-regulable gelation rate, adjustable mechanical strength within physiologically relevant values, and high materials homogeneity at the microscale. By incorporating biochemical cues (i.e., cell-adhesive and cell-degradable ligands) to the hydrogel network, cell behavior and cell-materials interactions were modulated. Our gels supported the culture of hMSCs: encapsulated cells showed high cell viability (demonstrating the good cytocompatibility of these gels) and maintained their proliferation capability. 3D cell spreading (volume expansion), accompanied by high degree of cell protrusion and F-actin stress fiber formation, was observed in the presence of both cell-adhesive and cell-degradable cues in the gels.
To further develop the firefly-inspired hydrogel system as an injectable platform, novel redox-triggerable hydrogel precursors were introduced to the molecular design. The cysteine-based precursor was modified with a protecting group at the thiol residue, thus blocking gel crosslinking. Upon addition of a biocompatible reductant, the cysteine group was deprotected and the crosslinking reaction was triggered with exquisite control of the reaction rate. The storage stability of precursors was also improved, which is convenient for future upscaling and translation [3].
Conclusions. Firefly-inspired gels are robust and provide versatility for easy adaptation to diverse biomedical situations. Molecular engineering confers higher user control for the fabrication of injectable biomaterials. These biomaterials are expected to become valuable platforms for tissue engineering.
References:
[1] Paez, J.I. et al. Biomacromolecules 22, 7, 2874 (2021).
[2] Jin, M. et al. ACS Appl. Mater. Interfaces 14, 4, 5017 (2022).
[3] Jin, M., Paez, J.I., unpublished results."
41883651579
"Introduction
Alginate is widely used in the biomedical field, particularly to build three-dimensional (3D) systems with ECM-like properties. Despite being a bio-inert biomaterial, alginate can be chemically modified to promote highly specific cell-ECM interactions[1]. We have shown that molecularly designed alginate 3D matrices recap key features of native ECMs supporting tissue morphogenesis[2-4]. Here, we developed a novel methodology to synthesize and characterize functional bioresponsive alginate hydrogels based on thiol–maleimide “click” chemistry. The potential of these peptides-conjugated hydrogels as ECM-like matrices for 3D cell culture was evaluated.
Methodology
Ultra-pure low viscosity alginate (Alg) with high guluronic content was modified with variable amounts of maleimide (AlgM) by amidation of alginate carboxyl groups with the amine groups of 1-(2-Aminoethyl)-maleimide. Degree of substitution was assessed by 1H-NMR. Thiol-flanked (bi-functional) protease-sensitive peptide (GIW-peptide, CGPQGIWGQC) and thiol-terminated cell-adhesion peptide (RGD-peptide, CGGGGRGDSP) were grafted to AlgM via thiol-maleimide Michael addition click reaction. To confirm peptide double-end grafting and consequent crosslinking, Alg solutions dynamic viscosity was analyzed by oscillation/viscometry rheology. Alterations on GIW-crosslinked AlgM viscosity and molecular weight were assessed by gel permeation chromatography (GPC). Grafting of RGD-peptide was quantified by BCA Protein Assay (Pierce). Primary human mammary normal (nFIB) and cancer-associated (CAF) fibroblasts mixed with alginate and peptide solutions were cast as hydrogels. 3D cell response to different GIW/RGD-peptide concentrations was studied at different timepoints. MMP production (gelatin zymography), cell viability (live-dead assay), morphology (F-actin staining), and mechanical properties were assessed.
Results
1H-NMR qualitatively confirmed successful alginate functionalization with different maleimide amounts (theoretical substitution degree from 1 to 10%). Maleimide presence was identified by the appearance of a new peak (~6.9ppm) corresponding to the protons in the double bond of the maleimide group. Reaction efficiency was approximately 10%. We observed that high degrees of maleimides lead to poor solubility of alginate derivatives, so we only used derivatives with up to 0.3% of modification. Addition of bi-functional GIW-peptide increased hydrogel viscosity due to the formation of a chemically crosslinked gel network. Viscosity increase and consequent gel formation was observed between 120-480µM of GIW-peptide. Higher concentrations (840µM) did not alter solution viscosity but increased alginate Mw, because GIW-peptides were preferentially bound only by one side, occupying the maleimides without bridging two alginate chains. nFIB and CAF (fibroblasts with different proliferative/metabolic profiles) were successfully embedded within GIW/RGD-AlgM matrices, presenting elongated morphologies and forming extensive multicellular networks, contrary to control MMP-insensitive hydrogels, where cells remained essentially round. Due to its nature, CAF formed more extensive cellular networks faster, without major differences regarding MMP production.
Conclusions
A novel methodology for the synthesis of alginate containing maleimide functional groups was established. Covalently grafted maleimides allow alginate biofunctionalization and in situ crosslinking by thiol-Michael addition reaction. Incorporating protease-sensitive peptides significantly enhanced 3D cell-cell interactions in alginate hydrogels, improving their performance as ECM-mimics.
[1] Neves, M. I. et al., Front. Bioeng. Biotechnol. 8, 665 (2020).
[2] Torres AL. et al., Biomaterials 228, 119554 (2020).
[3] Teixeira FC. et al., Front. Bioeng. Biotechnol. 9, 647031 (2021).
[4] Bidarra SJ. et al., Sci. Rep. 6, 27072 (2016)."
94238166968
"Injectable biomaterials for cell and drug delivery is a rapidly expanding field that may revolutionize medical therapies. Here we report on the fabrication of injectable electrospun microscaffolds used to deliver desired cargo through the needle. We observed an efficient attachment of cells to the scaffold's surface, creating cell-populated microscaffolds (MS) that could be injected or 3D printed through 23-26G needles.
Polymer nano- and microfibers were electrospun and then structured with a picosecond laser. To increase the hydrophilicity of the MS, nanofibers were first hydrolyzed with sodium hydroxide and then functionalized with natural polymers like chitosan or chondroitin sulfate. For morphological characterization of MS with and without cells, we used Scanning Electron Microscopy (SEM). Physico-chemical characterization was done to analyze the impact of laser processing on polymer nanofibers. We used the L929 cell line to assess the biocompatibility of produced MS and the possibility of injecting the construct into the desired tissue. Nucleus pulposus cells were used as the target cells to evaluate their survival after injection through different needle sizes.
The direct injection of cells into tissues faces several challenges, such as low survival and their retention at the injection site. With cell-protective MS, the survival rate can be significantly increased. When using laser processing, any shape of microscaffolds can be created, among others, MS with quadrilateral, triangular or circular base. Moreover, low melting of the fibres at the cut surfaces can be observed. The cytocompatibility assays show an increase in cell number with culture time. L929 cells populated MS at each side, resulting in the formation of agglomerates. The injectability studies through 24G and 23G needles showed that the ejection rate was 97% and 98%, respectively.
We developed a novel and straightforward method to fabricate microscaffolds from almost any type of electrospun material. MSs are compatible with living tissues and readily populated with cells. By designing the surface chemistry of nanofibers, the physical and chemical structure of MSs can be customized to improve cell-MS interaction. The injectability studies show that PLLA-based MSs are injectable through the tested range of needle sizes and could be well‐suited for minimally invasive cell delivery applications. One of the examined applications is intervertebral disc degeneration, where designed MS delivers active molecules that enhance the synthesis of glycosaminoglycans. A single administration of the drug in the MS to the tissue will result in several weeks of the release of the active substance, which may have a beneficial effect on the regenerative processes of the intervertebral disc.
Acknowledgements: This work was supported by the National Centre for Research and Development grant no. LIDER/14/0053/L-9/17/NCBR/2018 and National Science Centre no. 2015/19/D/ST8/03192.
References: Nakielski P. et al., Small, 18, 2104971 (2022)"
20941818568
The components of cellular microenvironments, especially the extracellular matrix (ECM), strongly regulate biological processes through biochemical and mechanical signaling. For this, hydrogels have been widely used to build artificial niches mimicking the native ECM. Strain-promoted azide-alkyne cycloaddition (SPAAC) is a biorthogonal reaction between strained cyclic ring-containing alkynes and azides, which proceeds under mild, copper-free, biocompatible conditions. Here, we developed an amphiphile SPAAC-clickable alginate derivative capable of forming ionic hydrogels with hydrophobic microdomains, which can be further functionalized in-situ and in the presence of cells. This system provides topographical cues to cells within a true 3D microenvironment and may sequester molecules with hydrophobic moieties while allowing on-demand dynamic switch of matrix properties.
Ultra-pure alginate was functionalized with a cyclooctyne (BCN-amine) by carbodiimide chemistry. Modification degrees of alkyne-alginates (ALK) were assessed by 1H-NMR. ALK derivatives were characterized by contact angle measurements, hydrophobic probes, scanning electron cryomicroscopy, and viscometry. ALK hydrogels (acellular and cell-laden) were prepared by ionic crosslinking with Ca2+ [4]. Mechanical analysis was performed by microindentation and rheometry. SPAAC conjugation with azide-functionalized compounds was performed at 37ºC, in pre-gel solutions (0.9% w/v NaCl) or pre-formed hydrogels (culture medium). Grafting kinetics and efficiency were estimated using fluorescent azido-tags. Clickable hydrogels laden with mesenchymal stem cells (MSC) were analyzed in metabolic activity and morphology, ECM protein expression by immunostaining, and gene expression for 14-d.
ALK with varying modification degrees was successfully produced. Increased modification produced ALK derivatives with less hydrophilicity, stronger interactions with hydrophobic probes, and increased viscosity in aqueous solutions. At higher modification degrees, ALK derivatives showed the ability to spontaneously establish concentration-dependent associations between polymer chains. Ionic ALK hydrogels with denser microstructures within a sparser 3D network were produced, showing spatial heterogeneity in stiffness, as expected. These regions not only added topographical features to the otherwise smooth bulk hydrogel but also provided binding regions for sequestering compounds with hydrophobic sites, such as proteins, as verified using an extrinsic hydrophobic fluorescent probe. In-situ and on-demand multi-functionalization was confirmed by performing consecutive SPAAC conjugations. Reactions proceeded rapidly (< 30 min) under physiological conditions (i.e., in culture medium at 37ºC). In MSC-laden ALK hydrogels, cells remained viable and metabolically active throughout culture time. Cell spreading and extracellular fibronectin expression were detected at the microdomain regions only, which worked as topographical harbors for cell anchoring.
By taking advantage of the intrinsic hydrophobicity of cyclooctyne groups, which can be conjugated with azido-conjugated compounds via SPAAC, we successfully formulated hydrogels that present topographical cues to cells in a true 3D microenvironment, while also allowing dynamic, on-demand, (bio)functionalized in the presence of cells.
References: [1] Kim, E, et al., Chem Sci 10(34) (2019) 7835-7851. [2] DeForest, CA, et al., Nature Materials 8(8) (2009) 659-664. [3] Jain, E, et al., ACS Applied Bio Materials 4(2) (2021) 1229-1237. [4] Torres, AL, et al., Biomaterials 228 (2020) 119554.
Acknowledgment: Portuguese Foundation for Science and Technology (FCT) for project EndoSWITCH (PTDC/BTM-ORG/5154/2020), fellowship SFRH/BD/129855/2017, and contract IF/00296/2015
62825474529
"
Injectable biomaterials have evolved from serving as simple structural fillers to acting as multi-functional systems capable of directing human mesenchymal stromal cell (hMSCs) response. However, the selection of appropriate design parameters for injectable polymeric microbeads for translational applications remains a challenge. We have demonstrated promising strategies to address this need by tailoring the architectural features of injectable microbeads to act as modulating moieties of attachment and osteogenic response in hMSCs [1, 2].
Topography offers a vital tool to be harnessed for guiding cell fate, since topographical features tend to be more robust than surface chemistry and can be modified in terms of size, shape and degradation rate. We have demonstrated that topographical patterning of polylactic acid microbeads offers cell-instructive 3D microenvironments to allow the modulation of hMSCs fate by eliciting the desired downstream response without adding exogenous bioactive supplements. Topographically-patterned microbeads of varying microscale features (acting as braille for cells) were produced by phase separation of a sacrificial component from polylactic acid during fabrication. We established that culturing hMSCs on dimpled microbeads recreates mechanical aspects of the endosteal niche and exhibited varying morphological, integrin-mediated adhesion and proliferation responses. Additionally, significantly increased expression of osteogenic markers in hMSCs cultured on dimpled microbeads relative to conventional smooth microbeads was observed in the absence of exogenous biochemical factors. The cells also exhibited significantly altered metabolic profiles on different microbeads designs and resulted in varying histological characteristics in vivo [1]. Surface-functionalised textured microbeads were used to investigate the relative importance of surface chemistry over topography on the formation of 3D hMSCs-microbeads aggregates for 3D culture applications [2].
Our work delivers new guiding principles for the design of 3D cell-material interfaces, and opens up new avenues for engineering tailored injectable materials for applications spanning regenerative therapies, disease models, cell culture and advanced cell delivery systems.
References:
[1] Amer, M., Alvarez-Paino, M., McLaren, J., Pappalardo, F., Trujillo, S., Wong J., Shrestha, S., Abdelrazig, S., Lee, JB., Stevens, L., Kim, D, Gonzalez-Garcia, C., Needham, D., Salmeron-Sanchez, M., Shakesheff, K., Alexander, M., Alexander, C., Rose, F. (2021) “Designing Topographically Textured Microparticles For Induction and Modulation of Osteogenesis in Mesenchymal Stem Cell Engineering”. Biomaterials, 266, 120450
[2] Alvarez-Paino, M., Amer, M., Nasir, A., Cuzzucoli Crucitti, V., Thorpe, J., Burroughs, L., Needham, D., Denning, C., Alexander, M., Rose, F., Alexander, C. (2019) “Polymer Microparticles With Defined Surface Chemistry And Topography Mediate The Formation Of Stem Cell Aggregates And Cardiomyocyte Function"". ACS Applied Materials & Interfaces, 11(38) 34560."
73296354639
"Extracellular vesicles (EVs) have garnered growing attention as promising acellular tools for bone repair. Epigenetic regulation through histone deacetylase (HDAC) inhibition has been shown to increase differentiation capacity. Although EVs efficacy has been shown, their short half-life in vivo hinders their therapeutic potency. Gelatin methacryloyl (GelMA) hydrogels functionalised with synthetic nanoclays have been demonstrated to improve growth factor retention. This study investigated the potential of combining epigenetically activated osteoblast-derived EVs with the GelMA nanocomposite hydrogel to stimulate bone repair.
GelMA/nanoclay composites were fabricated by combining 5wt% GelMA with different concentrations of LAP (0.5, 1 and 2 wt%) prior to visible light crosslinking. The hydrogels compressive modulus, shear-thinning behaviour, 3D printing fidelity and osteogenic potency was evaluated. EVs were derived from 5 nM TSA-treated or untreated osteoblasts over a 2-week period. The EVs size, morphology and concentration were assessed via nanoflow cytometry and transmission electron microscopy. The isolated EVs were incorporated within the composites and their release kinetics were determined using the CD63 ELISA. The osteogenic differentiation of human bone marrow stromal cells (hBMSCs) within the EV-functionalised hydrogel was evaluated by qPCR, biochemistry and histological analysis.
LAP improved GelMA compressive modulus and shear-thinning properties in a dose-dependent manner. Nanoclay incorporation enhanced the shape fidelity when 3D printed compared to LAP-free gels. Interestingly, GelMA hydrogels containing LAP exhibited increased mineralisation capacity (1.41-fold) over 14 days. EV release kinetics from these nanocomposites were strongly influenced by LAP concentration with significantly more vesicles released from LAP-free constructs. EVs derived from TSA-treated osteoblasts (TSA-EVs) enhanced proliferation (1.09-fold), migration (1.83-fold), and mineralisation (1.87-fold) of hBMSCs when released from the GelMA-LAP hydrogel compared to the untreated EV gels. Importantly, the TSA-EV functionalised GelMA-LAP hydrogel significantly promoted encapsulated hBMSCs extracellular matrix collagen production (≥1.3-fold) and mineralisation (≥1.78-fold) in a dose-dependent manner compared to untreated EV constructs.
Taken together, these findings demonstrate the potential of combining epigenetically-activated osteoblast-derived EVs with a nanocomposite photocurable hydrogel to enhance the therapeutic efficacy of acellular vesicle approaches for bone regeneration."
62825408706
"Introduction: Large bone defects are a challenge for orthopedic surgeons. BMPs are the most potent bone inductors and accelerators of bone growth. However, poor BMP-2 retention by scaffolds leads to its rapid clearance from implantation sites,1 and thus, require the use of supra-physiological doses. Serious concerns regarding BMP-2-related adverse effects have been reported (including osteolysis, ectopic bone formation, and inflammatory response)2, and prompt the need for new carrier materials for optimizing BMPs spatiotemporal delivery. Our study aimed to evaluate the regeneration of a critical-sized segmental bone defect in a sheep preclinical model, with a 3D architectured poly L-lactic acid (PLLA) scaffold coated with a biomimetic film containing BMP-2. The osteoinductive properties of these films deposited on different types of implantable materials have previously been demonstrated both in vitro3,4 and in vivo.5–7
Methodology: PLLA cylindrical scaffolds (25mm high, 14mm in diameter and with cubic pores of ~880µm) were 3D printed, coated with a biomimetic film previously developed by members of our team8,9 and subsequently loaded with BMP-2. A 25mm-long mid-diaphyseal segmental metatarsal bone defect was created and stabilized with a plate in 9 sheep. Defects were filled with either (i) PLLA scaffold loaded with (from 300 to 600 µg) BMP-2 (n=7) or (ii) BMP-2-free PLLA scaffold (n=2). Monthly radiographic follow-up was performed until animal sacrifice at 4 months. The newly-formed bone between the defect edges and within the scaffold was quantified in explanted specimens with micro-CT. Specimens were then processed for undecalcified histology to characterize bone formation/resorption, bone-scaffold interface, inflammatory response and vascularization of tissue.
Results: Consistent radiographic bone union was observed in 7/7 animals when BMP-2 containing film-coated PLLA scaffolds were implanted, whereas none of the 2 animals implanted with the BMP-2-free PLLA scaffold did achieve bone union. Neither abnormal bone resorption nor chronic inflammatory response were observed with the bioactive scaffold containing BMP-2. Dense newly-formed bone filled with numerous osteocytes was observed all around and in direct contact with the scaffold material pillars.
Conclusions: 3D printed PLLA scaffolds coated with a biomimetic films containing BMP-2 provided consistent radiographic bone union in a preclinical critical-size segmental defect without noticeable deleterious effects. This strategy opens new avenues for the replacement of segmental bone defects.
94238118639
"Introduction: Several molecules of natural origin are of great interest to add specific surface functionalities to implant biomaterials: phenolic compounds and keratin can be used for different targets, as an example. They are derived from industrial processing of respectively plants or animals derivatives through a valorization process of waste and a sustainable circular economy approach. The modified surfaces enhance and fast the tissue integration, fight or reduce the risk of infections, guide the tissue growth, modulate the inflammatory response.
Different processing can be followed: grafting of a molecular monolayer (functionalization), thin or thick continuous coatings. The selection of the processing must be guided by the chemistry of the surface and ligands as well as by the expected mechanism of action of the biomolecule: progressive release or permanent link to the surface. Functionalization or coating can be coupled to different surface topography of the substrate for a synergic chemical-physical effect on the host response.
Post-processing (packaging, sterilization, storage) of the final products must be adapted to the presence of the biomolecules, too.
Potential applications are in orthopedic, dental, cardiovascular implants.
Methodology: Polyphenols (phenolic acids, flavonoids, and condensed tannins) are extracted from organic red grape pomace. The functionalization process is performed at pH = 7.4 with the addition of calcium ions, which act as a bridge between the substrate and polyphenols. The presence, amount (semi-quantitative), distribution, release, and type of bonding to the surface of the grafted polyphenols have been assessed.
Using electro-spinning, mirror-polished Ti disks were uniformly coated with keratin fibers obtained from discarded wool via sulfitolysis; surface functionalization with keratin molecules has been released, too. The keratin modified surfaces were then doped with silver (Ag) to introduce antibacterial properties.
The resulting specimens were characterized in terms of morphology and chemical composition by FESEM, FTIR, zeta potential titration curves, KPFM, and XPS. The antibacterial properties of the Ag-doped specimens were tested against a multidrug-resistant Staphylococcus aureus biofilm through morphology (FESEM) and metabolic assay. Lastly, the cytocompatibility of the specimens was confirmed using human primary gingival fibroblasts and mesenchymal cells.
Results: The functionalized samples have a homogeneous distribution of polyphenols as a continuous layer and micro-sized agglomerates. The grafted polyphenols maintain redox chemical and radical scavenging ability. A fraction of polyphenols is released into water in one day, while a firmly grafted layer remains on the surface even after four weeks. A larger release can occur in case of an environment with pH of 4–5 (e.g. inflammation). The functionalized surfaces can be sterilized by gamma irradiation without significant damage of the grafted polyphenols.
Concerning keratin, the Ag surface enrichment was effective in reducing viability and maturation of S. aureus biofilm, without compromising human cell viability. The cell spread was found to be very sensitive to keratin fiber stimulation.
Conclusions: Both the strategies thus appear to be very promising to introduce surface features in line with the main requirements for transmucosal and bone implants and it is of great interest to compare them in terms of efficacy and target application."
62825444649
"Introduction
The bone morphogenetic protein-2 (BMP-2) is one of the most potent growth factors for bone repair. In the clinic, BMP-2 is widely used for spinal fusion, particularly in the product called InFUSE Bone Graft® (Medtronic). Despite its strong efficacy, the safety of BMP-2 remains questionable as some treated patients suffer from serious side-effects, such as ectopic bone formation, nerve damage, severe inflammation and cancer. In this project, we engineered a bridge protein that effectively slowed the release of BMP-2 from collagen sponges, the carrier material used in InFUSE Bone Graft®, thus allowing significant dose reduction of BMP-2 for bone repair.
Methodology
The bridge protein was designed for dual affinity to collagen I and to BMP-2. Specifically, it was made by the fusion of the fragment antigen-binding (Fab) of an anti-collagen antibody to the growth factor-binding domain of laminin, which displays high affinity to BMP-2. The bridge protein was produced in Human Embryonic Kidney (HEK) 293 mammalian cells and purified by affinity-mediated chromatography. In all experiments, the bridge protein was simply admixed to BMP-2 prior to incorporation into collagen sponges. We first tested the efficacy of the bridge protein in slowing BMP-2 release in vitro. Then, we assessed the therapeutic efficacy of BMP-2 ± bridge protein, delivered in collagen sponges, in two in vivo mouse models of bone regeneration, a critical-size calvarial defect model and an intervertebral defect model newly developed by us to mimic spinal fusion in mice. Bone regeneration was monitored via in vivo CT scan imaging.
Results
In vitro, the bridge protein strongly enhanced the retention of BMP-2 into collagen sponges. Indeed, BMP-2 was released over more than 7 days versus about 3 days in presence or absence of the bridge protein respectively. Upon single implantation in vivo, the bridge protein permitted significant improvement of bone regeneration at very low doses of BMP-2, as measured by the volume of newly formed bone, the defect coverage and the rate of spinal fusion. Positive results were consistently observed in both the calvarial and intervertebral defect models in mice [1]. In addition to retention in collagen sponges, we demonstrated that the bridge protein allowed local retention of BMP-2 in the endogenous collagenous extracellular matrix (ECM) of tissues.
Conclusion
We engineered a bridge protein that substantially improved the delivery of BMP-2 from collagen-based materials. Combining the bridge protein to BMP-2 significantly enhanced bone regeneration in vivo, therefore allowing good therapeutic efficacy at very low dose of BMP-2. Such protein engineering approach for growth factor delivery may be generalizable to many other applications of tissue engineering, considering the broad use of collagen-based materials in regenerative medicine (e.g., collagen sponges, collagen hydrogels, decellularized matrices).
20941847799
"Collagen scaffolds are well known for their regenerative potential. However, the relatively poor mechanical properties represent a problem (scaffold deformation, partial collapse of internal open pore structure) for the translation into a clinical setting. In this study, we present a novel approach: A Mechano-Hybrid-Scaffold (MHS) that combines a collagen-based biomaterial with highly aligned channel-like pores with a 3D printed poly(ε-caprolactone) (PCL) support structure [1], overcoming contradictory requirements for mechanical stiffness and scaffold architecture.
The previously described orientated collagen scaffold [2] was combined with a 3D printed PCL macro-porous support structure [3] that preserves the internal architecture of the collagen scaffold. Internal architecture characterization (scanning electron microscopy imaging) and mechanical characterization (monoaxial compression test) were performed. Crosslinking and degradability were assessed by determination of the denaturation temperature of the collagen (Td).
MHS were successfully produced with stiffness of 9.56 MPa (stiff) and 0.01 MPa (soft) for the supporting structure, respectively. MHS characteristics, e.g. Td (79.8 ± 0.1 °C) and pore size (78.1 ± 18.1 µm), remain the same as the ones of collagen reference scaffolds without a PCL supporting structure. Scanning electron microscopy images show full integration of the support structure inside the collagen structure and no alteration of the scaffold inner architecture.
With this approach, mechanical characteristics can be tuned independently at the micro-scale (cell-level) and the macro-scale (tissue-level). The MHS opens the door for new applications of collagen scaffolds in rigid tissue regeneration by solving the paradox of providing soft cell environment and high structural stability in implantable materials.
[1] Patent pending: DE102016007931.2; PCT/DE2017/000183; US 16/313,937
[2] Petersen et al. (2018), Nat. Commun. 9:4430.
[3] Tortorici et al. (2021), Mat. Science & Engineering C 123 (2021) 111986
The authors acknowledge financial support by the German Federal Ministry of Education and Research (BMBF) via grants no. 13XP5048A-D)."
83767245227
"From a mechanical point of view, bone demonstrates exceptional mechanical properties owing to its complex hierarchical composite structure. The human skeleton acts as a support for the whole body as it withstands stresses produced by daily routines and gravitational force. As a result of these stresses, the bone regulates its geometry and density through activating formation and resorption mechanisms (Pivonka, 2018). Different mechano-regulatory theories were developed to address the dependency of the bone remodeling mechanism on mechanical stimulation at multiple scales. At tissue scale, where the bone is considered as a homogeneous material, the strain energy density (SED), effective stress, octahedral shear strain and interstitial fluid flow have been examined as the driving force for bone formation and resorption mechanisms. Advances in computational power and numerical techniques have extended the ability to apply these mathematical schemes to large-scale problems involving the design and optimization of scaffolds for large bone defects. Numerical schemes, such as finite element method (FEM), boundary element method (BEM), and meshless methods have been used to simulate bone remodeling, among which, FEM is by far the most used method (García-Aznar et al., 2021). Design of scaffolds faces numerous challenges at different scales and physics. One of the ongoing challenges is to optimize the microstructure of the scaffold to maximize the efficacy of the scaffold as a supporting structure for bone formation. For this reason, functionally graded scaffolds (FGSs) are designed which closely resemble the mechanical, biological, and morphological properties of the bone structure (Zhang et al., 2018). In this study, an FEM-based approach (Shi et al., 2018) was adopted to investigate the effect of porosity variation on bone formation inside an FGS incorporating both degradation and regeneration. The SED-based feedback mechanism was employed to consider the effect of mechanical stimuli on bone formation of a FGS. Bulk, surface, and stochastic degradations were considered in modeling of the scaffold degradation for the first time for a FGS. The aim of this study is to evaluate the effect of microstructure on the bone formation inside an FG bone scaffold and establish the basis for potential future studies on optimization studies of FGSs for maximum performance. The reported results can be used as benchmark solutions for future numerical analysis of the bone formation inside FGSs and serve as a means to validate future in-vivo or in-vitro experimental results.
References
García-Aznar, J. M. et al., Bone. 116032 (2021).
Pivonka, P. (Ed.). Springer International Publishing. (2018)
Shi, Q. et al., Biomechanics and modeling in mechanobiology. 17(3), 763-775 (2018)
Zhang, X. Y. et al., Materials & Design. 157, 523-538 (2018)"
62825451306
Design and fabrication of optimal scaffolds for bone tissue repair is a multi-physics problem targeting osteoconductive, biodegradable material designs with loading of necessary growth factors. Therefore optimal tissue regeneration based on accurate analysis models including the degradation behavior of biomaterials is a critical design requirement (Byrne et al., 2007). In order to explore bone regeneration, cell response to growth factors and existing intracellular signaling pathways driving bone regeneration through transcription factors should be taken into consideration considering degradation of the scaffold (Sun et al., 2013). In this work, this problem is addressed by solving time dependent hydrolysis reaction equations coupled to a reaction-diffusion equation and a set of ordinary differential equations representing release of growth factors and intracellular signaling pathway, respectively. The evolution of bone scaffold degradation, growth factor release and intracellular signaling pathway are conducted as a parametric study based on the effect of pore size of the scaffold within a FEM based modeling environment. Parameters such as degradation rate constant, diffusivity of water through scaffold are tuned based on existing computational models and experimental data in literature. In addition to providing an efficient tuning ability for design parameters, computational models offer significant advantages such as time and cost savings of experimental design and validation processes (Wang et al., 2020). Therefore, the aim of this study is to analyze the effect of porosity on the scaffold degradation, growth factor release and cell response during the bone healing process for a regular structured 3D cubic scaffold with aligned pores based on coupled reaction-diffusion PDE equations. COMSOL Multiphysics® was used to create a unified modeling framework that should allow for additional multi-physical effects such as mechano-biology based regeneration, diffusion of MSCs and angiogenesis to be considered within future design studies. Degradation profiles for scaffolds with different pore sizes and porosities were analyzed and the effect of degradation on growth factor release profiles and cellular response were observed. It has been shown that the pore size and porosity of a bone scaffold affects the bone regeneration process through dynamic interaction between growth factor release profiles and their regulatory mechanism on transcription factors with scaffold degradation.
94238177319
"
3D-printed personalised scaffolds are an attractive approach for mandibular bone repair. Poor mechanical stability of medical grade ceramics is a disadvantage. Also, delivery and retention of regenerative cells within 3D-printed scaffolds remains a challenge. This work aims to create 3D-printed personalised scaffolds based on a novel combination of materials with enhanced mechanical and cell-adhesion properties and with a configurable layered composition including cell-laden collagen membranes for improved cell delivery. The printable ink is created in ethylene carbonate (EC) and consists of 40% (w/V) PLGA to support printability and mechanical strength, 20% (w/V) β-TCP to increase osteoconductivity and 10% (w/V) TPU for elasticity. Solvent-based printing is applied using the RegenHu 3D Discovery® Bioprinter. A 3D model of a mandibular defect is derived from CT scans, then sliced and modified with CAD to obtain LEGO®-like structures. The personalised scaffolds are printed as a series of layers incorporating an interlocking mechanism. Human bone marrow derived mesenchymal stromal cells (MSCs, obtained will full ethical approval) are seeded and kept for 21 days under osteogenic culture conditions on two commercially available collagen membranes: 1. Lyostypt (B. Braun) and 2. Collagen Cell Carrier (CCC) (Viscofan Bio Engineering) (N=3). Viability of MSCs on the collagen membranes placed in between 3D-printed scaffold layers is examined via live-dead staining after 8 days of culture (N=1). Water-mediated EC removal leads to surface microporosity and roughness, both confirmed by SEM, and both favourable properties for improved cell adhesion. Mechanical compression test (N=10) of the 3D-printed scaffold shows improved stiffness and ductility compared to the commercially available ceramic Osteoink®. Under osteogenic culture conditions, MSCs seeded on collagen membranes have increased alkaline phosphate activity at day 14 compared to controls, with an effect more profound for the CCC. When MSC are seeded on the collagen membranes, expression of bone sialoprotein mRNA is upregulated not only in the osteogenic medium but also in the control medium. Live-dead staining shows good cell survival on the Lyostypt cultured in between 3D-printed scaffold layers, while a higher number of dead cells are detected on the CCC. 3D-printed scaffold biocompatibility and cell proliferation is shown by live-dead staining over 8 days of cell culture. Large scale personalised mandibular implants can be successfully printed, assembled and combined with cell-laden collagen membranes. We propose a novel 3D printable ink for mandibular bone reconstruction as an alternative to ceramics. Ongoing tests aim to demonstrate that osteogenic capabilities of the 3D-printed scaffold and efficient seeding of biologics intraoperatively to promote osteogenesis and vascularisation."
41883649528
"Introduction
Most bone defects heal successfully, however, there is an increasing number of cases where bone self-healing is insufficient. Thus, there is a high need for scaffolds able to replace the clinical gold standard treatment autologous bone grafting, which entails donor site morbidity and lacks control over spatial architecture to match defect sites. Biofabrication offers great potential to produce constructs that provide control over shape, architecture and composition. Therefore, this study aims to develop a 3D-printable composite biomaterial-ink to fabricate patient-specific bone graft substitutes for bone regeneration. Based on the heterogenous nature of bone, the biomaterial ink combines inorganic osteoinductive calcium phosphate particles (CaP) with tyramine modified hyaluronic acid-Collagen type I (THA-Col) organic matrix for the delivery of chemically modified RNAs (cmRNAs) inducing nerve, vessel, and bone formation.
Methods
Biomaterial-ink formulations consisting of 17.5 mg/mL THA, 2.5 mg/mL Col with 1 U/mL horseradish peroxidase (HRP), and 0.02% w/v Eosin Y, were combined with a range of 0-30% w/v CaP of size 45-63 or 45-106 µm. 0.17 mM H2O2 was added for enzymatic pre-crosslinking, to create a viscoelastic gel with shear thinning properties. After extrusion of desired structure, further gelation was triggered by light crosslinking for 30 minutes (505 nm). 1% v/v Nanocapsules, as vectors for the cmRNA, were mixed into the pre-polymer solution, gelation and distribution of Nanocapsules within the ink was then analyzed. Composites were further characterized for printability, cohesion, swelling, degradability, and compression modulus. Printability of formulations was evaluated by printing a continuous strut, line spacing, lattice, and overhanging strut on a pillar structure. Further, biomaterial composites were assessed in vitro using a metabolic activity assay after 1, 3 and 7 days using human mesenchymal stem cells (hMSCs).
Results
All formulations were viscoelastic and extrudable, with the formation of a continuous strut, good shape retention and without waviness. The addition of cmRNA vectors resulted in homogeneous dispersion within the matrix and did not influence the gelation mechanisms. All printed formulations retained their original weight and macroscopic shape when lyophilized and rehydrated. Additionally, formulations of THA-Col showed higher metabolic activity compared to THA alone. The range of identified formulations is being assessed for in vitro osteogenesis of hMSCs (viability, mineralization, alkaline phosphatase (ALP) production, gene expression, and protein production).
Conclusion
Here, a 3D-printable composite THA-Col/CaP biomaterial-ink was developed that is suitable for the combination with cmRNAs/vectors and holds significant potential as bone graft substitute for bone regeneration."
94238145577
My research group is focussed on developing new bioengineering technologies that can provide control over the assembly of biomaterials and tissues. I have a long-standing interest in ultrasound: waves of pressure that can interact with living and non-living matter to instigate a range of chemical, physical, and biological processes. First, I will discuss a technology that uses high-frequency ultrasound standing waves to rapidly and remotely pattern living cell populations into tuneable geometric arrays. I will explore our progress in using biomaterials to encapsulate these cellular assemblies and show that these patterned biomaterials can be used to engineer a range of tissues displaying anisotropic structure, which in certain cases leads to directed functional processes. I will also discuss another ultrasound-based technology: the use of low-frequency ultrasound to trigger molecular processes, such as enzyme catalysis and hydrogelation. I will explore the design of this modular system and describe how this new technology opens up new opportunities in tissue engineering and regenerative medicine.
73387300964
Introduction
Nowadays, great attention is devoted to the development of 3D in-vitro neuronal models both for fundamental neuro-mechanobiology applications as well as for disease modelling. Typical approaches include either scaffold-free or scaffold-based strategies. Although the first ones, based on cell self-assembly mechanisms, lead to the formation of tissue-like structures called neuro-spheroids or neuro-organoids, they often suffer from batch-to-batch variability and development of early-stage necrotic cores. Scaffold-based approaches involve instead the use of manufacturing techniques such as fused deposition modelling, bioprinting and electrospinning. These methods however lack the possibility of creating precise micrometric or sub-micrometric geometries able to guide cell fate. In this presentation, I will highlight two recent investigations where we employed a high-definition light-based technology to fabricate for the first time: 1) 2.5D and 3D nanostructures cultured in presence of primary microglia extracted from the brain of rhesus macaque; 2) 3D engineered glioblastoma microenvironments in the context of proton radiobiology studies.
Methodology
All the 3D structures were fabricated by two-photon laser assisted polymerization (2PP), exploiting the two-photon absorption of near-infrared radiation by focusing infrared femtosecond laser pulses onto an organic pre-polymer material. This non-linear mechanism is tuned in order to induce the photopolymerization of the exposed material in extremely confined volumes of sub-micrometric size. In the first study, primary microglia were derived from isolated brain tissue (white matter) of adult rhesus macaque (Macaca mulatta) donors that were free from neurological diseases and cultured both on flat substrates, 2.5D micro/nano-pillars and 3D micro/nano-decorated scaffolds. The morphology of the microglia was then assessed by immunofluorescence and scanning electron microscopy. In the second study, human glioblastoma (GBM) U-251 cells were cultured on 3D architectures mimicking the brain blood vessel geometry and its vascular branching points where GBM cells naturally cluster and proliferate. The engineered glioblastoma microenvironments were then exposed to different proton radiation doses (2 Gy and 8 Gy) and the amount of DNA damage was assessed by using the fluorescence Gamma-H2AX marker.
Results
The combination of sub-micrometric topographies (close to the dimensions of cell filopodia) and mechanical cues (represented by a low effective shear modulus approaching the stiffness of brain tissue), induced a substantial increase in the numbers of microglia characterized by a ramified resting phenotype as compared to cells cultured on flat stiff substrates, mostly featuring an amoeboid morphology. Concerning the second study, upon proton irradiation, GBM cells consistently showed lower DNA damage in the 3D engineered microenvironments compared to 2D GBM cell monolayers, which correlates with the response of GBM cells in-vivo where a greater radioresistance is observed. We hypothesize that this difference in the formation of the number of foci is directly connected to the differences in terms of cytoskeletal properties, cell-matrix interactions and repair kinetics between 2D and 3D cell culture configurations.
Conclusions
We demonstrate how 2PP can be employed to create: 1) 2.5D and 3D micro- and nano-structures able to guide the fate of primary microglia towards ramified phenotype; 2) 3D engineered glioblastoma microenvironments, which can be used as a reliable benchmark tool for proton radiobiology.
94238111005
The current gold standard for peripheral nerve repair is autograft. However, the low availability of nerves and loss of function at the donor site are the major disadvantages associated with this procedure. Thus, to address the limited regenerative capability of the human nerves, nerve guidance conduits (NGCs) fabricated using biocompatible and biodegradable materials has proven to be a potential alternative. Various approaches have been explored so far to develop tubular structures for neural regeneration including bioprinting, self-assembly, micropatterning, electrospinning among others. Multiple reports in literature indicates potential of such NGCs in in vitro experiments, however, they fail to provide axonal outgrowth in in vivo studies. The underlying reason is the limitations associated with 3D printing approaches like low resolution of printing tubular structure, thin walled tubes for permeation of nutrients and waste products, high shear forces needed to print the material and difficulty in fixation of endings of ruptured nerves in the nerve conduits. 4D biofabrication based on fabrication of complex structures using 2D and 3D objects by desired shape transformation with response to external stimuli can provide potential solution to the above-mentioned problems. Furthermore, 4D biofabricated structures can be designed to mimic the human tissues like blood vessels, neural tissues etc.
In our group, we have designed and fabricated various shape-morphing systems towards tissue regeneration. We observed that fabrication of fibres using electrospinning technique incorporates high porosity which proved to be beneficial towards fast actuation in addition to better exchange of nutrients and waste products. [1, 2] Making use of our previous expertise, in our current project, we are fabricating NGC using smart materials which is expected to overcome limitations not accessible via state of art technologies. We have fabricated fibrous bilayer which is able to self-fold owing to the non-uniform swelling of the two layers. The inner layer of the fibrous bilayer consists of aligned fibres produced using coaxial electrospinning technique with active biomolecules like growth factors in the core and conductive material in the shell. [3] The shell of the fibres will help in providing electrical stimulation whereas the core will help in growth of nerve cells by sustained release of growth factors upon slow degradation. The thoughtfully designed NGCs using smart materials and advanced techniques have potential to overcome the current limitations associated.
[1] Apsite, I; Stoychev, G; Zhang, W; Jehnichen, D; Xie, J; Ionov, L. Biomacromolecules, 2017, 18, 3178
[2] Apsite, I.; Constante, G.; Dulle, M.; Vogt, L.; Caspari, A.; Boccaccini, A. R.; Synytska, A.; Salehi, S.; Ionov, L. Biofabrication, 2020, 12 (3), 035027.
[3] Yang Lu, Jiangnan Huang, Guoqiang Yu, Romel Cardenas, Suying Wei, Evan K. Wujcik1 and Zhanhu Guo. Nanomed. Nanobiotechnol., 2016, 8, 654–677
83767208488
Introduction
Chronic back and joint pain has been rated as a top risk factor of disability worldwide. In vitro culture of peripheral sensory nerve, namely dorsal root ganglion (DRG) neurons, is a useful model to investigate pain-associated biology and to discover novel regenerative medicine in terms of pain alleviation. Typical monolayer culture of DRG cells, however, losses the multicellular shape, which may cause a disturbed intercellular communication. For example, the multicellular structure provides the basis of synchronized cross excitation commonly observed in vivo [1]. We used the sound induced morphogenesis (SIM) method to aggregate DRG cells into a multicellular system [2]. Viability and calcium signal synchronization of the DRG cells were evaluated in the sound induced multicellular system.
Methodology
DRG cell line ND7/23 was aggregated using SIM in a 0.5 mg/mL collagen solution with a cell seeding density of 0.2 M cells/ml. Cells randomly distributed in collagen gel without SIM served as control (non-SIM control). The DRG cell viability was analysed using live dead staining. After 2 days of culture, the neuronal discharge was evaluated using calcium imaging (Fluo4) [3]. In each culture, 5000 pairs of neurons were randomly sampled to investigate the synchronization of their calcium signalling which was quantified by the ratio dividing their synchronized calcium event number by total calcium event number (synchronization ratio).
Results
Cells in the SIM-induced aggregates displayed higher viability comparing to those outside the aggregate in the same culture (viability 91.9% vs 50.6%) and cells in the non-SIM control (viability 91.9% vs 77.1%). Higher calcium synchronization was found in the SIM group compared to non-SIM control. The synchronization ratio was elevated from 20.4% to 28.1% comparing SIM to non-SIM control. Interestingly, in the SIM culture, cells outside the aggregate also displayed elevated synchronization ratio (29.9%), indicating that the SIM-induced intercellular communication did not depend on an intercellular apposition. Generation of multicellular systems by patterning primary DRG neurons from large animal is ongoing. Omics studies will be performed to unravel their physiological relevance.
Conclusions:
The multicellular system allows a better inter-neuronal communication to form the synchronized neuronal discharge which has been formerly observed in vivo [1]. Thus, our multicellular culture system not only reconstruct the complexity of in vivo morphology but is also important to recapitulate the in vivo function.
Reference
1. Kim, YS. et al., Neuron. 91(5), 1085-1096 (2016).
2. Petta, D. et al., Biofabrication. 13, 015004 (2020).
3. Ma, J. et al., Neurospine. 17(1), 42–59 (2020).
94238136546
Introduction:
3D in vitro systems are an envisioned alternative to animal models especially in drug testing for research of antitumor treatments (1). Within native tissue microenvironments, the vascular system supports the physiological organ growth with nutrients and growth factors, but also plays important role in pathological conditions such as treatment-resistant tumor progression. Among other cancer types, malignant pleural mesothelioma (MPM) is an example of the latter implication. Reproducing vascular organization within in vitro models of cancer is therefore highly needed for more reliable in vitro drug testing platforms. Therefore, bottom-up approaches and novel biofabrication methods have been increasingly considered for the creation of functional tissues in a layer-by-layer approach. Sound patterning allows the spatial arrangement of biological materials such as individual cells or spheroids (2). The sound-driven hydrodynamic forces induce contactless condensation of cells within hydrogels into defined and reproducible patterns (3). This fast, mild, and simple process can be applied to assemble endothelial cells into a microcapillary network which could serve as vascularization system for functional 3D models at the centimeter scale.
Methods:
Green fluorescent protein expressing human umbilical vein endothelial cells (gfp-HUVEC), human pericytes (hPC) from placenta were used for sound patterning of the microcapillary network layer in fibrin. The patterning procedure was realized with the sound patterning device (mimiX biotherapeutics, Switzerland). The microcapillary network was characterized with fluorescence microscopy. A proof-of-concept of tumor microenvironment (TME) was realized by adding a heterotypic tumor spheroid to the assembled microcapillary network. MPM cells and human fibroblast were used for the tumor spheroid preparation by spontaneous assembly in low adhesion well plates. The growth of the microcapillary network was quantified from the fluorescence image analysis. Ultimately, the effects of anticancer (Cisplatin, Platinol®) and antiangiogenic (Bevacizumab, Avastin®) drugs on the model were evaluated.
Results:
Sound-patterned microcapillary ring networks were created. The high cell packing density induced by sound into the pattern's line facilitated cell-cell connection to form microcapillary structures with the expression of VE-cadherin and lumen formation. The microcapillary ring pattern had a diameter of 1781 ± 142 µm and a thickness depending on the cell seeding density (100-400 ± 30 μm). The presence of the tumor spheroids induced 100 % more area covered by the network compared to the capillary network cultured alone, and further 50 % increase in presence of anticancer drug and tumor spheroid.
Conclusion:
We demonstrated that a microcapillary network layer can be fabricated by sound patterning technology. Thereby, we created a 3D in vitro model used to assess the crosstalk between different biological components as proven by drug-induced biological response. Overall, in this study a novel concept for biofabrication of vascularized models is presented as an alternative to overcome present limitations. In subsequent studies, the system will be transferred to a custom-built open chamber to host a centimeter scale multicellular construct, allowing for perfusion of the vascular network.
References:
1) S. Perrin et al. Nature, 2014
2) D. Petta et al. Biofabrication, 2020
3) A.G. Guex et al. Materials Today Bio, 2021
20941830404
Melt electrowriting (MEW) technique is a manufacturing technology used to fabricate scaffold with user-oriented design. Main distinctive compared to additive manufacturing technique is its ability to fabricate the diameter of few micrometers to sub micrometers range while maintaining high surface to volume ratio. However, this technique has certain drawbacks, such as inaccurate large volume scaffold and fiber placement, sagging and fiber pulsing behavior, and thermal damage to the material properties.
We introduce a controlled way of modulating fiber formation in MEW by optimized installation of a second heater in the vicinity of the Taylor cone, thus presenting additional processing parameters that help to tune the scaffold design parameters more robustly and mitigate some of the current drawbacks of this technique. The primary function of the second heater is to control the solidification rate of the polymer by increasing the ambient temperature surrounding the nozzle. The study is divided into four different sections. 1) Non-isothermal modelling and simulation using COMSOL are performed to optimize the location of the second heater to the nozzle axis and predict the temperature distribution along the spin line region with varying second heater temperatures. 2) Critical speed and fibre pulsing under different conditions are analyzed to evaluate the effect of the second heater on jet stability. 3) Mechanical testing of the stacked fibres is characterized to model/predict the fusion between layers under different conditions adn at last 4) Spinning of thermosensitive and high molecular weigth polymers are investigated, with a low syringe temperature to mitigate degradation and increasing spin line temperature to facilitate processing.
Overall, with the help of the second heater, we were able to fabricate scaffolds from a high molecular weight medical-grade polycaprolactone, poly L-lactide and PVDF, thus expanding the range of materials processable by this exciting technology.
52354535444
Introduction
Within the scope of personalised healthcare in the field of regenerative medicine, patient-derived cells are key players. Their successful application is, however, often hampered by low cell numbers at the expense of donor-site morbidity and lengthy in vitro expansion. Novel biofabrication methods requiring lower initial cell numbers are therefore timely to address this unmet clinical challenge. In vitro, local cell density enhancement by use of sound induced morphogenesis (SIM) at low frequency of <100 Hz was shown to induce increased microvasculature formation at lower cell concentration than conventional methods.1 Based on this, we are developing cell-hydrogel biografts with local cell density enhancement and evaluate their performance after subcutaneous implantation at the back of nude mice. Our research is driven by the hypothesis that local cell density enhancement can improve the therapeutic efficacy in various clinical scenarios such as anastomosis within wounds or bone formation of non-union fractures.
Methodology
We followed a twofold approach, assessing on the one hand anastomosis of implanted human umbilical vein endothelial cells (expressing green fluorescent protein, GFP-HUVEC) and on the other hand ectopic bone formation of mesenchymal stem cells (MSC). To assess anastomosis, HUVEC and MSC were mixed at a 1:1 ratio and resuspended in PEG-based or Dextran-based hydrogels at a final concentration of 2×106 cells per mL. To assess ectopic bone formation, MSC were resuspended in PEG-based hydrogels at a final concentration of 2×106 or 5×106 cells per mL, with or without BMP-2. Cells were then placed on a custom-made acoustic bioprinter and assembled into distinct patterns at a frequency of 60 Hz. Four cell-hydrogel biografts of approximately 4 x 9 mm2 were implanted at the back of nude mice and harvested after 2 or 8 weeks, respectively. Explants were fixed and either imaged as whole constructs or embedded in paraffin for subsequent histological analysis.
Results
By adjusting formulations of PEG-based and Dextran-based hydrogels, time to gelation was increased from 4 minutes to 7 minutes, which proved essential for successful pattern formation by sound. During a 3-day in vitro culture, endothelial cells assembled into pre-vascular structures of tight cell-cell contacts. The animal experiments were conducted with zero mortality during the time of implantation and no other complications. Microscopic evaluation and visualisation of the GFP signal indicated that HUVEC were retained within the PEG-hydrogel after 2 weeks of implantation and formed a pre-vascular network. Further analysis will investigate anastomosis between the host vasculature and implanted HUVEC. Based on visual inspection, ectopic bone formation was more pronounced in samples and regions of higher cell density. In future experiments, the extent of bone formation will by quantified by micro-CT, followed by decalcification and histological evaluation.
Conclusions
Our results provide evidence that sound induced morphogenesis is a versatile method to produce cell-hydrogel biografts for subsequent pre-clinical evaluation. We demonstrated that local cell density enhancement by sound requires a lower initial cell concentration than conventional methods to achieve comparable microvasculature structures or local osteogenesis.
References
1 Petta et al., Biofabrication 2021, 13, 015004
94238122328
"Introduction:
Pericytes reside outside of capillary blood vessels. In the brain, pericytes, brain microvascular endothelial cells, and astrocytes form the neurovascular unit (NVU). A normally functioning NVU regulates cerebral blood flow and the permeability of the blood-brain-barrier (BBB). Breakdown of the NVU and dysfunction of the BBB are increasingly recognised early biomarkers of dementia, including Alzheimer’s disease. An isoform of Apolipoprotein E (APOE), namely APOE4, which is the strongest genetic risk factor for Alzheimer’s disease1, has a particular importance in BBB dysfunction compared to the E3 variant. Pericyte dysfunction has received increasing recognition in Alzheimer’s disease where pericytes contract capillaries and thus reduce cerebral blood flow, in a downstream response to amyloid-β2. However, pericytes in health and disease remain poorly understood, partially due to a lack of adequate models that can recapitulate the complexity of the multi-cellular NVU. The aim of this study is to investigate the effect of APOE4, compared to APOE3, on the contraction of human pericytes in vitro.
Methodology:
iPSCs from a donor diagnosed with Alzheimer’s disease carrying the APOE e4/e4 allele (UKBi011-A, EBiSC) as well as the isogenic control with the APOE e3/e3 isotype (UKBi011-A-3, EBiSC) were differentiated into pericytes3. Success of differentiation was confirmed by immuno-histochemistry of pericyte markers. The labelled calcium indicator Fluo4 was used to measure increase in fluorescence, thus increase in calcium release, within the iPSC-derived pericytes.
Results:
APOE3- and APOE4-pericytes stained positively for NG2, PDGFR-β, calponin, and SM22. Genomic sequencing confirmed the isoforms of APOE3 and APOE4. Fluo4 staining was used to observe calcium release, which was triggered by two known vasoconstrictors Endothelin-1 and U46619, the latter one being a thromboxane A2 receptor agonist. Calcium firing was observed for a period of at least 10 minutes. Change in cell area and calcium increase were quantified using an automated image analysis pipeline in CellProfiler.
Conclusion:
These results suggest that functional pericyte-like cells were obtained and that their calcium release could be triggered. We will build on these findings to develop a robust model to study the effect of APOE4 on pericyte function and dysfunction in health and Alzheimer’s disease.
References:
1. Blanchard J.W., et al., Nat Med. 26(6):952-963 (2020).
2. Nortley R., et al., Science 365(6450):eaav9518 (2019).
3. Faal T., et al., Stem Cell Reports. 2019;12(3):451-460 (2019)."
73296323247
"Introduction
Despite the availability of safe and effective vaccines against COVID-19, new variants of concern (VOC) such as Delta and Omicron are emerging. In these surges, governments and institutions around the world urgently need ways to rapidly respond to outbreaks of new VOCs or other infectious respiratory diseases with pandemic potential.
In this context, tissue-engineered models serve as valuable tools for immediate preclinical and translational research regarding therapeutics and prophylactics. Models faithfully mimicking human respiratory mucosa and submucosa could support research and development regarding COVID-19 and other diseases caused by pathogens entering the body by the respiratory route.
Methodology
Human bronchial fibroblasts (HBFs) isolated from cryo-biopsies were combined with human umbilical vein endothelial cells (HUVECs) inside 3D fibrin hydrogels. Primary human bronchial epithelial cells (HBEs) were seeded on top of the gels, which were then grown on transwell inserts for one week as submerged culture followed by 4 weeks of air-liquid interface culture. The models were fixed and CD31/PECAM-1 and the SARS-CoV-2 entry receptor hACE2 were analyzed by 2-Photon Laser Scanning Microscopy (2P-LSM) and immunohistochemistry, respectively. Furthermore, cultures were examined by Scanning Electron Microscopy (SEM).
SARS-CoV-2 viruses were cultured in Calu-3 cells, isolated, and added from either the apical side, the basal, or both. Cultures were fixed 8 days post-treatment and immunostained for the viral Nucleocapsid (N) protein to visualize the infection. 2D monocultures of HBFs and HUVECs were also infected and analyzed for spike (S) and N protein expression by a newly developed in-cell-ELISA1.
Additionally, a clinical isolate of the VOC Delta was cultured in Calu-3 cells to prepare virus particles that were inactivated by ultraviolet light (UV) irradiation (“UV-Delta”). UV-Delta was added to the aforementioned airway 3D models, which were incubated for 4, 24, or 48 hours, fixed, and examined by histological periodic acid Schiff´s (PAS) reaction and SEM.
Results
Similar to native tissues, histology of the airway tri-cultures showed high expression of the SARS-CoV-2 receptor hACE2 mainly in the epithelium layer. The 2P-LSM 3D stacks of the CD31/PECAM-1 staining showed the HUVECs arranged in a network of capillary-like structures, while the SEM confirmed the presence of a fully developed ciliated epithelium on the apical side of the model.
While the icELISA detection in 2D monocultures verified that both HUVECs and HBF are in principle susceptible to SARS-CoV-2 infection, the N protein immunostaining of tri-cultures revealed that complex models are only permissive to SARS-CoV-2 when exposed to the virus from the epithelium side. The PAS reaction showed a weakening and thinning of the epithelial barrier when exposed to UV-Delta, while the SEM analysis revealed an increase in ciliation of the epithelium after 24 and 48 hours.
Conclusion
Our airway tri-cultures represent a complex model for research regarding the pathogenesis of SARS-CoV-2 and therapeutics against COVID-19. The treatment of the tri-cultures with inactivated viral particles from the VOC Delta induces an increase in ciliation, a counterintuitive effect that differs from the findings with live SARS-CoV-2 and other coronaviruses.
References
20941816389
"Introduction: Craniosynostosis (CS) is a bone developmental condition that affects 1 in 2100 children worldwide, characterised by premature ossification of the cranial sutures. Particularly, non-syndromic-CS (NS-CS) has been associated to microenvironmental causes. However, little is known about the signalling pathways that govern this skull suture premature ossification. Thus, we hypothesize that by investigating the role of microenvironmental cues in NS-CS, we can identify novel ossification therapeutic targets that could be utilised to develop novel biomaterials-based therapeutic treatments for bone fracture healing in adults.
Methodology: Cells were isolated from patent (unfused) sutures, fused sutures and calvarial bone of children (5-28 months) diagnosed with NS-CS. Tissues were collected during cranial vault remodelling –standard CS surgical procedure- at CHI at Temple Street after parental consent and ethical approval were obtained[1]. To evaluate their osteogenic potential, alkaline phosphatase (ALP) activity and extracellular matrix mineralization of cells cultured for 7-21 days in growth (GM) and osteogenic medium (OM) were quantified. Subsequently, to understand how variations in the substrate stiffness affect premature ossification, cells were cultured on soft (10 kPa) and stiff (300 kPa) collagen-coated polyacrylamide substrates[1]. Then, their osteogenic potential and morphological responses were evaluated. The differences in the mechanoresponse of these cells were further investigated with a 96 gene PCR array to identify potential therapeutic targets[1].
Results: Cells from patent and fused sutures expressed similar ALP activity and extracellular matrix mineralisation at the different evaluated time-points, when cultured with GM. Interestingly, when cultured with OM, cells from fused sutures expressed higher mineralisation levels and ALP activity. Thus, suggesting that cells from fused sutures have a stronger osteogenic response than cells from patent sutures when biochemically stimulated. Furthermore, when cultured with GM on soft and stiff substrates, cells from both patent and fused sutures exhibited morphological changes and increase in their spreading area, in a stiffness-increasing manner. Particularly, cells from fused sutures showed a bigger and rounded shape, resembling osteoblasts while cells from patent sutures were elongated, resembling mesenchymal stem cells. Finally, when combining variations in the substrate stiffness and OM, a stiffness-dependent upregulation of genes mediating bone development (TSHZ2, IGF1), activation of inflammation (IL1β), involved in the breakdown of extracellular matrix (MMP9) and controlling osteogenic differentiation (WIF1, BMP6, NOX1), was observed in cells from fused sutures. These findings suggest that the increased osteogenic potential of cells from fused sutures might be associated to the activation of the BMP6, IGF1 and/or MAPK-associated non-canonical WNT pathways.
Conclusions: Our results further suggest that NS-CS may be linked to an abnormal mechanical environment. Understanding the changes in the regulation of genes associated with the premature suture ossification in CS opens up avenues to not only understand better this developmental condition but also will help us to design novel therapeutic strategies to accelerate non-union bone fracture healing in adults.
Reference:
1. Barreto, S. et al., Sci. Rep. 7, 11494 (2017).
Acknowledgements:
This work was funded in part by the Children's Health Foundation Temple Street (RPAC-2013-06 and RPAC-19-01), and by the European Research Council (ERC Advanced Grant ReCaP project #788753)."
20941813149
"
Introduction
The intervertebral disc (IVD) distributes multiaxial loads applied to the spine, namely axial compression, tension, lateral bending, and torsion. The effect of mechanical loading on IVD health and degeneration is commonly investigated in bioreactors used for ex vivo culture of IVD organ models. Currently available bioreactors have mainly integrated one or two degrees of freedom (DOF), while thus far developed multiaxial simulators with 6 DOF lack control of biological conditions. A new generation of bioreactors will integrate both six DOF loading and sterile culture of IVD organ models. Such a multiaxial system requires the implementation of the holding mechanism that must enable proper transmission of the loads from the bioreactor onto the specimen and sufficient nutrition through the cartilaginous endplate. We developed and validated a sample holder and new ex vivo IVD organ model according to the requirements for multiaxial bioreactor culture.
Methodology
A customized, circular IVD holder with a central cross pattern was designed. An ex vivo bovine caudal IVD organ model was adjusted to maintain more vertebral bone than the standard model (5-6 mm instead of 0.5 mm) to machine the cross counterpart and a central hole for nutrient access. The new and standard models were compared for long-term maintenance in a bioreactor under physiological conditions by alternating cyclic compressive uniaxial loading (0.02-0.2 MPa, 0.2 Hz, 2h/day) and overnight free swelling recovery. The disc height changes were measured daily, and cell viability was assessed with histology after 1, 2 and 3 weeks of culture (n= 2 for each time point) in comparison to day 0 samples (n= 3). The interface of the new IVD model and sample holder was enhanced with tightening of side screws onto the bone, or a combination of side screws and top screws, or side screws and adhesive, and was tested for failure point in compression, tension, torsion, and lateral bending (n=3 for each test).
Results
The new model retained a high level of cell viability during three weeks of in vitro culture (standard versus new model after 3 weeks: outer annulus fibrosus 82% and 84%, inner annulus fibrosus 69% and 64%, nucleus pulposus 75% and 73%). In both models, the decrease in IVD height after loading was in the range of typical physiological conditions (≤ 10%). When differently directed motions were applied, the holder-IVD interface with side screws transmitted compression and torsion above reference values (average obtained values were 320.37 N and 1.64 Nm, respectively), while the combination of side and top screws improved the resistance to tension and bending compared to the targeted values (average obtained values were 431.86 N and 0.79 Nm, respectively).
Conclusion
We have developed a mechanically reliable holding system for application in a new generation of multiaxial bioreactors and demonstrated that the new ex vivo IVD organ model can be maintained in long-term culture. Additional studies are envisaged to validate the system in the new bioreactor. Such a unique bioreactor will enable overcoming the gap between preclinical in vitro cultures, animal models, and clinical trials."
20941829709
Cancer continues to be a leading cause of mortality in modern societies; therefore, improved and more reliable in vitro cancer models are needed to expedite fundamental research and anti-cancer drug development. Here, we describe the use of a miniaturized continuous stirred tank reactor (mCSTR) to first fabricate and mature cancer spheroids (i.e, derived from MCF7 cells, DU145 cells, and a mix of MCF7 cells and fibroblasts), and then to conduct anti-cancer drug assays under continuous perfusion. This 3 mL mCSTR features an off-center agitation system that enables homogeneous chaotic laminar mixing at low speeds to support cell aggregation. We incubated cell suspensions for 3 days in ultra-low-adherence (ULA) plates to allow formation of discoid cell aggregates (~600 µm in diameter). These cell aggregates were then transferred into mCSTRs and continuously fed with culture medium. We characterized the spheroid morphology and the expression of relevant tumor biomarkers at different maturation times for up to 4 weeks. The spheroids progressively increased in size during the first 5 to 6 days of culture to reach a steady diameter between 600 and 800 µm. In proof-of-principle experiments, we demonstrated the use of this mCSTR in anti-cancer drug testing. Three drugs commonly used in breast cancer treatment (doxorubicin, docetaxel, and paclitaxel) were probed at different concentrations in MCF7 derived spheroids. In these experiments, we evaluated cell viability, glucose consumption, spheroid morphology, lactate dehydrogenase activity, and the expression of genes associated with drug resistance (ABCB1 and ABCC1) and anti-apoptosis (Bcl2). We envision the use of this agitated system as a tumor-on-a-chip platform to expedite efficacy and safety testing of novel anti-cancer drugs and possibly in personalized medicine applications.
62825463608
Introduction
Plasma cell malignancy - multiple myeloma (MM), occurs primarily in the bone marrow (BM) and appears to be strongly regulated by interactions with BM mesenchymal stromal cells (MSCs). In addition, collagen is an important constituent of the BM environment and provides not only structural, but functional support to many of its cells. Improved understanding of MM pathology requires a development of 3D models that can recapitulate the BM-supported MM survival and intercellular communication. Therefore, we aimed to establish a collagen-based 3D co-culture model of MM cells and MSCs.
Methodology
MSCs were obtained from BM of patients undergoing total hip arthroplasty. For 3D co-culture, MSCs and semi-adherent MM cells (MM1.S) were grown in hydrogels generated using rat-tail collagen type I. For mono-cultures, 3 x 105 cells were cultured per 100 µl of collagen, whereas in co-culture, the cells are seeded in an initial one to one ratio, giving a total of 3 x 105 cells (2D controls were performed in parallel). Viability and metabolic activity of encapsulated cells were examined performing an MTT assay and measuring ATP concentration at 1, 3 and 7 days of culture. Gene expression was assessed by qPCR and proliferation evaluated using flow cytometry. Immunohistochemistry was performed to identify functional marker changes. Finally, clonogenic capacity of both cell types was determined after retrieval from collagen gels.
Results
Both MM1.S cells and MSCs were successfully encapsulated and cultured in collagen gels. Upon day 7, the portion of MM1.S cells exceeded the percentage of MSCs in the 3D co-culture system, where MM1.S cells accounted for about two-thirds of total retrieved cells. The viability of the cells did not seem to be affected by a collagen-based environment. However, the proliferation of MM1.S cells appeared changed, with a decreased Ki-67high fraction of MM1.S cells growing in 3D culture, suggesting a attenuated proliferation potential, regardless of the presence of MSCs. Reduced metabolic activity and ATP production were observed in 3D cultured MM1.S cells and MSCs in comparison to standard 2D culture. In addition, MM1.S cells recovered from 3D collagen cultures showed a higher clonogenic potential when compared to 2D cultures.
Conclusion
Here, we established a physiologically relevant model system to co-culture semi-adherent MM cells and MSCs. The collagen-supported 3D co-culture of BM myeloma cells and MSCs might alter their behavior, governing MM cell viability and clonogenicity, in a manner different to conventional 2D systems. Further optimization of the 3D co-culture will provide significant insights in the behavior of MM cells and, ideally, their response to anti-cancer treatments.
73296342489
Epithelial Ovarian cancer (EOC) is the most lethal malignancy in women. Despite de-bulking surgery, chemotherapy and radiotherapy almost 90% of EOC patients will relapse and succumb. One of the main causes of drug resistance and metastasis is the tumor microenvironment (TME) consisting of cancer-associated fibroblasts (CAFs), endothelial cells (ECs), anergic immune cells, adipocyte stem cells (ASC) and other components1,2. Mounting evidence suggest that microRNAs (miRNAs) play critical roles in shaping the TME by inhibiting gene transcription in cancer cells3.
We have previously shown that miR-200c was able to contrast the induction of the immune checkpoint PD-L1, c-myc and β-catenin oncogenes expression by combinatorial therapies in EOC cell lines and biopsies4. Another study showed that miR-200c inhibited epithelial-to-mesenchymal transition (EMT), by targeting ZEB15. Recently, ASCs were discovered as key players in EMT of OC1,6. Since miR-200c targets multiple genes and de-regulates different molecular pathways, more physiologically relevant human preclinical models are required to unravel its role in TME.
In this work, we 3D printed a biomimetic human 3D printed-OCTME model where miR-200c transfected tumor cells interact closely with immune cells and ASCs. The proposed model conveys unprecedent physiological features to better evaluate the effects of miR-200c on i) anti-tumor T-cell response, ii) ASC immunomodulation and iii) EMT in OC cells.
For the 3D TME model, a microfluidic-based extrusion bioprinter was used to deposit two GelMa-based bioinks containing miR-200c-transfected SKOV3 cells and ASCs isolated from healthy donor. The microfluidic spatial control on the material deposition results in the fabrication of cylindrical constructs with radial cell concentration gradient (i.e. tumor cells in the core, ASCs in the shell). After 3 days, PBMCs, isolated from healthy donors, were added on top of the constructs and let for 3 hours to penetrate the gel. The same bioprinting conditions were also used for the parental cell line transfected with the empty vector. Confocal microscopy of cell-labeled constructs at day 5 revealed the formation of a TME exhibiting the physiological architecture found in vivo. ASCs cells surrounded the tumor site enabling the interaction with tumor cells. T-cells traveled across the stromal tissue and reached the tumor site, demonstrating their successful inclusion in the structure. Cells viability and functionality were addressed by live/dead assay and immunostaining of activated T-cells and SKOV3.
The proposed study aims to develop a 3D TME in vitro preclinical model to better understand the interactions between tumor cells, immune cells and ASCs in the presence/absence of miR-200c. Our technology is a proof-of-concept assay toward a precision stratification of EOC patients before undergoing miRNA-based and drug therapies, to test the therapeutic effectiveness and avoid side effects of these treatments.
62825424005
Introduction. In recent years tumor microenvironment has been recognized as one of the main actors in cancer relapses and metastasis. Indeed, the crosstalk between malignant cells and the niche, as well as the extracellular matrix (ECM) remodeling, not only favor tumor mass growth, but are also preliminary mechanisms to dissemination. These processes have been mainly investigated in tumors of epithelial origin, for which many 3D models have been produced, while little is still known about sarcoma microenvironment. This is true in particular for rhabdomyosarcoma, the most frequently diagnosed soft tissue sarcoma in pediatric patients. Thus, the development of a suitable 3D model to recapitulate the onset of metastasis, which is mostly driven by an altered ECM deposition, is in need. Macromolecular crowding (MMC) has been shown to enhance the process of ECM deposition. The aim of this work was the application of MMC to rhabdomyosarcoma cell lines. A 2D monolayer culture and a 3D spheroid model were compared with a 3D bioprinted construct.
Methodology. RH30 and RD cell lines were cultured under MMC in 2D in presence of a Ficoll cocktail. MMC was also applied to spheroids produced in ultra-low attachment (ULA) plates. Finally, a 3D bioprinted model was produced using the inkjet printing technology and Matrigel as scaffold, to which also MMC was applied. ECM deposition in these samples was analyzed by immunofluorescence and qRT-PCR, and shape parameters were described for the 3D samples.
Results. MMC induced ECM deposition in both cell lines in 2D, which resulted in increased fibronectin signal in immunofluorescence images. MMC-treated spheroids displayed not only augmented ECM protein presence, but also reduced dimensions and higher solidity compared to untreated spheroids. Interestingly, only RH30 spheroids treated with MMC attached to ULA culture surfaces, depositing collagen and fibronectin on the bottom of the culture well. Also, the 3D printed constructs treated with MMC were smaller and more compact than the untreated ones, probably due to the increased amount of ECM components; furthermore, after day 10 of culture small multicellular aggregates detached from the core of these samples, mimicking a dissemination mechanism. This release was less evident in the MMC-treated condition compared to control, but interestingly in both conditions RH30 samples released a higher number of spheroids compared to RD. Of note, CXCR4 levels increased in RH30 samples under MMC stimulation, a gene involved in metastasis promotion.
Conclusions. These observations indicate that MMC stimulate ECM deposition in rhabdomyosarcoma cells and induce a metastatic behavior. As future perspective, the MMC-treated and 3D printed model developed in this work could find applications in drug testing.
20941866339
Introduction: Dystrophic epidermolysis bullosa (DEB) is a genodermatosis caused by mutations in the COL7A1 gene, in which patients exhibit mechanically fragile skin (1). The underlying mutation determines the clinical phenotype, which can range from a severe condition characterized by widespread blistering associated with the development of chronic wounds-RDEB (recessive subtype) - to a relatively mild disorder associated with localized blistering-DDEB (dominant subtype). To date, the impact of the different COL7A1 mutations on ECM biological and mechanical properties is largely unexplored. Hence, this work aimed to investigate the differences in ECM that occur amongst DEB subtypes and, for the first time, to establish a correlation between these alterations and ECM mechanical properties.
Methodology: Immortalized cells from patients representing three DEB subtypes (different COL7A1 mutations and disease aggressiveness) were obtained from EB House Austria. As a control, healthy primary fibroblasts were used. The cells were cultured for 14 days with 50µg/mL ascorbic acid to promote maximum ECM deposition. Mass spectrometry-based label-free quantification was used to assess changes in the deposited ECM. Western blot and immunocytochemistry were used to further dissect the abnormal ECM features in the different DEB subtypes. Furthermore, the influence of distinct COL7A1 mutations in ECM mechanical properties was addressed at nanoscale by Atomic Force Microscopy (AFM) and at macroscale by uniaxial tensile testing.
Results: Extracellular proteome analysis revealed that fibroblasts from each DEB subtype have their own unique proteomic fingerprint. Independently of the disease subtype - and its associated clinical aggressiveness - down-regulation of structural proteins that impact ECM tensile strength and compression to resistance were identified. Additionally, all DEB subtypes demonstrated a decrease in the dermal-epidermal junctional proteins collagen IV and laminin, as well as an increase in ECM proteins closely associated with wound healing and scarring (tenascin C and vimentin). Furthermore, those differences strongly impact the ECM mechanical properties. At the nanoscale, data indicated a significant reduction in the mechanical stability of the native ECM produced by the different DEB subtypes, which was simultaneously associated with a significant decrease in macroscale stiffness. Interestingly, the more severe subtype (RDEB) results in a significant loss in ECM tensile strength when compared to a healthy control, whereas the milder form (DDEB) shows no difference.
Conclusion: This study demonstrates that different COL7A1 mutations in DEB have a significant impact on the overall dermal ECM features, with structural proteins involved in the mechanical stability being down-expressed and proteins involved in pathological ECM remodeling of wound healing and scarring – hallmarks of DEB - being over-expressed. Furthermore, these changes in the biological features of the ECM have a major effect on the native ECM’s mechanical integrity at both the macro and nano scales. Overall, this work contributes to the advancement of DEB knowledge, being the first to correlate alterations in ECM composition with its mechanical properties in disease scenario.
Acknowledgements: Financially supported by ERC Consolidator Grant ERC-2016-COG-726061,FCT project MImBI-PTDC/EME-APL/29875/2017 and LAETA project UIDB/50022/2020. FCT for grant SFRH/BD/137766/2018(MDM),grant SFRH/BD/147807/2019(AA) and contract Grant No.Norte-01-0145-FEDER-02219015(MTC).
References:
1.G.Tartaglia et al. Int. J. Mol. Sci. 22 (2021).
73296308004
Artificial cells have been topic of intensive investigation over the past years. Although in first instance they have been mainly developed as a platform to attain a better fundamental understanding how living cells operate, they have recently also been recognized as interesting structures in biomedical research. In this lecture I will discuss an artificial cell system based on complex coacervates as developed in our group. These are formed by the mixing of two oppositely charged amylose derivatives that spontaneously interact and phase separate into a polymer-rich and polymer-depleted water phase. The coacervate microdroplets that are in first instance created are prevented from coalescence and further growth by a stabilizing polymer membrane. Coacervates droplets are interesting cell mimics since their crowdedness shows similarities to the dense cytoplasm of living cells. Furthermore, they have the ability to effectively sequester cargo. The polymer membrane we have created not only provides stabilization, but also allows exchange of molecules with the outside environment. We have demonstrated that biocatalytic cascades can be efficiently executed within one artificial cell, as a result of the high accumulation (150 fold) of the enzymes in the coacervate core. Furthermore, we have also been able to achieve uptake of protein cargo using affinity tagging; by incorporation of a Ni-NTA moiety in the coacervate, His-tagged protein were taken up highly effectively. By enzymatic removal of the His tag the proteins could also be controllably released. We have extended this uptake and release behavior to a DNA mechanism, which now allows for reversible shuttling of proteins in and out of the artificial cell, and between artificial cells. We have also investigated the biocompatibility of our artificial cells when in contact with living cells. After appropriate purification procedures we could demonstrate that the artificial cells are well tolerated. These developments have now opened up the way to combine artificial cells with living cells in the formation of organoids, in which the artificial cells are used as sending units of growth factors with spatiotemporal control.
62903402107
Artificial cells are biomimetic systems used to study properties of biological cells and to explore new possible applications in place of biological cells. Tissue engineering aims at replacing or recovering damaged or diseased tissues, or reconstituting tissues in vitro for disease modelling and drug development. Artificial cells can be combined with modern biofabrication strategies like bioprinting to generate bottom-up 3D tissues and organs. The Duarte Campos Bioprinting Lab investigates biofabrication technologies and biomaterials suitable for tissue and organ engineering, and their impact on the structure and function of natural and synthetic living tissues. This lecture will show our major achievements toward engineering lab-grown tissues and in vitro models using biological cells, and report on our first steps on the path to bioprinting tissues with artificial cells.
52419501284
"Tendon injuries are the top reported musculoskeletal injuries with Achilles, patellar, and rotator cuff tendons as the most common types of tendon injuryy (Ackermann & Institutet, 2013). Tendon injuries are a common cause of disability and account for decreased productivity. Both chronic and acute injuries can result in long-term pain and disability(Pabón & Naqvi, 2021).Extracellular vesicles, specifically exosomes, treatment has proven to enhance regenerative strategy by improving ; biomechanical properties, accelerating tenocyte proliferation and migration, reducing inflammation, and facilitating the healing at tendon–bone interface(Ren et al., 2021).
A THP-1 cell line activation model used to quantify the effects of tenocyte conditioned media (CM) on the proliferation and morphology of the activated THP-1 cells. When THP-1 cells are activated, they polarize into macrophages and monocytes depending on environmental signals. Flow cytometry was used to accurately characterize specific macrophage phenotypes in the presence of tenocyte CM by using CD-markers (CD14,CD25,CD197,CD204 CD206 and HLADR) to understand if the modulation triggered inflammation(M1) or supressed inflamation (M2) phenotype Exosomes isolated from tenocyte CM cultured at 21% and 2% oxygen concentrations then filtered (0.22um) before differential centrifugation (100,000g 90mins), pellets were analyzed for protein concentration and particle size then added to a density gradient prepared with Optiprep solution and spun at 100,000g for 16 hours. Protein concentration was obtained for 12 fractions, iodixanol content(via UV vis 230nm) and particle size (zetasizer). Fractions 4-7 (1.08g-1.22g) were spun again for 2hrs at 120,000g to remove idoxoanol ,finally the presence of exosomes was confirmed via Scanning electron microscopy and zeta sizer particle size data.
Tenocyte CM obtained at 21% and 2% oxygen concentrations, have higher levels of inflammatory cytokines when compared with serum free DMEM. Cytokine levels obtained via quantitative cytokine array (eve technologies Canada). Significant differences observed in cytokine levels (IL-2, IL-6 and IL-8) with 21% CM displaying higher levels (28ug,22,20ug/ml) than 2% oxygen.(19,16,15ug/ml) .The most abundant cytokines were; IL-2 IL-6, IL8, TNFa in CM in both oxygen concentrations. Flow cytometry utilizing; CD197, HLADR and CD86 we were able to demonstrate that activated THP-1 cells cultured in tenocyte CM showed a phenotypic shift towards higher levels of M1 macrophages (inflammatory state), at 21%. In 2% tenocyte conditioned media a lower level of expression of all but CD25 when compared to 21%, pointing towards hypoxia inducing a suppression of M1 and M2 macrophages. However, 2% CM THP-1 cells demonstrated higher levels of dendritic cells when compared to 21% (established by CD25 expression. Exosome preparations confirmed a protein concentration of 120ug/ml after density gradient ultracentrifugation, we further characterized via zetasizer which confirmed particles from 30nm-150nm and SEM imaging to confirm the presence of exosomes (via morphology and size).
Futhur characterisation using ; tetraspanins and CD markers. Exosome preparations will be added to the THP-1 activation model in order to observe how exosomes influence the macrophage phenotype. Tenocyte conditioned media is able to modulate the immune system and stimulate healing."
62825469128
MATRIX-BOUND NANOVESICLES AS SELECTIVE MODULATORS OF THE IMMUNE RESPONSE
Introduction: Matrix bound nanovesicles (MBV) have recently been identified as an inherent component of the extracellular matrix (ECM) and possess the ability to mitigate the proinflammatory activation state of macrophages. While the “anti-inflammatory” properties of MBV have several potential clinical applications, it is unknown if there is an associated compromise of the broader immune system. Stated differently, the systemic effects of MBV, and more specifically the effects of MBV upon the adaptive immune system and the ability to mount a protective immune response to pathogens is unknown and has not been explored.
Objective: To investigate the effects of MBV at systemic level and upon the adaptative immune response to pathogens.
Methodology: Biodistribution of MBV was assessed by fluorescence tracking after systemic administration intraperitoneally (IP) and intravenously (IV) in mice. Antibody and blood work analysis were carried out to assess biosafety. MBV modulation of pro-inflammatory activation was assessed in vitro in macrophages and in vivo in a psoriasis model in mice.
To assess the effect of MBV on the immune humoral response to pathogenic infection, mice were vaccinated with the pneumococcal vaccine PneumoVax™23 at day 0. Then, for 5 weeks, MBV (1012/mouse) were injected IP weekly, while a weekly dose of methotrexate was used as immunosuppressor control. Anti-pneumococcal polysaccharide IgG and IgM antibody titers were measured at days 7 and 28. At week 5 mice were infected with Streptococcus pneumoniae and the survival of the animals was recorded over 2 weeks.
Results: Antibody levels and bloodwork in healthy animals treated with MBV showed no relevant fluctuations nor biosafety concerns, whereas biodistribution showed accumulation in depurating organs such as liver, kidneys and spleen. MBV downregulated inflammatory markers in pro-inflammatory macrophages in vitro and meliorated the symptoms of imiquimod-induced psoriasis in mice corresponding with a downregulation of the IL-17/IL-23 axis, which corroborated the anti-inflammatory properties of MBV.
Pneumococcal vaccine-immunized mice with and without MBV treatment presented similar anti-S. pneumoniae polysaccharide IgG and IgM titers at days 7 and 28, demonstrating that MBV do not compromise the adaptative immune response. Contrary, immunized mice treated with methotrexate showed lower IgG and IgM titers. The functionality of the humoral immune response was further confirmed with the higher survival in vaccinated mice with and without MBV treatment, conversely to unvaccinated and methotrexate treated animals. Interestingly, when infected with a lethal dose of S. pneumoniae (107 CFU/mouse), 50% of mice treated with MBV after vaccination presented complete recovery after 14 days, whereas the rest of the groups showed no survival after 2 days of infection. These results suggest a boosting effect on the adaptative immune response elicited by MBV.
Conclusion: The anti-inflammatory properties of MBV do not compromise the ability of the adaptative immune system to build up a response against pathogens. Moreover, preliminary results suggest that MBV could further enhance the humoral immune response.
94238103084
Purpose/Objectives: Rheumatoid Arthritis (RA) is an autoimmune disease characterized by chronic inflammation and destruction of synovial joints that affects approximately 7.5 million people worldwide. Disease pathology, while multifactorial in etiology, is driven by an imbalance in the ratio of pro-inflammatory vs. anti-inflammatory immune cells, especially macrophages. Modulation of macrophage phenotype, specifically an M1 to M2, pro- to anti-inflammatory transition, can be induced by biologic scaffold materials composed of extracellular matrix (ECM). The ECM-based immunomodulatory effect is thought to be mediated in part through recently identified matrix-bound nanovesicles (MBV) embedded within ECM. While it is known that an M1:M2 disequilibrium contributes to RA disease progression, there are no therapies available that specifically modulate macrophage phenotype to promote disease remission through an M2, anti-inflammatory phenotype. There is thus a clear unmet need for developing approaches to modulate rather than suppress the immune response for the treatment of autoimmune diseases such as RA. The evidence supporting ECM- and MBV-mediated immunomodulation of macrophage phenotype, combined with the clinical evidence of pro-inflammatory M1 macrophages as a key mediator of RA, provides the premise of the present research. Using the pristane-induced, pre-clinical rat model of RA, it was hypothesized that MBV would reduce inflammatory arthritis disease development, decrease synovial inflammatory cell infiltration, prevent adverse cartilage remodeling, modulate synovial and systemic macrophage populations from a pro-inflammatory M1 phenotype towards an anti-inflammatory M2 phenotype, and thus promote disease resolution.
Methodology: Isolated MBV were delivered via intravenous (i.v.) or peri-articular (p.a.) injection to rats with pristane-induced arthritis (PIA). The results of MBV administration were compared to those following intraperitoneal (i.p.) administration of methotrexate (MTX), the clinical standard of care, using disease scoring, microCT imaging, histopathology, multiplex cytokine analysis, and multi-parameter flow cytometry.
Results: Relative to the vehicle treated animals, i.p. MTX, i.v. MBV, and p.a. MBV reduced arthritis scores in both acute and chronic phases of pristane-induced arthritis, decreased synovial inflammation, decreased adverse joint remodeling, and reduced the ratio of synovial and splenic pro-inflammatory M1 macrophages to anti-inflammatory M2 macrophages (p<.05). Both p.a. and i.v. MBV, but not MTX, reduced the serum concentration of RA and PIA biomarkers CXCL10 and MCP-3 in the acute and chronic phases of disease (p<.05). Flow cytometry dimensional data reduction with Uniform Manifold Approximation and Projection (UMAP) revealed the presence of a systemic CD43hi/His48lo/CD206+, immunoregulatory monocyte population unique to p.a. and i.v. MBV treatment associated with disease resolution.
Conclusion/Significance: The results show that the therapeutic efficacy of both systemic and local administration of MBV is equal to that of MTX for the management of acute and chronic, pristane-induced arthritis, and further, this effect is associated with modulation, not suppression, of local synovial macrophages and systemic myeloid populations. The findings suggest that the immunomodulatory properties of ECM-based materials, specifically the MBV component of ECM-based materials, have therapeutic potential for diseases driven by a dysregulated immune system such as RA. The anti-inflammatory effects of ECM-based products have been well documented, and the expanded clinical applications made possible by MBV are worthy of further investigation.
20941808655
Introduction. Matrix-bound nanovesicles (MBV) are nanometer-scale extracellular vesicles secreted by cells and found embedded within the extracellular matrix (ECM)[1]. MBV are similar to exosomes in size and shape, but MBV have distinctly different lipid profiles and RNA cargo[2]. MBV have demonstrated the ability to induce an M2-like pro-healing macrophage phenotype, promote neuronal stem cell differentiation, and suppress pro-inflammatory astrocyte signaling in optic nerve repair[3,4]. MBV have the potential to be used as a therapeutic and diagnostic tool, much like exosomes; however, an understanding of MBV biogenesis is lacking and hence their full potential depends on developing an understanding of the mechanisms of action. Our goal is to determine MBV biogenesis by answering the questions of the intracellular biogenesis of MBV, the mechanism of transport and binding to the ECM, and the mechanisms that govern their production.
Methods. To evaluate the intracellular origin of MBV, we used fluorescent lipid dyes to stain for lipids specific to cellular compartments. MBV and exosomes were isolated from the same cell source (fibroblasts) and were stained with lipids related to various cellular compartments (ie. Golgi) to determine if MBV or exosomes stained positively for these lipids and if we could live-cell image MBV production. To evaluate how MBV bind to the ECM we assess ECM-related surface markers on both MBV and exosomes and use high-resolution imaging to visualize vesicle location related to collagen production. Finally, we assessed the mechanisms that govern MBV production by sequentially deleting ESCRT-independent and -dependent subunits, a pathway in which exosomes use for their production, and assessing the impact of this on MBV production and collagen formation.
Results. We report a process for isolating MBV and exosomes from the same cell source, exosomes from the liquid phase and MBV from the deposited ECM. Ongoing work will be presented on the lipid staining differences between MBV and exosomes, and we expect that MBV and exosomes will have staining differences, as previous work demonstrates MBV have different lipid profiles from exosomes[2]. Ongoing data will be presented on ECM-binding surface proteins on MBV and exosomes, and whether MBV bind to collagen fibrils during or after collagen production. Finally, we show that GW4869, an ESCRT-independent pathway inhibitor, does not inhibit collagen and MBV production, possibly indicating that MBV do not use the ESCRT-independent process for production. We will further present the impact of ESCRT-dependent deletions on MBV and collagen production.
Conclusions. The present work will expand on our knowledge of a new class of extracellular vesicle, MBV. By understanding the mechanisms of their production, the origin of their production, and how they bind to the ECM, this will allow us to better realize their theranostic potential.
References:
[1] Huleihel, L. et al., Sci Adv. 2, e1600502 (2016).
[2] Hussey, G. S. et al., Sci Adv. 6, eaay4361 (2020).
[3] Huleihel, L. et al., Tissue Eng Part A. 23, 1283-1294 (2017).
[4] van der Merwe, Y. et al., Sci Rep. 9, 3482 (2019).
[5] Laulagnier, H. et al., Blood Cells Mol Dis, 35, 116-21 (2005).
20941802564
"PLATELET-DERIVED EXTRACELLULAR VESICLES SHOW THERAPEUTIC EFFECTS ON A 3D TENDON DISEASE MODEL
Ana Luísa Graça1,2, Rui M. A. Domingues1,2, Isabel Calejo1,2, Manuel Gómez-Florit1,2 and Manuela E. Gomes1,2
13B's Research Group, I3Bs – Research Institute on Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, AvePark, Parque de Ciência e Tecnologia, Zona Industrial da Gandra, 4805-017 Barco, Guimarães, Portugal;
2ICVS/3B’s–PT Government Associate Laboratory, Braga/Guimarães, Portugal;
Introduction: Tendon diseases are common clinical problems that can dramatically affect the quality of life of individuals across the demographic spectrum. Current clinical approaches do not tackle the etiology of the disease, underlined by an unresolved inflammatory scenario that provokes hypercellularity, neovascularization, and a dysregulation of the critical balance between extracellular matrix (ECM) remodeling proteases and their inhibitors. In this regard, extracellular vesicles (EVs), a diverse group of nanosized membrane-enclosed particles actively released by all types of cells with key roles in communication, are being considered as very attractive therapeutic agents to trigger repair/regenerative processes in injured tissues. Thus, herein, the therapeutic potential of EVs derived from platelets was evaluated using a pre-establish 3D tendon disease in vitro model.
Methodology: First, bioengineered tendon disease models consisting of electrospun isotropic nanofibrous scaffolds coated with cell-laden hydrogels encapsulating human tendon-derived cells (hTDCs), were produced. Then, different platelet-derived EVs populations were isolated by differential centrifugation, added to hTDCs culture media, and their influence in cells phenotype and ECM remodeling was assessed over culture time.
Results: As expected, after 14 days of culture, a disease-like phenotype was observed in hTDCs of the miniaturized 3D tendon units. We verified that although EVs do not have a remarkable influence in hTDCs morphology, these are able to influence their biological response. Interestingly, the addition of EVs reestablish the expression of tendon-related markers like MKX, SCX, and TNMD in diseased hTDCs and decreasing the expression of osteogenic and fibrotic markers. Moreover, EVs increased the expression of different ECM components such as COL31A and DCN, and the expression of MMP-3, important factors in the balance between the synthesis and degradation of tendon ECM. Moreover, the presence of EVs was found to modulate the inflammatory response, as demonstrated by an increase of anti-inflammatory mediators, like IL-4, which might contribute to blunt the inflammatory processes occurring in damaged tissue.
Conclusions: Overall, we showed that platelet-derived EVs have a positive influence on tendon cells cultured on a disease-like in vitro model, not only by increasing the expression of healthy tendon cells markers and promoting ECM remodeling, but also by increasing the expression of anti-inflammatory cytokines. The beneficial effects of these vesicles are worthy to be explored in further studies to provide more insights on how EVs interact with tendon cells, becoming a promising therapeutic tool for tendon injuries recovery.
Acknowledgments: ERC CoG MagTendon grant agreement 772817; EC Twinning project Achilles 810850; FCT for PhD grant PD/59/2013 and PD/BD/135255/2017, Post-Doc grant SFRH/BPD/112459/2015, CEECIND/01375/2017 and 2020.03410.CEECIND.
"
83767236666
TBA
TBA
Macrophages are known as the most dominating cells at the wound site, and they coordinate the transition between tissue repair phases during the entire wound-healing process. Especially, anti-inflammatory macrophage (M2) subtypes, namely M2a and M2c, are reported to modulate the tissue repair process tightly and chronologically by modulating fibroblast differentiation state and functions. To establish a well-defined three-dimensional (3D) cell culture model to mimic the tissue repair process, we utilized THP-1 human monocytic cells and a 3D collagen matrix as a biomimetic tissue model. THP-1 cells were differentiated into macrophages and activated using IL-4/IL-13 (MIL-4/IL-13) and IL-10 (MIL-10). Both activated macrophages were characterized by both their cell surface marker expression and cytokine secretion profile. Our results demonstrated that surface markers and cytokines secretion profile of MIL-4/IL-13 and MIL-10 is akin to M2a and M2c macrophages derived from human PBMC, respectively. To mimic the initial and resolution phases during the tissue repair, both activated macrophages were co-cultured with fibroblasts and myofibroblasts. We showed that MIL-4/IL-13 can modulate tissue repair by controlled secretion of TGF-β1 to induce fibroblast differentiation, while MIL-10 macrophages secrete high amounts of IL-10 to resolve inflammation and tissue repair processes. Besides, we demonstrate that IL-10 can reverse myofibroblast into fibroblast phenotypes. By neutralizing IL-10 with antibody in co-culture with MIL-10, no dedifferentiation of myofibroblast could be observed, emphasizing the role of IL-10 in resolution of the tissue repair phase. Overall, our results pinpoint the importance of the co-culture model of fibroblast and macrophages for biomimetic wound healing, instead of fibroblast monoculture. In addition, our established biomimetic model can guide the development of well-defined high-throughput platforms for improving tissue healing and anti-fibrotic drugs testing, as well as other biomedical studies.
41883603644
"Introduction
The use of human bilayer tissue-engineered skin substitutes (hbTESSs) for the treatment of dermatological pathologies is a promising therapy, especially for severe burn patients where there is a lack of donor tissue and wound healing process is disrupted, increasing risk of infection and mortality. In search of personalized medicine, several hbTESSs are under research; their comparison would help understand which technique is more appropriate according to the patient’s pathology and condition, however, even at in vitro level, such comparison is complicated because of the high costs or specific requirements. Among hbTESSs, two models have been already applied on more than ten patients as part of respective clinical trials1,2. On the one hand, the Self-Assembly (SA) approach (LOEX-Canada) uses appropriate culture and mechanical conditions to induce fibroblasts to secrete significant amount of extracellular matrix (ECM) as during organogenesis3. On the other hand, human plasma fibrin-based strategy (UPCIT-Spain) generates a dermal layer of fibroblasts embedded into a hydrogel composed, mainly, of human plasma (clotting factor: fibrin) which can be mixed with biomaterials such as hyaluronic acid or collagen4. The aim of this study was to compare these two hbTESSs models.
Methodology
Three human skin samples were collected, and fibroblasts and keratinocytes were extracted for manufacturing both hbTESS models (N=3). Skin substitutes produced by SA approach were composed of three dermal (fibroblasts+ECM) and one epidermal (keratinocytes) layers. Using human plasma fibrin-based strategy, fibroblasts were embedded into three different dermal matrices (fibrin only, fibrin-hyaluronic acid (HA) or fibrin-collagen (COL)) and epithelialized with a layer of cultured keratinocytes on top. Mechanical properties were analyzed using a tensile testing machine. Immunofluorescence (Ki67, Keratin (K) 19, Collagen-IV, K10, Loricrin), western blot (Collagen-I and -IV) and PrestoBlueTM assay (cell metabolic activity indicator) were performed to compare the results. The same culture media were used for both protocols, but initial number of cells and time of culture followed the original clinical guidelines of each process.
Results
SA approach generates skin substitutes more resistant to tensile forces and with higher adhesion at the dermo-epidermal junction (2 times higher), however plasma-based hbTESSs are thicker, more elastic, and their production is less time-consuming (18 vs. 32 days). Higher number of cells and proliferative cells (Ki67+) is found in SA substitutes although their metabolic activity is lower. After epidermal differentiation, no significant differences were observed between both models, for the number of epidermal stem cells (K19+), and the K10 and Loricrin expression. Overall, production of collagen (I and IV) is higher in SA substitutes, but Collagen-IV is more specifically located at the basement membrane for plasma-based hbTESSs. Finally, properties of plasma-based subtypes are quite similar and only in some specific studies, significant differences are observed (higher amount of Collagen-I in fibrin-COL substitutes -p<0.01-).
Conclusions
Our study characterizes two hbTESS models, demonstrating that manufacturing time as well as mechanical and some biological properties are different, however previous clinical studies have already shown their safety. Future in vivo experiments should compare their wound healing potential and long-term persistence after grafting to complete their characterization."
83767208046
"<div>Introduction. The dermal white adipose tissue (dWAT) is the population of intradermal adipocytes within dermal part of the skin which actively participates in physiological and pathological processes i.e. hair regeneration, thermoregulation, immune response in skin infections and wound healing. Despite the growing interest in this population of adipocytes and identification of their impact on skin physiology, dWAT regulatory pathways have not been fully recognized. It has been shown that activation of epidermal Wnt/β-catenin pathway correlate with dWAT thickness and stimulate adipogenic differentiation by induction of pro-adipogenic ligands: BMP2 (Bone Morphogenetic Protein 2) and IGF2 (Insulin-like Growth Factor 2). Our previous study revealed that epidermally expressed transcription factor Foxn1 regulates homeostasis of epidermis and affects the phenotype and functional characteristics of dermal fibroblasts (DFs). In the present study we investigated the role of transcription factor Foxn1 on intradermal adipocytes differentiation and lipid metabolism in intact and post wounded skin.
Methodology. Experiments were performed on young (8-11 weeks old) Foxn1+/+ (Balb /c; with active Foxn1 factor) and Foxn1-/- (with inactive Foxn1 factor) mice. For skin wound healing model, four (4 mm diameter) full-thickness excisional wounds were made on the back of mice. The skin samples were collected from intact skin (day 0) and during the process of healing. At post- injured days 1, 3, 5, 7 and 14, mice were sacrificed and skin samples from the back of the mice were collected using biopsy punches with a diameter of 8 mm. Tissue samples were frozen and stored in liquid nitrogen until RNA and protein isolation or fixed for immunohistochemical and immunofluorescence analyses.
Results. Histological analysis of intact skin showed an increase in the adipocyte number and the percentage of dWAT area in Foxn1+/+ mice compared to Foxn1-/- mice. Immunofluorescence staining pattern of LipidTOX fluorescence dye displayed lipid accumulation exclusively in the lower layer of the dermis, particularly in the skin of Foxn1+/+ mice. The expression profile of genes related to the process of adipogenesis and lipid metabolism demonstrated increased levels of lipolysis markers in Foxn1-deficient mice. Injury increased levels of adipogenic and lipid metabolism genes exclusively in mice with active Foxn1. Interestingly, Foxn1-deficient mice were characterized by lower in comparison to Foxn1+/+ mice expression of adipogenesis regulators (Pparγ, Fabp4 and Mest) during the entire healing process. In contrast, the expression levels of lipogenic and lipolytic genes were elevated at later stage of wound healing (day 14) in Foxn1-/- mice in comparison to Foxn1+/+ animals. Western Blot and immunofluorescence analyses of two elements of adipogenic stimulatory pathway revealed higher BMP2 and IGF2 protein content in the skin of Foxn1+/+ mice. Additionally, Foxn1+/+ animals demonstrated peak of Bmp2 expression at 14 day post injury which corresponded with increased Foxn1 mRNA levels during wound healing process.
Conclusions. The results indicate that: (i) Foxn1 modulates dWAT morphology and lipid profile; (ii) stimulated by wounding Foxn1 affects intradermal adipocytes activation during early phase of wound healing; (iii) Foxn1 participates in transcriptional regulation of lipogenesis and lipolysis; (iiii) Foxn1 contributes in stimulation of pro-adipogenic pathways: BMP2 and IGF2.</div>"
20941826257
"Cutaneous chronic wounds are characterized by the absence of healing after six weeks. The classic treatment is the debridement of the wound bed followed by a compression method. When the treatment is not efficient enough, the application of wound dressings is required. To date, no dressings are appropriated to treat the different kinds of wounds. Nowadays, research orientation is towards medicated wound dressings incorporating therapeutic molecules within biomaterials in order to favor skin repair. In this study, dense collagen/PLGA composite hydrogels have been developed to deliver dexamethasone or spironolactone in a controlled manner to modulate inflammation and favor wound healing. To evaluate composite hydrogels as a novel medicated wound dressing, their physical properties, drug loading and release kinetic were analyzed. Then, the in vivo performance of composites was evaluated in a pig model of impaired wound healing.
Dense fibrillar collagen hydrogels concentrated at 40 mg/mL were incubated in PLGA solutions containing dexamethasone or spironolactone for 24 hours. Different chain lengths from 7 to 60 kDa were tested. Then, the mixtures were incubated in PBS to trigger in situ PLGA nanoprecipitation within the collagen network. The ultrastructure and the mechanical properties of composite hydrogels were analyzed. Last, the drug release kinetic from composites was studied over one month and their cytotoxicity evaluated on fibroblasts and keratinocytes using a live/dead assay. Composite hydrogels loaded with spironolactone were then applied onto full thickness wounds of a pig model. Their effect on wound closure and re-epithelialization was evaluated.
The nanoprecipitation enabled the immobilization of a large amount of PLGA regardless of the chain length (50 % of the total mass). The presence of PLGA negatively impacted the swelling properties but all hydrogels exhibited a high degree of hydration (over 80%). Unlike PLGA 28 and 60 kDa, PLGA 7 kDa did not altered the hydrogel deformability and doubled the hydrogel stiffness. The ultrastructure analysis revealed the presence of polydispersed nano/microparticles at the surface of collagen fibrils. Compared to pure collagen hydrogels, the drug loading in all composite hydrogels was 5 times higher. The release kinetic of spironolactone and dexamethasone from collagen/ PLGA 7kDa hydrogels was quasi constant over the first two weeks and complete after a month. Unlike pure collagen hydrogels, no burst release was observed. Increasing the chain length negatively impacted the drug delivery as only 20% of the initial dose was released at day 28 for PLGA 60 kDa. Cell viability experiments showed the absence of cytotoxic effect of composite hydrogels on fibroblasts and keratinocytes regardless of the PLGA type used. The in vivo experiment in pig revealed a high performance of collagen/PLGA composite hydrogels on wound healing. Spironolactone loaded composite hydrogels improved wound closure by 50% and permitted a complete re-epithelialization after 6 days.
Taken together, these results show that dense collagen/PLGA composite hydrogels are promising medicated wound dressings for the treatment of chronic wounds as they deliver constant doses of drugs favoring skin repair, possess good physical properties and promote wound healing in vivo."
83767202946
"Introduction: Mesenchymal stem cells (MSCs) can improve chronic wound healing, and recently it was suggested that the therapeutic effect of MSCs is mediated mainly through the growth factors and cytokines secreted by these cells. However, MSCs still are not the standard of care in wound healing due to several limitations such as patient-specific difference in MSCs, poor survival of transplanted cells, and technical considerations such as standardization of isolation, characterization, expansion and delivery. To overcome difficulties related to the translation of cell therapy into clinic we propose an innovative, standardized skin treatment option, a conditioned medium (CM) from recently established by our research group Human Adipose Tissue Mesenchymal Stem Cell (HATMSC) line (1). In this study we evaluate the biological activity of HATMSCs-produced factors following incorporation into collagen hydrogel as a potential treatment for chronic wounds (2).
Methodology: Biocompatibility and biological activity of hydrogel-released HATMSC2-origin bioactive factors were investigated in vitro by assessing the proliferation and metabolic activity of human fibroblast, endothelial cells and keratinocytes. Hydrogel degradation was measured using hydroxyproline assay while protein released from the hydrogel was assessed by interleukin-8 (IL-8) and macrophage chemoattractant protein-1 (MCP-1) using ELISAs. Pro-angiogenic activity of the developed treatment was assessed by tube formation assay while the presence of pro-angiogenic miRNAs in the HATMSC2 supernatant was investigated using real-time RT-PCR.
Results: The results showed significant 3-fold increase in metabolic activity of fibroblast (p < 0.001) and 2-flold of endothelial cells and keratinocytes (p < 0.01) following 3 day culture in the presence of HATMSC2-origin growth factors loaded hydrogels compared to unloaded gels. The supplementation of hydrogel with HATMSCs supernatant improves the tube formation process in angiogenic test in vitro. Moreover, we have confirmed the expression of pro-angiogenic miRNA (miR210, miR126 and miR296) in the HATMSC2 secretome indicating that supernatant can support proangiogenic processes in tissue regeneration. Hydrogel release study showed that there is a substantial difference in the levels of IL-8 and MCP-1 between unloaded hydrogels and supernatant–loaded hydrogels. For example, on day 3 for MSU-1.1 cells the levels of MCP-1 and IL-8 in hydrogel treated groups were 0.4 pg/mL and 156.2 pg/mL, while in cells treated with supernatant-loaded hydrogel the these level were much higher, 45.5 pg/mL and 1723.7 pg/mL, respectively. This suggest that the hydrogel used in this study is an appropriate carrier of HATMSC-originated trophic factors.
Conclusions: This study demonstrated that the therapeutic effect of the HATMSC2-produced bioactive factors (IL-8, MCP-1, proangiogenic miRNAs) is maintained following incorporation into collagen- hydrogel as confirmed by increased proliferation of skin-origin cells and improved angiogenic properties of endothelial cells. These results suggest the possible beneficial effect of dressing, composed of hydrogel loaded with HATMSCs bioactive factors, on the wound healing process in the context of restoration of proper angiogenesis.
References:
1. Kraskiewicz, H. et al., Stem Cell Res. Ther. 11, (1), 29 (2020).
2. Kraskiewicz, H. et al., Int. J. Mol. Sci. Nov 12;22(22):12241 (2021)."
52354525386
TBA
TBA
Mesenchymal stem/stromal cells (MSCs) are considered a disease-modifying treatment for osteoarthritis (OA). However, the precise molecular mechanisms of actions under which MSCs exert their therapeutic effect have not yet been identified in OA. Since MSCs actively interact with their environment, most likely the inflammatory OA milieu will stimulate their response. To identify these mechanisms, we retrieved GFP+ bone marrow-derived MSCs after intra-articular (IA) delivery in a murine collagenase induced osteoarthritis (CIOA) model. The transcriptome of retrieved cells and control in vitro licensed GFP+ BM-MSCs were analyzed to identify the predicted secretome and potential novel therapeutic factors activated by the OA microenvironment.
CIOA was induced in C57BL/6 mice (n=8) and 2x105 mouse syngeneic GFP+ bone marrow derived MSCs (BM-MSCs) were IA injected at D14 and D56 representing early acute and late OA. For cell retrieval, knee joints were digested and isolated by FACS-coupled for RNA sequencing. Samples were compared to GFP+ BM-MSCs retrieved from SHAM joints, where the knee joints were injected with saline solution. BM-MSCs licensed in vitro with a single dose of interleukin 6 (IL-6) and a combination of IL-6, monocyte chemoattractant protein-1 (MCP-1) and interferon gamma (IFN- γ) were also analysed. After 72 hours, cells were processed for RNA sequencing. Validation of BMP/retinoic acid-inducible neural-specific protein 3 (BRINP3) as a new MSC marker was performed using indirect immunofluorescence staining in healthy and OA murine cartilage, mouse embryonic limb and in vitro chondrogenic differentiation in human MSCs and articular progenitor cells (ACPs).
BRINP3 was identified as a common element between the four groups and as a novel protein associated with MSC modulation. BRINP3 protein expression was validated, identifying positive signal in meniscal cells in healthy murine cartilage, and was also detected on the meniscal and articular cartilage surfaces in mouse models of OA and expressed in joint-forming locations and in the periosteal sleeve in the developing mouse limb. We further investigated BRINP3 expression during in vitro chondrogenic differentiation of MSCs and ACPs. Positive expression was identified in the cytoplasm of MSCs and at later stages of chondrogenic differentiation external to the cells suggesting active secretion. However, ACP signal was confined solely in the cell cytoplasm throughout all stages of cell differentiation.
We generated a database of predicted secreted genes that can be a valuable resource for identification of small molecules with potential therapeutic efficacy for OA treatment. Furthermore, the data provided insights of the therapeutic mechanisms of action of MSCs in the context of OA. Among secreted genes, we identified for the first time BRINP3 as a new protein expressed during embryonic limb development, in vitro chondrogenic differentiation and on the meniscal and articular cartilage surface in vivo where chondroprogenitor cells are located. The data also highlights a mechanism of action of with surviving MSCs taking on a chondroprotective role and future studies are needed to validate the potential of BRINP3 as a local treatment of OA.
83767210269
"EFFECT OF DIFFERENT LIGHT WAVELENGTHS ON ADIPOSE TISSUE-DERIVED MESENCHYMAL STEM/STROMAL CELLS
Introduction: Treatment of cells with electromagnetic irradiation (light) can affect their proliferation and differentiation ability. Exposure of cells to light sources with different wavelengths (wavelengths around 415 nm/blue, 540 nm/red, and 810 nm/infrared are most common) appears to have different effects depending on the wavelength, energy intensity and the duration of exposure. Cytochrome c oxidase is believed to be one of the main photon acceptors. In this study, we have developed an experimental setup suitable for irradiating cells (with adipose tissue-derived mesenchymal stem/stromal cells/adMSC) with light of different wavelengths. With this setup, we are investigating the effect of light exposure on the regenerative capacity of adMSC.
Methodology: Since the cell culture media and growth surfaces have specific absorption properties, we measured absorption spectra of different media (with and without fetal calf serum/FCS, with and without phenol red) with different plate formats (clear bottom, with or without lid) and different volumes (50 µl, 100 µl, and 150 µl) using a microplate reader. Using the cell culture medium optimized for light treatment, the adMSC suspension was irradiated with the different wavelengths (blue (430 nm), red (660 nm) and IR (810 nm)) and different exposure times (5, 10 and 15 min). Subsequently, the phenotype, viability, cell number, cell cycle, mitochondrial membrane potential and differentiation ability of the cells were determined.
Results: Since the measurement with black plate with a flat transparent bottom without lid, showed little light scattering, these plates were used for the further analyses. The analysis of absorption spectrum of cell culture media showed that phenol red absorbs light with wavelengths below 600 nm. The media additive FCS showed similar absorption properties. The studies of different filling volumes showed that the absorption capacity increased in direct proportion with increasing filling volume. After optimizing the experimental setup, it was possible to carry out the first experiments on the irradiation of adMSC. Depending on the light source used, different effects occurred. These ranged from changes in cell morphology to reductions in cell number and metabolic activity. We also showed that the effect of light exposure on adMSC also depended on duration and energy input of the light. Further analyses to understand the energy and wavelength-dependent effect on cellular properties are still pending.
Conclusion: Since different materials and compounds have different absorption effects, the experimental setup needs to be adapted to the irradiation conditions. The composition of the media has a direct influence on the light absorption by adMSC, so it has to be adjusted accordingly. Preliminary experiments confirm that the viability and proliferation capacity of adMSC changes depending on the light wavelength and the energy input. With this background knowledge, we can now examine the study of light effects on differentiation and migration ability. These examinations could help to transfer the light treatment to clinical application.
References:
[1] Wang, Yuguang et al, Scientific reports vol. 7,1 7781. 10 Aug. 2017
[2] Chen, Hongli et al, Lasers in medical science vol. 34,4 (2019)
"
20941845455
"Introduction
The wide spectrum of brain injuries experienced in neonates and preterm newborns and the potential plasticity of the CNS prompts us to seek solutions in the field of neuroregeneration in this group of patients and to prevent the worst effects of prematurity and perinatal problems. Drug-resistant epilepsy remains one of the biggest problems of prematurity and its consequences. There is a need among patients suffering from drug-resistant epilepsy (DRE) for more efficient and less toxic treatments than long time pharmacotherapy.
Method
The purpose of this study was to evaluate the safety and potential efficacy of multiple administrations of HE-ATMP comprised of 3x107 WJMSCs. A study group was composed of six patients, qualified for the treatment with a diagnosis of chronic hypoxic – ischemic encephalopathy and inflammation (including sepsis, and systemic inflammatory reaction) with diagnosis of DRE. All the patients underwent repeated rounds of HE-ATMP administration to the CSF via LP.
Results
There were no adverse events, and the therapy was safe and feasible over 2 years of follow-up. The therapy resulted in neurological and cognitive improvement in all patients, including a reduction in the number of epileptic seizures (from 40 per day to 2-5 per week) and an absence of status epilepticus episodes (from 4 per week to 0 per week). The number of discharges on the EEG evaluation was decreased, and cognitive improvement was noted with respect to reactions to light and sound, emotions, and motor function.
Conclusion
After two years of follow-up examination, we demonstrated the safety and beneficial effects of WJMSCSs transplantation, including neurological improvements and reduction of functional neurodeficits. We are aware that the samples size of this study is relatively small, therefore data need to be further tested in larger groups."
41883635884
Cell-based therapies in the clinic are limited by the number of cells that can be produced quickly and inexpensively. Whereas about one million cells are isolated from a single donor, existing cell-based therapies can require hundreds of millions to billions of cells. Rapid, exponential expansion of cell number would allow faster delivery of life-saving treatments, such as bone marrow transplants, to a greater number of patients.. Chaotic printing is a novel, patent-pending, high-resolution biofabrication technology that could dramatically improve cell expansion capabilities. It produces layered filaments with significantly higher Surface Area per unit Volume (SAV) than existing cell proliferation systems. Cell-laden hydrogel layers can be interspersed with “fugitive ink” layers. The fugitive ink dissolves and evacuates to leave open channels in between the cell-laden layers. The higher SAV should facilitate an exponentially larger interface between nutrient media and cells cultured in these filaments. We predicted this would dramatically improve the speed and yield of cell proliferation by improving nutrient availability and waste removal. In a first experiment, Bone Marrow-derived human Mesenchymal Stem Cells (BM-hMSCs) were cultured for 1 month in chaotically printed hydrogel filaments. To observe the open channels’ impact on cell expansion, hydrogel filaments with alternating cell-laden layers and open channels were compared to a control group of hydrogel filaments without any open channels. The group with open channels had significantly more cells than the control group on days 1, 7, 14, and 21 of culture. The largest difference between the groups occurred on day 7, the open channel group having 2.1 times as many cells as the control group. This experiment showed BM-hMSC viability for 1 month with our hydrogel formulation and chaotic printing method while also demonstrating increased cell expansion rate with open channels present. We also observed a peak expansion rate within the first week with the open channel design. We proceeded to develope and validate a novel bioreactor design for perfusing the open layers in our chaotically printed hydrogel filaments with flowing nutrient media. We hypothesized that adding flow to the system would further improve nutrient availability and waste removal. Co-axially extruding calcium chloride along with the hydrogel bioinks through our printhead allowed for chaotically printed hydrogel filaments to be solidified and extruded directly into small polystyrene tubes. This method results in consistent hydrogel filaments that contain open channels and are flush to the edges of the polystyrene “bioreactor” tubes into which they are extruded. This well-fitting bioreactor system ensures the flow of nutrient media through the open channels. These tubes can then be cultured within an incubator while connected to a flow circuit driven by a peristaltic pump located outside the incubator. A second experiment demonstrated that BM-hMSCs continued expanding in number for 1 week in chaotically printed hydrogel filaments housed in polystyrene bioreactor tubes when media was flowed through (i.e., replaced via flow) twice per day. Cell-laden filaments in tubes without flow regimens had virtually no viability by day 4. Work is ongoing on a flow regime that will optimize BM-hMSC expansion.
94238144705
"<div>Endochondral bone regeneration (EBR) recapitulates natural development of long bones during embryogenesis and fracture repair through implantation of a cartilaginous template into a defect, which is eventually remodeled into bone. Typically, autologous multipotent mesenchymal stromal cells (MSCs) are exploited to generate such cartilage constructs. However, the use of patient-own cells is associated with donor-site morbidity and donor-to-donor variability with respect to chondrogenic differentiation potential. Non-autologous MSCs are a promising alternative and offer an off-the-shelf solution due to pre-selection of chondrogenically potent donors, but could possibly cause immune rejection [1]. Previously, we demonstrated that by devitalization of allogeneic cartilage constructs, an adverse immune reaction was avoided, and full defect bridging of a critical size rat femur defect was achieved [2]. The next step to bring this approach into the clinic is the transition from small animal research to pre-clinical studies in large animals. However, successful bone regeneration through EBR using allogeneic constructs has not been achieved in large animal models before. This study aimed at proof of concept of devitalized, allogeneic cartilage constructs for EBR in a critical size defect in a large animal model.
Goat MSCs (gMSCs) were isolated from the iliac crest of Dutch Milk goats, encapsulated within a collagen hydrogel and differentiated in chondrogenic medium for 28 days, followed by devitalization [2]. Constructs were implanted into bilateral, critical size iliac crest bone defects. Each defect was divided into three equal compartments by a titanium spacer. Experimental groups consisted of allogeneic devitalized cartilage constructs. Controls contained gold standard bone autograft, empty carrier control and empty defect (n = 6 for each group). One and two months post-implantation, fluorochromes (calcein and oxytetracycline) were administered intravenously to mark sites of active bone formation at the time of administration. Animals were sacrificed 3 months post-implantation and explanted samples were evaluated for mineralization and new bone formation via microCT, histology, histomorphometry and presence of fluorochromes.
Preliminary results demonstrate the feasibility of EBR using allogeneic cartilage constructs in a large animal model for the first time. Moreover, a critical size defect at the cm3-scale was regenerated by a cell-based implant. Devitalized allogeneic constructs induced more bone formation than the empty (2x more) and carrier (1.5x more) control. Further, the constructs induced new bone formation comparable to the gold standard autograft, as shown by microCT and histological analysis. These findings indicate that our novel approach can achieve bone regeneration, at least comparable to bone autograft, without associated drawbacks such as donor-site morbidity and a second surgical intervention. As this is the first time an allogeneic EBR approach has performed successfully in a pre-clinical large animal model, these results contribute to the clinical translation of EBR in the form of an off-the-shelf product. Further, these results allow us to take the next steps towards clinical translation of our approach by evaluating its safety and efficacy in further pre-clinical models, before proceeding to first in-man trials.
1 Longoni, A. et al., Frontiers in bioengineering and biotechnology 8, 651 (2020).
2 Longoni, A. et al., Advanced science (2021).</div>"
94238119124
STEM CELLS IN BONE REGENERATION, A RANDOMIZED CLINICAL TRIAL
Cecilie Gjerde1, Mariano Sanz2, Markus Rojewski3,4, Pierre Layrolle5, Hubert Schrezenmeier3,4, Kamal Mustafa1
1Institute of Clinical Dentistry, Faculty of Medicine, University of Bergen, Norway.
2Department of Dental Clinical Specialities (DECO), Faculty of Odontology, University Complutense of Madrid, Spain
3Institute of Transfusion Medicine, Ulm University, Ulm, Germany.
4Institute for Clinical Transfusion Medicine and Immunogenetics Ulm, Red Cross Blood Service
Baden-Württemberg—Hessen and Institute for Transfusion Medicine, University Hospital Ulm, Ulm, Germany.
5INSERM, UMR 1238, PHY-OS, Laboratory of Bone Sarcomas and Remodeling of Calcified Tissues, Faculty of Medicine, University of Nantes, Nantes, France.
Introduction: In a non-controlled recent clinical study, autologous bone marrow derived stem cells combined with biomaterial induced new bone formation. This has been reported as promising new approach for reconstruction of atrophied posterior alveolar mandibular ridges. We aimed in the present study to demonstrate the efficacy of this therapeutic ATMP approach in a randomized multicenter controlled clinical trial.
Our research group has pioneered the field of bone tissue engineering and demonstrated feasibility, safety, and efficacy of the combination of Biphasic Calcium Phosphate (BCP) granules and autologous bone marrow derived stem cells (MSC) in preclinical studies and early phase human clinical trial (n= 11 patients). Successful regeneration of the alveolar bone in the pilot trial was evident in radiographic and histological findings [1]. The findings were supported by the ability of the newly formed tissue to accommodate a dental implant and withstand the forces of mastication on daily bases. Therefore, the present work is aimed to perform phase II multicenter randomized controlled clinical trial for regeneration of mandibular bones of patients prior to dental implants using autologous MSC.
Methodology: Patients with a need for bone reconstruction of residual edentulous ridges in both the mandible and maxilla due to bone defects with a vertical loss of alveolar bone volume and/or knife edge ridges (≤ than 4,5 mm) unable to provide adequate primary stabilization for dental implants were included in the clinical study. Autologous bone marrow MSC were expanded, loaded on BCP (MBCP+™; Biomatlante, France) and used to augment the alveolar ridges. After five months bone biopsies were harvested at the implant position site and implants were installed in the regenerated bone. The implants were loaded after 8 -12 weeks. Safety, efficacy, quality of life and success/survival were assessed. Five clinical centers, 4 different countries participated. Bone grafts harvested from the ramus of the mandibles were used as control in the study.
Results: 41 patients have so far been screened and enrolled in the study. 21 patients have been treated in the test group, 9 in the control group, 6 are waiting for treatment, and 5 withdraw before treatment.
Conclusions: The results this far indicates that the use of bone marrow derived stem cells in the applied protocol for augmentation of the atrophied mandibular ridge have results comparable to the gold standard; autologous bone transplantation with predictable longtime results.
References: Gjerde C et al. Stem Cell Res Ther. 2018 Aug 9;9(1):213
83767248587
Insufficient vascularization is a major obstacle for clinical application of tissue engineered transplants including bone. The ambition is to provide an environment rich in vascular networks to achieve efficient osseointegration and accelerate functional restoration after implantation. Of particular interest is the microvasculature that is crucial for oxygen and nutrient delivery. Microvascular networks in 3D can be formed in vitro through the co-culture of endothelial cells (ECs) with supporting pericytic cells. Mesenchymal stem/stromal cells (MSCs) derived from bone marrow (BMSCs) and adipose tissue (ASCs) are an attractive choice for pericytes due to their natural perivascular localization and ability to support formation of mature and stable microvessels. Furthermore, they are most used cell types for bone tissue engineering and clinical trials focusing on bone regeneration.
Here, our aim was to explore the vasculogenic potential of human ASCs and BMSCs in a perfusable microfluidic device.
BMSCs and ASCs were co-cultured with ECs in a fibrin hydrogel in a microfluidic chip. We compared the capacity of BMSCs and ASCs to induce the formation of mature microvascular networks by ECs and to differentiate into pericytes. We studied the effect of MSCs on vessel characteristics such as area, diameter, length, and perfusability. Interstitial flow across the hydrogel area was measured daily in EC-BMSC and EC-ASC cocultures using fluorescence imaging. We assessed MSCs pericytic differentiation in terms of pericyte area and pericyte coverage by immunohistochemical staining and quantitative analysis. Furthermore, we evaluated the expression of main vasculogenesis related genes.
We demonstrated that using MSCs of different origin resulted in vascular networks with distinct phenotypes. Both types of MSCs supported formation of mature and interconnected microvascular networks. However, BMSCs induced formation of fully perfusable microvasculature with larger vessel area and vessel length compared to ASCs. Co-culture with ASCs resulted in only partially perfusable microvascular networks. Immunostainings revealed that BMSCs had greater potential to differentiate towards pericytes than ASCs. The gene expression analysis revealed significant differences in the expression of endothelial-specific and pericyte-specific genes, as well as genes involved in vasculature maturation and remodeling.
Overall, our study provides valuable knowledge on the properties of BMSCs and ASCs as vasculature supporting cells and highlights their distinct directing role in the regulation of microvascular phenotype that might have implications in bone tissue engineering applications.
94238140506
Background:
Mesenchymal stem cell (MSCs) -based therapy has a promising potential in bone tissue regeneration. Although, growing evidence has suggested that paracrine mechanisms may be involved in the underlying mechanism of MSC transplantation, and extracellular vesicles (EVs) are an important component of this paracrine role. However, information on the influence of different microenvironmental stimuli of MSCs culture conditions on the osteogenic effects of EVs is scarce. The main purpose of this study was to determine whether EVs derived from MSCs under normoxic (Normo-EVs), Hypoxic (Hypo-EVs) and chemically osteogenic induced MSCs (Osteo-EVs) show greater effects on osteogenic differentiation potential in vitro and on the bone formation of calvarial defects in vivo, and whether findings are associated with various proteins profile.
Methods:
Undifferentiated MSCs were incubated under normoxic and hypoxic culture conditions, and 7-days of chemically osteogenic induced MSCs were incubated under normoxic conditions, for 72 h. Conditioned media were collected and concentrated onto 100 kDa centrifugal filters (UF), followed by separation of EVs using size-exclusion chromatography method (SEC). The Normo-EVs, Hypo-EVs, and Osteo-EVs recovery were characterized by size distribution using DLS, morphology using TEM, and flow cytometry analysis of tetraspanin CD63 and CD81. The proteomic composition of different groups of EVs was characterized by LC-MS/MS. We evaluated the in vitro effect of EVs groups (10 μg/ml) on the proliferation, scratch assay, and osteogenic differentiation of human bone marrow-derived mesenchymal stem cells (hMSCs) by Alkaline phosphatase (ALP) staining, Alizarin red S (ARS) staining, and qRT-PCR for osteogenic related genes, respectively. Furthermore, Osteo-EVs (50 μg/ml) were combined with collagen membrane scaffolds (MEM) to repair critical-sized calvarial bone defects in rats, and the efficacy was assessed using in vivo and ex vivo µCT, and histological examination. The in vitro release of Osteo-EVs from MEM scaffolds and their internalization by cultured MSCs were also examined.
Results:
Using UF-SEC, we could isolate and characterize EVs from all groups. We found that all EVs groups could profoundly enhance the proliferation, and migration of cultured hMSCs. However, Osteo-EVs were shown greater effects on the in vitro osteogenic differentiation of hMSCs as detected by higher mRNA expression levels of late markers of osteogenesis-related genes, BSP and OC, and calcium deposit using ARS. In addition, Osteo-EVs/MEM combination scaffolds could enhance greater bone formation after 4 weeks as compared to native MEM loaded with serum-free media. In vitro assay showed that the Osteo-EVs could release from the MEM scaffold and could be internalized by cultured hMSCs.
Conclusions:
We suggest that EVs derived from chemically osteogenic induced MSCs can significantly enhance both the osteogenic differentiation activity of cultured hMSCs and the osteoinductivity of MEM scaffolds. These results indicate that Osteo-MSC secreted nanocarriers-EVs combined with MEM scaffolds can be used for repairing bone defects.
31451708199
Introduction
Endothelial cells (ECs) have potential in bone tissue engineering due to their important role on vascularization. For repairing bone defects, co-culturing of ECs and bone marrow stem cells (BMSCs) is suggested to induce both the capillary network formation and bone regeneration. The communication between the two cell types has been experimented on settings based on cell number ratio, culture medium [1-2] and culture distance [3]. With regards to 3D printing technology, EC suspensions have been previously flushed into engineered lumens or canals to initiate vessel formation in existing structures [4-5]. However, bioprinting is yet a rarely used form of technology for delivering ECs. Vascu-ink, containing fibrinogen and gelatin, aimed for soft, elastic and dynamic 3D environment, enabling the rapid maturation of bioprinted ECs. The aim of this study was to investigate the influence of a multimaterial bioink, vascu-ink, in a co-culture setting on angiogenic tissue maturation.
Methodology
The performance of the vascu-ink with ECs was characterized as single bioink but including the co-culture effect by seeding BMSCs outside the bioink structures. To investigate the potential of vascu-ink as an EC carrier, the bioink was mixed with the ECs (1x107/ml) and bioprinted in a four-layered structure using a 3D-Bioplotter (EnvisionTEC) with a 250 μm metal needle. The structures were crosslinked externally with thrombin (2.5U/ml) and CaCl2 (100mM). BMSCs were seeded on the bottom of the well plate and the crosslinked vascu-ink samples were lifted on top. The co-cultures were then followed for structural integrity, cell viability (Live/Dead staining), metabolic activity (Cell Counting Kit -8) and tubular formation, via immunostaining, for up to 21 days. Endothelial growth media was used for the first three days and then switched into osteogenic media. After that, the media was changed three times a week.
Results
The developed vascu-ink was very compliant as desired. The viability of the ECs was high throughout the culture period, cells were spreading and migrating, and the metabolic activity of ECs was maintained with the BMSCs. The printed structures survived the culture period but had gradually lost their fidelity as the material was preferred by both of the cell types. The desired tubular formations and organization of the ECs was recorded by both Live/Dead staining and immunostaining.
Conclusions
The promising results indicate that the developed bioink serves as a tissue specific cell carrier and culture environment. The co-culture of the two cell types was also beneficial in tubular formation.
References
1. Ma, J., et al., Tissue Eng Part C Methods. 17(3), 349-57 (2011).
2. Bidarra, S.J., et al., Stem Cell Res. 7(3), 186-97 (2011).
3. Piard, C., et al., Biomaterials 222, 119423 (2019).
4. Kolesky, D.B., et al., PNAS 113(12), 3179-84 (2016).
5. Miller, J.S., et al., Nat. Mater. 11(9), 768-74 (2012).
94238130844
INTRODUCTION: Mesenchymal stromal cells (MSC) combined with biphasic calcium phosphate biomaterial (BCP) is a promising clinical strategy to repair and regenerate lost bones [1]. Further, MSC derived extracellular vesicles (MSC:EV) are established factors of paracrine inter-cellular communication with various cell types, including immune cells, which impacts their regenerative potential [2] . However, scarce studies have explored the immune modulation behavior of EV isolated from MSC+BCP constructs (MSC+BCP:EV). The aim of this study was to isolate and characterize MSC+BCP:EV, including from inflammatory primed cells (MSCp+BCP:EV).Isolated EVs from all MSC groups were exposed to primary human macrophages to determine change in macrophage maturation and polarization states.
METHODS: EV isolation was done by using size exclusion chromatography (SEC) columns (PURE-EV, HansaBioMed Life Sciences Ltd., Estonia). After confirming the particle size in eluted EV fractions via dynamic light scattering (DLS) and protein amount by BCA, fraction no. 8-15 were selected for further analysis. EVs were characterized by TEM and flow cytometry. A human cytokine 27-plex assay was used with a Bio-Plex® 200 System (both from Bio-Rad Laboratories) to measure the cytokine content of EVs. Macrophages were obtained from differentiation of primary blood derived monocytes, which were isolated from donor buffy coats via magnetically activated cell sorting (MACS, Miltenyi Biotec GmBH).
RESULTS: Size and morphologies of EVs from different MSC system were found comparable. Further, MSC+BCP:EV showed less proinflammatory, whereas MSCp+BCP:EV showed more immunomodulatory and angiogenic cytokine profile compared to MSC:EV. Functional macrophage analysis revealed increased potential of MSCp+BCP:EV to induce unpolarized/naive macrophages (M0) into an anti-inflammatory phenotype (M2), as compared to EVs from an unprimed construct (MSC + BCP:EV) and MSC alone (MSC:EV). Further, it was found that MSCp + BCP:EVs also have an enhanced potential for bi-directional macrophage polarization switching (i.e., from pro- to anti-inflammatory state and vice-versa).
CONCLUSIONS: This study established methods to isolate and characterize EVs from a MSC and biomaterial constructs. We showed that both priming and biomaterials have differential effect on EV-cytokines and hence immunomodulation by EVs. EVs derived from primed MSC + BCP constructs showed enhanced potential to modulate both naïve (M0) and pro-inflammatory (M1) human macrophage subsets towards an anti-inflammatory (M2) type. Such properties were attributed to the higher levels of immunomodulatory cytokines present in the MSCp + BCP derived EVs. Thus, our study provides new insights into role of EVs in MSC-biomaterial induced bone regneration [3].
REFERENCES:
[1] C. Gjerde, K. Mustafa, S. Hellem, M. Rojewski, H. Gjengedal, M.A. Yassin, X. Feng, S. Skaale, T. Berge, A. Rosen, X.-Q. Shi, A.B. Ahmed, B.T. Gjertsen, H. Schrezenmeier, P. Layrolle, Cell therapy induced regeneration of severely atrophied mandibular bone in a clinical trial, Stem Cell Research & Therapy 9(1) (2018) 213.
[2] D. Medhat, C.I. Rodríguez, A. Infante, Immunomodulatory Effects of MSCs in Bone Healing, International journal of molecular sciences 20(21) (2019) 5467.
[3] N. Rana, S. Suliman, N. Al-Sharabi, K. Mustafa, Extracellular Vesicles Derived from Primed Mesenchymal Stromal Cells Loaded on Biphasic Calcium Phosphate Biomaterial Exhibit Enhanced Macrophage Polarization, Cells 11(3) (2022) 470.
52354540746
Introduction: Adipose tissue-derived stem cells (ATSCs) have been used as an alternative to bone marrow mesenchymal stem cells (BMSCs) for bone tissue engineering. However, several studies have reported that the efficacy of ATSCs in bone regeneration in comparison with BMSCs remains inferior 1,2. The aim of this study was to investigate the mechanism underlying differences in ATSCs versus BMSCs osteogenicity in tissue-engineered constructs by focusing on the innate immune response mediated by the ATSCs.
Methodology: Bone formation induced by transplanted human BMSCs and ATSCs combined with calcium carbonate ceramic granules (named tissue constructs) was evaluated in vivo in an ectopic mouse model. Explants were analyzed by µCT and histology. Kinetic analyses of both the expressed human and murine genes pertaining to osteogenesis, angiogenesis, and inflammatory response in tissue constructs explanted at 0, 7, 14, and 28 days post-implantation were performed. The gene expression and secretome profiles of pro-inflammatory cytokines/chemokines in both ATSC and BMSC were analyzed.
Results: All constructs containing BMSCs induced ectopic bone formation and histological observation of explants revealed the presence of new bone with the presence of osteoclastic (TRAP +) multinucleated cells on contact with the ceramic particles. On the contrary, the constructs containing ATSCs did not generate (or minimally) bone tissue; they were infiltrated with fibrous tissue, and numerous TRAP- multinucleated giant cells (MNGC) were observed. Gene expression analysis of explants revealed that implanted human BMSCs differentiated into the osteogenic lineage in vivo concomitantly with the osteogenic differentiation of host murine progenitors. In contrast, the osteogenic differentiation in construct-contained ATSCs started later than in BMSCs, when only less than 1% of implanted ATSC were present; no osteogenic differentiation in host murine cells occurred. Expressions of genes pertaining to vascularization were not significantly different between both groups. Regarding the inflammatory response, compared with BMSCs, the expressions of human IL1b and IL6 genes were highly upregulated in implanted ATSCs during the first-week post-implantation and then decreased; In parallel, murine IL1b was also upregulated in ATSC-containing constructs as were the M1/MNGC-associated iNOS and CD86 murine genes. An extensive analysis of gene expression of human cytokines and chemokines comparing the ATSC and BMSC contained in constructs at day 0 (before implantation) was conducted. This revealed up-regulation of 23 inflammatory mediators out of 84 tested in ATSC compared to BMSC (the highest (> 30-fold) upregulated genes were CSF3, CXCL10, CXCL5, CXCL11). Only CXCL12 (SDF1), RANKL, and BMP4 were slightly (3-7 fold) upregulated in BMSCs. Such a pro-inflammatory profile of ATSCs was confirmed at the protein level after quantification in the construct-contained cell supernatant.
Conclusion: In contrast to BMSCs, ATSCs display no/weaker osteogenic potential in vivo. ATSCs are a transient source of proinflammatory cytokines and chemokines that promote an inflammatory environment within the cell-containing constructs. This event correlates with impaired osteogenic differentiation of both implanted ATSCs and host osteoprogenitors.
1. Brennan, M. A. et al. Stem Cells Transl. Med. 6, 2160–2172 (2017).
2. Mohamed-Ahmed, S. et al. Stem Cell Res. Ther. 9, 168 (2018)
94238122617
Introduction
Mesenchymal stem cells (MSC) regulate their behavior by sensing mechano-environmental factors.
Accumulated evidence indicates that appropriate mechanical force including fluid shear stress enhances the osteogenic property of MSC on 3D polymeric scaffolds even without the presence of osteogenic cocktail (i.e., dexamethasone and beta-glycerophosphate). However, despite a common understanding of how cytoskeleton transmits mechanical stimuli, a detailed role of cytoskeleton in fluid flow-induced osteogenesis is not fully understood. The aim of the present study was therefore to assess cytoskeletal modulation under fluid force and then explore causal relationship with altered cell growth and flow-induced osteogenic differentiation by using a perfusion bioreactor.
Methodology
MSC from Lewis rat bone marrow (rBMSC) were seeded on 3D microporous scaffolds made of Poly(L-lactide-co-trimethylene carbonate) and subjected to laminar flow excreting shear stress at 1 mPa on an average for 14 days in a perfusion bioreactor. Cytoskeletal modulation was assessed by, but not limited to, RT-qPCR array, cell morphological analysis, enzymic activity of Rho-associated protein kinase (ROCK), and level of phosphorylation of myosin light chain. Transcriptional profile of osteogenesis-related markers in rBMSC under fluid stimuli was compared with that induced by the osteogenic cocktails and a static control without osteo-inducement. To evaluate the role of cytoskeletal modulation in flow-induced osteogenesis, pharmacologic inhibition of cytoskeletal modulators, namely, Rho GTPases, ROCK, myosin light chain kinase and non-muscle myosin II ATPases, was performed to induce cell relaxation during perfusion culture. With the inhibitors, cell growth and osteogenic differentiation were further evaluated.
Results
Under the fluid flow, rBMSC significantly altered the expression pattern of mRNA related to cell morphogenesis and focal adhesion including Pkt2, Prkca, Rock1 and Rock2. This was accompanied with cell morphological alternation characterized by cell contraction and actin stabilizayion, and the enhanced phosphorylation of myosin light chain was observed. In such a condition, rBMSC upregulated a number of osteogenic markers including Runx2, Sp7, Col1a1, Bmp2, ALPL and Spp1. Interestingly, the mRNA expression pattern of osteogenic markers differed from dexamethasone-induced osteogenesis. The inhibition of cytoskeletal modulation cascade from Rho activation to actomyosin contraction at different levels suppressed the flow-induced upregulation of the osteogenic markers. Despite the fact that cell proliferation was suppressed by fluid flow, the inhibition aggravated cell growth even further while the inhibitors did not show a notable suppressive effect on proliferation in the static control.
Conclusion
The present study using a perfusion bioreactor demonstrated that rBMSC responded to a low level of fluid stimuli by cytoskeletal moduration, which was associated with altered cell growth and osteogenic differentiation on 3D polymeric scaffolds. The inhibition of cell contraction revealed that cytoskeletal modulation under fluid stimuli was required for maintaining the proliferative state while it directed rBMSC fate towards an osteogenic lineage.
94238131255
Engineered materials that integrate advances in polymer chemistry, nanotechnology, and biological sciences have the potential to create powerful medical
therapies. Dr. Khademhosseini is interested in developing ‘personalized’ solutions that utilize micro- and nanoscale technolgoies to enable a range of therapies for organ failure, cardiovascular disease and cancer.In enabling this vision he works closely with clinicians (including interventional radiologists, cardiologists and surgeons). For example, he has developed numerous techniques in controlling the behavior of patient-derived cells to engineer artificial tissues and cell-based therapies. His group also aims to engineer tissue regenerative therapeutics using water-containing polymer networks called hydrogels that can regulate cell behavior. Specifically, he has developed photo-crosslinkable hybrid hydrogels that combine natural biomolecules with nanoparticles to regulate the chemical, biological, mechanical and electrical properties of gels. These functional scaffolds induce the differentiation of stem cells to desired cell types and direct the formation of vascularized heart or bone tissues. Since tissue function is highly dependent on architecture, he has also used microfabrication methods, such as microfluidics, photolithography, bioprinting, and molding, to regulate the architecture of these materials. He has employed these strategies to generate miniaturized tissues. To create tissue complexity, he has also developed directed assembly techniques to compile small tissue modules into larger constructs. It is anticipated that such approaches will lead to the development of next-generation regenerative therapeutics and biomedical devices.
"Regenerative implants are most ideally suited for individualized production using additive manufacturing. For regulatory market access, if not containing cells or tissue compounds making them ATMP’s, the choice is between serially produced scaffolds, patient-adapted designs or custom-made devices that require a prescription. While it seems helpful that custom-made products do not get a CE mark and leave much of the risk with the surgeon, the procedure is cumbersome to the clinical staff and reimbursement often prone to discussion. This might be the reason why entire sets of prefabricated size-ranges are still taken into the sterile area every day. In other regulatory spheres like Australia and USA, more practical approaches towards medical product registration of AM implants exist, while the MDR relies on the freshly enhanced custom-made path. A more viable solution for the European regenerative implant market that fits into reimbursement seamlessly might unleash the potential of AM in modern medicine, a field in which Europe is struggling to maintain its leading position.
With the current situation, and despite all the hype, AM regenerative implant manufacturers have to feel rather exotic and miss a secure embedding in the daily clinical routine, despite all the magic they can produce especially in cooperation with the right clinical partner, usually a university hospital. The discrepancy stems from traditional CE marking as medical product registration path, simultaneously creating clinical evidence or at least acceptance and thus the base for reimbursement by public and private health plans. Individually manufactured implants do not fit into this registration scheme, making it necessary to defend their application from case to case. However, it appears that, by copying from software medical product registration, and proving efficacy and safety similar to patient-adapted implants, a more pragmatic regulatory approach would be possible. Today, the development of AM production chains has come to a point where an individual implant, designed by constraints from the treating medical authority, can be produced and delivered on the push of a button. It seems possible that reimbursement, be it for operational models, implants or tissue engineering constructs, can be equally integrated into daily clinical practice – making the magic regulated and risk controlled.
An important aspect remains data handling and privacy in the light of the GDPR. First mobile solutions for something as simple as CT data transfer are emerging. A standardization of data handling, respecting the patient’s private sphere and at the same time gathering information important for AM on gender, personal heritage and behavioral patterns in a modern way might even be the base for a sound regulatory scheme, if integrated into intelligent information management as integral part of AM regenerative implant design."
83871206328
Medical additive manufacturing (aka medical 3d-printing) has received increasing attention in the past years with research applications in many clinical domains, particularly in surgical disciplines (e.g. orthopedic, oral, cardiovascular). In some domains medical 3d-printing has established itself in daily clinical practice, however the question remains if is it ready for broad clinical use. In this talk a short survey about the state-of-the-field will be presented, followed by examples from several research & development projects at the Medical University of Vienna. The selected projects will highiligh the medical background, the technological options employed as well as the challenges we have been encountering towards a successful establishment of 3d-printing in the clinics.
94355103928
Introduction
Additive manufacturing approaches have the potential to address a number of major challenges in the field of meniscus tissue engineering (TE), in particular the development of anatomically defined grafts with a spatial architecture and composition mimetic of the native tissue. Here, we report a novel method to engineer organized soft tissues, with a collagen architecture and mechanical behaviour similar to native meniscus. We compared the capacity of two direct material writing techniques, specifically fused deposition modelling (FDM) and melt electrowriting (MEW), to generate guiding structures for cells that are deposited using inkjet bioprinting. We hypothesised that by fabricating polymeric scaffolds with specific architectures it is possible to control collagen fibre organization and the mechanical behaviour of the engineered fibrocartilaginous tissue, thereby better recapitulating the native meniscal tissue
Materials and methods
FDM and MEW were employed to fabricate polycaprolactone (PCL) scaffolds with various defined geometric architectures. Furthermore, large volume MEW scaffolds with micro-fibrous features were fabricated to replicate the complex wedge-shaped macro-architecture of the human meniscus. A custom alginate based bioink was developed and inkjet bioprinting was used for the dispensing of cell-laden bioinks into scaffolds. After inkjetting, the cells were cultured in presence of TGF-β3 to induce chondrogenesis. Mechanical testing was conducted to determine the compressive and tensile properties of the engineered tissues.
Results
First, we assessed the suitability of the developed bioink for cartilaginous TE applications, finding that its rapid degradation allows cells to condense and begin the process of generating new tissue while exhibiting high levels of cell viability. The multicellular aggregates which formed within the defined boundaries provided by the PCL fibres generate a neo-tissue where the organization is determined by the architecture of the scaffold. Both FDM and MEW fibrous architectures facilitated the formation of anisotropic collagen networks resembling those of the native meniscus. However, PCL scaffolds fabricated using FDM displayed mechanical properties that are unsuitable for meniscus replacement, as they are too stiff in compression. By using MEW as a fabrication technique, the mechanical strength of the engineered tissues after 5 weeks of in vitro culture was similar to the native tissue. After demonstrating the benefit of using MEW to create scaffolds for meniscus regeneration, we fabricated larger (5 mm height) scaffolds replicating the shape of the meniscus. The pore architecture of these large scaffolds remained open even at the highest sections, enabling the bioprinting of cells and facilitating tissue growth throughout the entire scaled-up construct.
Discussion
In the present work, we have successfully developed a biofabrication approach that allows precise control over the orientation of the deposited collagen tissue. By using MEW as a fabrication technology we could better mimic not only the collagen network architecture but also engineer tissues with similar anisotropic mechanical behaviour. We have also succeeded in the fabrication of large volume MEW scaffolds of up to 5 mm height with well-defined micro-fibrous and macro-architectural features. This work demonstrates the potential of integrating MEW and bioprinting to engineer structurally organised soft tissues.
52354514949
Introduction:
Melt Electrowriting (MEW) is a novel 3D printing method that allows fabricating scaffolds with different designs, including structural gradients. The gradient scaffolds can be useful, for example, in the engineering of tissue interfaces, which are characterized by gradually changing mechanical and biological properties [1]. Nonetheless, the prediction of such scaffolds’ properties is a challenge.
The aim of this project is the development of a computer simulation that will allow predicting the mechanical properties of the scaffolds produced with MEW. This will ultimately facilitate the designing process for researchers working with MEW.
Methodology:
Polymeric scaffolds were fabricated using the MEW technique using polycaprolactone. the mechanical properties of scaffolds with different pore sizes and designs were analyzed in tensile testing. COMSOL Multiphysics® was used to simulate the tensile testing of printed scaffolds.
Results:
The scaffolds with varying designs, including gradients, were printed with a fiber diameter of 16 µm. SEM images showed good accuracy of printing and high precision of layer deposition. Tensile test results revealed the dependency of mechanical properties on scaffold design. The developed computational models allowed for accurate simulations of scaffolds’ mechanical performance, i.e. Young’s Modulus and deformation.
Conclusions:
The mechanical properties of MEW printed scaffolds can be tuned by the scaffold’s design. The proposed computer simulation helps predict the mechanical properties of the scaffolds with high accuracy, at the deformation range of up to 2% (elastic region). In the next steps, computer simulations will be evaluated for higher deformations. Such models will accelerate the development of MEW scaffolds with tissue-specific properties.
[1] Bayrak E, Huri PY. Engineering Musculoskeletal Tissue Interfaces. Frontiers in Materials 5, 24, 2018
20941813806
Introduction
Spinal cord injury (SCI) induces paralysis by severing the long axons of neurons and recovery is inhibited by poor regrowth rates. As neural cells are electroactive, electrical stimulation (ES) may present a promising method of promoting axonal regrowth when applied in conjunction with electroconductive (EC) biomaterials1. To efficiently deliver ES to regrowing motor and sensory axons, it is essential to have precise control of scaffold geometry.
This work focused on producing novel EC scaffolds for spinal cord injury by coating 3D-printed polycaprolactone (PCL) with polypyrrole (PPy) and assessing its suitability as a substrate for neuronal growth.
Methodology
PCL scaffolds consisting of multiple interlocking ‘axon’ channels were 3D-printed (Allevi 2 printer). PPy was then polymerised in situ to form an EC coating2. Electroconductivity was measured via the 4-point method and surface morphology and coating thickness were assessed using SEM.. Biocompatibility was tested by seeding SH-SY5Y neurons on PPy/PCL films and measuring metabolic activity and total cellular DNA. Neurons were immunostained for beta-III tubulin, counterstained with DAPI for nuclei and imaged using a Nikon 90i fluorescent microscope to determine neurite outgrowth. Images were analysed using ImageJ.
Results
A method was developed to coat complex 3D-printed PCL structures with a biocompatible, EC PPy layer. SEM images of coated films show that PPy forms a network of particles over the PCL surface. Conductivity of the PPy coating was 15 ± 5 S/m, rendering the scaffold suitably electroconductive for biological applications. In cultures of SH-SY5Y neurons on 2D PCL and PPy/PCL films, metabolic rate and total cellular DNA increased significantly (p<0.05) in both groups between day 1 and 3, with no significant difference in either metric between groups, showing the PPy coating is as biocompatible as uncoated PCL, providing a suitable substrate for neural proliferation. Neurons cultured on both film types (7 days) exhibited robust neurite outgrowth and typical morphology with no significant difference in cell number or neurite length between groups, confirming neuronal viability.
Conclusion
Biocompatible, 3D EC scaffolds with complex architectures were produced for SCI repair. Coating 3D-printable PCL with EC PPy allows printing of biocompatible EC structures with precise, controlled geometries and porosities that can match the organisation of grey and white matter tracts in the cord. Conductivity of the PPy coating is over 30 times higher than central nervous system grey matter, and 8 times higher than cerebrospinal fluid, potentially allowing for efficient direction of electrical stimulation3. Taken together, these data indicate that PPy/PCL is biocompatible, supports neuronal growth and is a suitable substrate for growth of spinal cord neurons. Ongoing optimisation work will determine the ability of the EC scaffold to increase efficacy of ES to further promote neurite extension as a prerequisite for SCI applications.
References
ACKNOWLEDGEMENTS
Research was funded by Irish Rugby Football Union Charitable Trust and Science Foundation Ireland Advanced Materials and Bioengineering Research (AMBER) Centre (SFI/12/RC/2278_P2).
62825410404
Combining different materials or material-properties in 3D printing is garnering widespread attention due to the wide range of possibilities that it provides to produce parts which are more functional and have improved properties. This paper presents the combination of lithography-based ceramic manufacturing, a vat photopolymerization technology capable of realizing high resolution 3D printing for ceramics with the mentioned multi-material approach. The presented approach not only enables the combination of different ceramics in different layers of the printed component, but also the spatially resolved combination within the same layer and hence, paves the way to the realization of complex bi-phasic ceramic components. First successful trials that will be presented include the combination of alumina and zirconia-toughened alumina (ZTA) and zirconia and hydroxyapatite. Especially, the latter combination possesses big potential in implants, as it allows combining the mechanical properties of zirconia ceramics with the biological performance and activity of hydroxyapatite. In this way it is possible to create implants that combine different mechanical, chemical, and biological properties in different areas and hence, would be very interesting candidates as bone replacement materials.
Moreover, this multi-material 3D-printing approach also allows the localized introduction of different levels of porosity and in such a way functionally-graded properties. Using so-called porogens or fugitives it was possible to create density gradients between almost fully dense areas and areas with a volumetric porosity of around 50%. In combination with the design freedom of 3D printing and the possibility of manufacturing complex cellular or lattice designs, this allows to fabricate implants or scaffolds with hierarchical porosity.
The paper will not only present the actual multi-material 3D printing process, but also focus on the results and current challenges in terms of co-sintering of different ceramic materials. The initial results show that this technological approach holds great potential to path the way from classical single material structures to bi-material components and subsequently multi-material and functionally-graded ceramics.
52354555566
Additive manufacturing allows for a wide range of freeform and complex shapes to be made with little or no manufacturing limitations. The most significant benefit of additive manufacturing in medical applications is that it allows for the creation of patient-specific medical products such as implants. Individualized implants are considered to provide greater comfort, precise fit, user acceptance, and may result in fewer revision surgeries. Additive manufacturing allows for tool-less production, which can lower prototyping & tooling costs as well as reduce medical product development time. Many challenges arise while designing for patient-specific implants, as each product has its own distinct characteristics. There is no one-size-fits-all approach that allows to infinitely reproduce the same result as with traditional procedures. A fast design process, verification, and validation of the implant design for the mechanical stability, biocompatibility, and printability, are among the challenges.
The meniscus, a fibrocartilage structure in the knee joint, plays a very significant role in load transmission, shock absorption, lubrication, and nutrient supply to the articular cartilage. Meniscus damage or wear occurs as a result of accidental injuries or aging, and may necessarily require partial or total replacement of the meniscus. This study focuses on the design of individualised meniscal implants that would relieve pain and restore knee joint functionality. The research aims to explore the load bearing capacity of meniscus implants using three different lattice structure designs. Material properties, cell topology and shape, and relative density of structures all influence the properties of lattice structures. In current study, a shell- core type meniscus geometry is analyzed where the lattice structures in core serves as a strengthening component, while the shell binds the meniscus geometry together and keeps it in shape. The beam diameters and lattice size of each individualized implant can be altered to better meet the strength requirements and production constraints. The implant design proposed here could also be used to create a multi-material meniscus implant that combines the strength of different materials.
52354565457
Current medical devices certification is challenging due to the update in the regulatory norms and the appearance of gaps (grey zone) due to advances in both the materials and the fabrication technology. A key aspect when reaching a higher level of technology readiness is to have a comprehensive view of the entire process that will lead to the conformity of the devise with the medical device regulation. To have a focus perspective, it is interesting to work on the research project to have defined several key steps for future medical device certification like the definition of the intended use, the documentation of risk analysis and the iterations of risk management, the documentation of design and manufacturing iterations, and the identification of the materials that are included in the medical device. This will lead to the definition the final device that shall be assessed for the biological characterization for conformity assessment under the medical devise regulation. The ISO 10933 series and the MDR devices that are composed of substances or of combinations of substances are discussed in an integrative approach to provide a focus perspective on the biological characterization of a medical device. Furthermore, the concpect of “equivalent” medical devise could be an interesting option for the additive manufacturing of medical devices. For example, custom-made approach could be used as a transition toward certification of series production of medical devices by additive manufacturing.
73387302597
Introduction: Vascular grafts are implanted daily, whether it is as leg or coronary bypasses or as arteriovenous shunts. Autologous blood vessels are the gold standard but have limited availability while synthetic materials are prone to thrombosis, intimal hyperplasia and infections. To overcome these limitations, our team produced a biological Tissue-Engineered Vascular Graft (TEVG) woven from yarn of Cell-Assembled Extracellular Matrix (CAM). This textile-based approach is very versatile because it gives fine control over the geometrical and mechanical properties of the TEVG. The goal of this study is to establish how changes in production parameters (e.g.: yarn count, yarn density, etc.) affect the properties of the TEVG (e.g.: mechanical properties, wall thickness, surface waviness, etc.).
Methodology: CAM sheets were produced by sheep dermal fibroblasts seeded in 225 cm2 flasks and cultured in DMEM/F-12 with 10% FBS and 0.5 mM Na L-ascorbate. Threads were cut with a custom motorized device composed of rolling blades spaced at the desired width (5 mm). Woven grafts were assembled on a circular loom and composed of a series of longitudinal threads, called “warp”, and a circumferential one that spiraled along the length of the vessel, the “weft”. The latter was made of two threads twisted together at 5 revolution/cm. The effects of yarn thickness and warp count (number of longitudinal threads) were tested. The influence of weft thread production parameters and its tension during weaving were also studied. Transmural permeability, suture retention strength, compliance and burst pressure were evaluated. Geometrical properties including TEVG inner diameter, wall thickness and graft lumen surface profile were assessed macroscopically and by X-ray microtomography. For each condition, n=3 TEVGs were woven.
Results: A lower yarn thickness decreased wall thickness and suture retention strength. A lower warp count had the same effect on wall thickness, while the compliance increased linearly with a decrease in warp count. In addition, the surface profile, which may have an impact on cellular infiltration and blood compatibility, was influenced by both parameters. Narrower weft ribbons decreased weft diameter which resulted in thinner walls and TEVGs with lower strength. Finally, a lower tension in the weft during weaving resulted in a significantly higher compliance while burst pressure was decreased.
Conclusion: We have demonstrated the influence of a number of parameters on the geometric and mechanical properties of a TEVG woven from CAM yarn. Investigation of the influence of weft twist is underway to improve our control over the properties of the TEVG, focusing on increasing its compliance. These results are helping to build a toolbox that will allow the production of the TEVG with the most relevant properties for implantations as an arteriovenous graft in sheep.
73296309639
Cell encapsulation in biomaterial microcompartments is a useful tool to deliver protected cell cargo into defective tissues. However, the processing of micrometric structures is often dependent on the use of typical emulsion agents, including oils and organic solvents. Those are often related with poor cell viability, or require complicated washing procedures to retrieve oil-free cell-laden materials. All-aqueous processing methodologies arose as promising strategies to process biomaterials with high cytocompatibility. Aqueous two-phase systems have been used as surrogates of classical water/oil-based emulsions to enable phase separation to enable the formation of, for example, microparticles. Experimental approaches similar to the ones widely explored for classical emulsion systems for biomaterial fabrication – including their adaptation into electrospraying or microfluidics-based techniques - have led to the formation of mostly spherical structures, which mostly comprise either continuous biomaterial beads, or hollow capsules obtained upon interfacial reactions. Concerning the latter, the interfacial complexation of polyelectrolytes at the interface of millimetric and micrometric droplets has enabled the cytocompatible encapsulation of mesenchymal stem cells along with adhesive microparticles [1]. However, unlike the preparation of thermodynamically stable spherical objects at the interface of emulsions, the preparation of soft compartments with other shapes has proven challenging. Although previous attempts have been made to fix the shape of objects at the interface of aqueous emulsions, structures capable of being handled outside of the emulsion interface could not be obtained. We here report an advance on the all-aqueous fabrication of continuous, non-leaky self-standing biomaterial tubes, capable of withstanding cell adhesion on their walls. Another versatile aspect of materials prepared using all-aqueous emulsions is related to the formation of multicompartment complex hollow structures assembled upon the establishment of specific processing parameters. Those were useful in spontaneously separating micrometric objects in different compartments enabling, for example, the encapsulation of viable cells in specific locations of multiwalled structures. The versatility achieved with the use of all-aqueous emulsions may be used to enable the fabrication of advanced biomaterials that may combine tailored release profiles of bioactive molecules with the exact positioning and protection of cells.
Reference:
[1] Vilabril, S., Nadine, S., Neves, C. M. S. S., Correia, C. R., Freire, M. G., Coutinho, J. A. P., Oliveira, M. B., Mano, J. F., One-Step All-Aqueous Interfacial Assembly of Robust Membranes for Long-Term Encapsulation and Culture of Adherent Stem/Stromal Cells. Adv. Healthcare Mater. 2021, 10, 2100266.
Acknowledgements:
This work was financially supported by the European Research Council grant agreement ERC-2014-ADG-669858 (project ATLAS), by the Programa Operacional Competitividade e Internacionalizacã̧o, in the component FEDER, and by national funds (OE) through FCT/MCTES, in the scope of the projects ‘TranSphera’ (PTDC/BTM-ORG/30770/2017) and “CellFi” (PTDC/BTM-ORG/3215/2020), and developed within the scope of the project CICECO-Aveiro Institute of Materials, UIDB/50011/2020, UIDP/50011/2020 & LA/P/0006/2020, financed by national funds through the FCT/MEC (PIDDAC).
41883625124
INTRODUCTION:
Transplantation of non-autologous β-cells is currently regarded as a promising therapy for the treatment of type 1 diabetes, caused by massive β-cell destruction, consequently resulting in insulin shortage. To evade the host’s immune responses, new materials are being developed to encapsulate and shield implanted β-cells. Several immunoprotective material formulations have been developed, but their clinical translation is challenged by their impermanent nature, development fibrous capsules upon implantation1, which is insufficient to produce therapeutical signifficance2. Additionally, such materials should be capable of inhibiting the diffusion of large immune molecules (i.e., IgG) whilst enabling diffusion of small molecules (i.e. Insulin and glucose) in a semi-permeable fashion3. In this work, we have developed non-immunogenic, immunoprotective, and enzymatically crosslinked, hollow, polyethylene glycol-tyramine (PEG-TA) microgels for β-cell delivery therapies.
METHODS:
Polyethylene glycol 8 arm – tyramine (PEG-TA) conjugates were synthesized in a two-step reaction and optimized for enzymatic crosslinking using horseradish peroxide and non-cytotoxic levels of hydrogen peroxide4. Droplet generation was optimized for production of micrometer-sized hollow PEG-TA microgels laden with beta cells using microfluidics5. Hollow microgels containing MIN6 pancreatic cells were extensively characterized in vitro based on variables such as cytocompatibility, cyto-immunity, permselectivity, and glucose responsiveness. Furthermore, in vivo performance was assessed by implanting diabetic STZ-mice to investigate immunoprotectiveness of the implant and its ability to re-establish normoglycemia.
RESULTS & DISCUSSION:
Consistent production of ~120-150 µm hollow microcapsules with ~20 µm shell thickness was achieved and characterized. Immunoprotection was confirmed by exposing cell-laden gels by absence of diffusion of IgG-FITC molecules into the microgels and by co-culture with natural killer NK-92 cells without inducing cell death.
Encapsulated cells were shown to sustain viability and capable of secreting insulin in vitro over a period of one month without showing significant shell burst. Immunological assessment by multiplexed ELISA analysis on live blood immune reactivity was performed, demonstrating little pro-inflammatory cytokine release in response to microgel presence, confirmed by gene expression analysis and immunostainings of inflammatory markers.
Intraperitoneal implantation of β-cell laden microgels in diabetic mice showed restoration of normoglycemia within the first 5 days and good tissue integration, with histology revealing alive aggregates at the time of sacrifice (14 days)
CONCLUSION:
Consistent high throughput microfluidic production of immunoprotective PEG-TA microgels was achieved. The produced microgels revealed good suitability for shielding and delivering non-autologous beta-cells within a living host.
ACKNOWEDGEMENTS: Financial support was received from the European Research Council (ERC, Starting Grant, #759425) and JDRF.
BIBLIOGRAPHY:
1Robitaille R, Leblond FA, Bourgeois Y, Henley N, Loignon M, Hallé JP, Journal of Biomedical Materials Research 44, 116-160, 2000;
2Golpanian S, Schulman IH, Ebert RF. Stem Cells Translational Medicine 5: 186–191, 2016; 3B, Lewińska D, Biomaterials Science, 8, 1536-1574, 2020;
4Kamperman T, Henke S, Zoetebier B, Ruiterkamp N, Wang R, Pouran B, Weinans H, Karperien M, Leijten J. Journal of Materials Chemistry B 5(25), 2017;
5van Loo SR, Salehi S, Henke S, Shamloo A, Kamperman T, Karperien M, Leijten L.Materials Today Bio, 6:100047 2020
83767252008
Symbrachydactyly is a rare congenital upper limb anomaly, that occurs in 1/30,000- 1/40,000 live births resulting in children born with short boneless fingers. Nowadays, these pediatric patients are treated with phalangeal bone transfer from the foot. However, morbidities are occurring at the donor site which result in unstable toes with significant disfigurations that worsen with the child growth.
In this project, we used a developmentally-inspired strategy to engineer osteogenic grafts for phalanx reconstruction via endochondral ossification (ECO). Human adipose-derived stem cells (ASC) isolated from the stromal vascular fraction (SVF) were seeded into collagen sponges and exposed to chondrogenic and hypertrophic factors to generate in vitro clinically-pertinent osteogenic grafts (100-200 mm3) in the form of hypertrophic cartilage templates (HCTs).
Specifically, we evaluated in vitro the impact of (i) the cell source (freshly isolated SVF cells or expanded ASCs), (ii) the scaffolding material (collagen type I sponge crosslinked or not), (iii) the cell seeding density (3x105 to 3x106 cells/construct) and (iv) the duration of exposure to chondrogenic (3 to 5 weeks) and hypertrophic factors (1 to 2 weeks) on the maturation of the HCTs generated. The mineralization (by alizarin red staining) and the cartilage maturation (evidenced by the cell morphology and the uniformity and intensity of glycosaminoglycan (GAG) revealed by Safranin-O staining) of these HCTs were evaluated on histological sections. Next, the bone forming capacity of these HCTs was assessed in vivo in an ectopic nude (immunocompromised) mouse model for up to 12 weeks, reflecting the clinical scenario of phalangeal soft tissue pocket
In vitro, we were able to generate HCTs of pertinent clinical sizes. As expected, the maturation of the HCTs was dependent on the duration of exposure to chondrogenic and hypertrophic factors. In addition, we observed that the cartilage formation was obtained more rapidly when using freshly isolated SVF cells rather than expanded ASCs. Overall, the best in vitro outcome was obtained for the crosslinked collagen sponge loaded with SVF cells at the highest cell density.
In vivo, we observed that the bone formation was correlated with the degree of hypertrophic cartilage maturation. Interestingly, even the HCTs that had very limited cartilage at the time of implantation were capable of generating bone once implanted, suggesting that the cells primed in vitro are capable of forming cartilage before the bone remodeling occurs in the early stages of the implantation. Similarly, across all the conditions tested, the quantity of bone tissue obtained in vivo was superior to the quantity of cartilage tissue obtained in vitro. Finally, while inferior to the freshly isolated SVF cells-based constructs, ASCs-based constructs remained capable of generating clinically pertinent bone tissue in vivo.
Taken together, these results demonstrate the feasibility of using SVF cells or expanded-ASCs to generate osteogenic grafts of pertinent clinical size in the context of symbrachydactyly. Moreover, despite the limited amount of donor-tissue available in pediatric patients, the data obtained for the expanded ASCs suggest that an autologous approach to generate osteogenic phalanx grafts for children born with symbrachydactyly would remain possible.
83767208404
INTRODUCTION
The role of gut microbiota in neurodegeneration is becoming a very interesting topic nowadays, and innovative in vitro and in vivo tools are becoming increasingly a need to help in dissecting the biochemical pathways involved in microbiota-brain interaction. An innovative engineered multiorgan-on-a-chip platform able not only to mimic microbiota-gut-brain connection but also to reproduce in vitro biological and mechanical stimuli to represent the so-called microbiota-gut-brain axis (MGBA) is the aim of our ERC project “MINERVA”. MINERVA platform is based on a 3D printed device compatible with commercially available tissue culture inserts and characterized by optical accessibility and capability to be connected to other devices. In the present work, we present our preliminary results related to the development of the gut epithelium modelling unit of our MGA engineered platform.
METHODOLOGY
Computational fluid dynamic simulations were performed with the software COMSOL Multiphysics® testing different flow rate values to obtain suitable oxygenation and shear stress values.
Biological validation was performed using human Caco2 cells seeded on collagen-coated inserts. After 7 days in static conditions, we assembled the seeded inserts into MINERVA devices under perfusion for other 7 days. As control we used seeded inserts maintained in static culture for 14 days. Cellular viability was tested by MTS test, FITC-dextran permeability and Trans-epithelial electrical resistance (TEER) evaluation. To assess cell layer morphology and maturation, immunofluorescence tests were performed with anti-mucin, anti-occludin, FITC-phalloidin and Hoechst dyes.
RESULTS AND DISCUSSION
From computational simulations we set flow rate at 30µL/min to guarantee oxygen supply and suitable shear stress value.
While MTS test showed no difference in terms of viability between static and dynamic condition, TEER values of static samples showed significant differences in line with apparent permeability (Papp) value, that resulted enhanced in dynamic condition.
Occludin and mucin reactivity are comparable between static and dynamic samples and their expression changes along the villus vertical axis. Morphological analysis confirmed mature cell layer formation in both static and dynamic samples with significantly higher villi in dynamic condition.
CONCLUSION
The selected flow rate set to mimic optimal physiological condition, maintained cell viability, promoted villi height and induced TEER value comparable to those of in vivo human intestinal epithelium [1]. Lower TEER values and higher permeability in dynamic condition are coherent with immunofluorescent data that confirmed mature cell layer formation characterized by lower cellular differentiation near to the villus base, likely required to guarantee villi turnover [2].
Overall our results confirm MINERVA device suitability for gut epithelium modelling in MINERVA MGA multi-organ platform.
ACKNOWLEDGMENT
MINERVA project has received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (Grant Agreement N° 724734).
REFERENCES
[1] Srinivasan B, et al. (2015) J Lab Autom. 2015;20(2):107-126.
[2] Gommers, L. et al. (2019). Acta biomaterialia, 99, 110–120.
62825428926
Introduction:
Axial vascularisation of tissue constructs is essential to maintain an adequate blood supply for a stable regeneration of clinically relevant 3D tissues. In the last 10 years, our research group has been investigating the efficacy of the arterio-venous loop (AV loop) as a model of axial vascularisation. The versatility of the AV loop could be demonstrated in various small and large animal models even after applying high doses of ionizing radiation comparable to those applied for adjuvant radiotherapy after head and neck cancer.
Methodology and Results:
We will report about our experience with axially vascularized tissue engineering constructs in the last 10 years including the in vivo irradiation of the rat and rabbit implantation chambers as well as the mandibular goat model. Furthermore, our most recent results showed a correlation between a state of controlled hypoxia inside the AV loop-constructs and its ability to attract progenitor cells from the systemic circulation. We will also present the early results of our recent clinical trial (NCT04001842) regarding axially vascularized mandibular regeneration of extensive defects after recurrent Ameloblastoma otherwise requiring free flap reconstruction.
Conclusions: Axially vascularized tissue engineering constructs represents a convenient method to reconstruct extensive mandibular defects otherwise requiring free bone transfer. Despite their proven versatility after irradiation in pre-clinical trials, human application after cancer ablation and irradiation remains a major challenge that should be further investigated in well-designed clinical trials.
83767214766
Introduction
Kidney failure happens due to two conditions; acute kidney injury (AKI) and chronic kidney disease (CKD). These lead to the deterioration of the glomerulus, the filtering unit in the kidney, leading ultimately to end-stage renal failure (ESRF). [1] Current treatments for ESRF, haemodialysis and kidney transplantation are inadequate since haemodialysis replaces filtration but not all other kidney functions, and transplantation is limited due to the shortage of donor organs. Therefore, to provide new treatment options, we are using a highly biocompatible bacterial polymer called Polyhydroxyalkanoates (PHAs) as a scaffold material for human kidney cells to develop a bioartificial glomerular filtration barrier.
Methodology
Bacterial fermentation was carried out to produce Polyhydroxyalkanoates using a selected bacterial strain, fed by specific fatty acids to produce a medium chain-length polyhydroxyalkanoate (mcl-PHA). The polymer produced was thoroughly characterised using; Gas Chromatography (GC) for monomer composition, Gel Permeation Chromatography (GPC) for polymer molecular weight, and Thermal Gravimetric Analysis (TGA) and Differential Scanning Calorimetry (DSC), both for thermal properties. Two types of glomerular cells, conditionally immortalised human podocytes (CIHP) and conditionally immortalised glomerular endothelial cells (ciGEnC), were used to test the biocompatibility of the mcl-PHA using the resazurin assay [2]. In addition, live dead assays were carried out using calcein green and ethidium bromide. The mcl-PHA was subjected to 3D printing (Fused Deposition Modelling) to engineer a kidney bioartificial filtration barrier. The CIHP and ciGEnC cells were separately bioprinted, using alginate [3] as the encapsulating agent, onto the polymer scaffold to introduce spatial separation.
Results
Mcl-PHA was produced with a yield of around 60 g of polymer per 100 g of dry cell weight (~60% dcw). [3] The polymer monomer composition revealed a higher 3HO percentage than 3HD monomers, with trace amounts of 3-hydroxybutyrate (3HB) and 3-hydroxyhexanoate (3HHx) monomers. From the GPC, the average molecular weight, Mw was around 100,000 g/mol. Tensile testing confirmed the elastomeric nature of the polymer, and a low melting temperature (Tm) enhanced the printability of the polymer. Cytocompatibility test showed for the first time that mcl-PHA was highly compatible with both the glomerular cells, CIHP and ciGEnC, comparable to tissue culture plastic (TCP). However, the cells are slightly less viable with alginate as an encapsulation agent, which needs improvement in enhancing the bioactive properties of the encapsulation agent.
Conclusion
This work aims to bio-mimic the human glomerulus to ultimately develop a bioartificial kidney using a tissue engineering strategy and bioprinting. In addition, we have shown that the polymer supports the adherence and growth of human glomerular cells despite the well-known hydrophobicity. In future, the bioartificial filtration barrier developed using the combination of the mcl-PHA along with glomerular cells will be assessed for its' ability to conduct haemofiltration, eventually, leading to the development of a complete bioartificial kidney.
References
1. Ferenbach, D.A. & Bonventre, J.V., Nephrologie & therapeutique. 12, S41-S48 (2016).
2. Kitching, A.R. & Hutton, H.L., Clinical Journal of the American Society of Nephrology. 11, 1664-1674 (2016).
3. Hinchliffe, J. D. et al., MDPI Polymers. 13, 1-48 (2021).
52354515966
"INTRODUCTION: Collagen I has been the gold standard material to generate many in vitro tissue models, including organotypic skin [1]. The rate of collagen remodeling by fibroblasts incorporated in the dermal part of the model, leads to significant dimensional changes during its development, which can be associated to additional hurdles when cultured within dynamic culture systems. These ones, are particularly relevant for skin tissue due to its multilayered nature, by providing the necessary fluid flow for tissue development and interlayer communication for biofunctionality mimicry. Thus, in this work, a different biomaterial, gelatin methacrylate (GelMA), is proposed to validate a dimensionally stable organotypic skin model developed and maintained in a custom-made bioreactor [2], generating a dynamic in vitro testing platform.
METHODS: Two solutions of 5% and 7.5% (wt/vol) GelMA were prepared and crosslinked for 30 seconds under 7.2 mW/cm2 UV intensity [3]. Rat tail collagen I hydrogels were prepared from 0.26% (wt/vol) solutions that were polymerized for 2 hours at 37°C. Human dermal fibroblasts (hDFbs) were encapsulated in the hydrogels at a density of 2.3×105 cells/ml and cultured in α-MEM medium up to 14 days. The elastic (G’) and viscous modulus (G’’) of the hydrogels were measured using a rotational rheometer and an uniaxial compression test allowed the determination of the compression modulus of the hydrogels. Cell viability was assessed after Calcein/PI staining and ECM deposition (collagen type I and laminin) was analyzed through immunocytochemistry.
RESULTS: The G’ of 5 and 7.5% GelMA hydrogels did not significantly vary along the culture, presenting respectively mean values of 0.9kPa and 1kPa, independently of the day of culture. Likewise, the compression modulus did not vary, being within the range of 5kPa and 18kPa, respectively for the 5% and 7.5% GelMA hydrogels. In opposition, the G’ mean value of the collagen I hydrogels, increased from 0.5 to 1.2kPa from day 1 to day 14 of culture. The compression modulus, also showed significant differences at day 14, being collagen about 3-fold stiffer than GelMA hydrogels. Moreover, the shrinkage ratio of GelMA and collagen hydrogels was circa 20% and 83%, respectively. Independently of the mechanical properties, GelMA didn’t negatively affect cellular viability being also capable to support ECM deposition.
CONCLUSION: From the results achieved so far, the use of GelMA as a substitute of collagen for generating organotypic skin models has demonstrated to possess several potentialities that surpass the struggles associated with the shrinkage of collagen. Future experiments will focus on the incorporation of human keratinocytes (hKCs) on the top of the constructs, to replicate the epidermal compartment and the in vitro dynamic culture of the full skin equivalent will take place in the recently developed sandwich-like bioreactor.
ACKNOWLEDGEMENTS: Financially supported by ERC Consolidator Grant ERC-2016-COG-726061.
REFERENCES
[1] Gangatirkar, P. et al., Nat. Protoc. 2, 727-746 (2007).
[2] Gasperini, L. et al., Provisional Patent Application 116901, (2020).
[3] Loessner, D. et al., Nat. Protoc. 11, 727–746 (2016)."
31412720605
"Introduction
linical translation of tissue engineering-based therapies is currently limited by the difficulty in inducing essential vascularisation for tissue viability after transplantation. Thick and metabolically demanding engineered tissues require a defined microvascular network to provide sufficient nutrient and gas exchange. Laser ablation has emerged as a promising technology to fabricate custom-made perfusable microfluidic channels that mimic capillary beds and aid both anastomosis and vascularization of tissue engineered constructs. So far, the proposed laser-driven methods use expensive laser systems or involve heavy in house customization. In this work, we developed a multistep patterning method to precisely create hierarchical vascular trees using a commercial, affordable and widely available 355 nm laser ablation system.
Methodology
n order to design physiologically relevant capillary networks that consider tissue geometry, physical constraints, and structure stability, vascular trees were generated using a constrained constructive optimization-based method [1]. More particularly, vascular trees were generated using Accelerated Constrained Constructive Optimization as arterial/venous matched pairs meeting at simple anastomoses. Batch optimization was used to minimize a combination of network volume and pump work, with post-build bifurcation asymmetry correction. Inter- and intra-network collisions were resolved, including padding to ensure vessel spacing. Vessels were smoothed and new collisions resolved before export. A Zeiss/Rapp-Opto commercially available laser ablation system was then used. A slicing and tiling algorithm was developed to bridge the gap between 3D CAD model and laser software specific formats. Also, an optimization of the working parameters of laser manufacturing tools (e.g., beam intensity, z-step, overlap, etc.) was required to precisely reproduce the 3D CAD model within a diversity of low stiffness hydrogels.
Results
Ablated features were sliced, imaged and measured through light microscopy. Channels were perfused with 2µm fluorescent beads or injected with commercial silicone rubber and were assessed through confocal microscopy or micro computed tomography, respectively. Complete ablation and formation of open lumen were achieved. Networks of different dimensions created through constrained constructive optimization– including networks larger than the objective were successful recreated. Precise control down to 10µm in resolution was also reported.
Conclusions
In conclusion, this work provides an efficient tool to create customized hollow networks within transparent hydrogel scaffolds in an automated manner. The resulting vascular trees can be used to obtain capillary beds for tissue engineering applications and the development method can be adapted to a multitude of other bio- inspired systems.
Acknowledgements
EU Horizon 2020 research and innovation programme under the ERC grant CapBed (805411), IF/00347/2015 and FCT (S. Queirós, CEECIND/03064/2018).
References
1. Guy, A. A. et al., IEEE Trans. Biomed. Eng. 67, 1650-1663 (2019)"
94238148339
Introduction:
Injectability is one of the most desirable features of biomaterials. The combination of injectability and photocuring can provide materials to be easily and safely delivered using minimally invasive procedures. This is especially important for tissue sealants which allow to exclude the use of sutures or staples or for patches supporting the weakened tissue. Therefore, the aim of presented work is to create new injectable amphiphilic polymer-polymer hybrid system, which can be UV-cured in situ in vivo to form flexible patches for soft tissue regeneration. Such systems, with sufficient adhesive properties can be used without the need of using sutures or tacks and by applying them via minimally invasive procedures to provide support for the tissue.
Materials and Methods:
The polymer-polymer hybrid networks were prepared from fatty acid-modified precursors (Pr) bearing methacrylic groups and from PEGylated fibrinogen. For the synthesis of telechelic fatty acid-based macromonomers, three catalytic systems were tested: bismuth tris(2-ethylhexanoate) (BiHex) and zinc (II) acetyloacetonate (ZnAc), both in 4 mol%, and organo-Mg-Ti catalyst (1 mol%). The obtained liquid monomers were characterized by NMR and GPC. The UV-curing has been performed at the wavelength λmax of 385 nm (LED source) turning the liquids into flexible solids with use of 2% w/w photoinitiator (Omnirad 819). The gel fraction has been determined by refluxing materials in DCM. Cytotoxicity tests were performed on extracts using L929 cell line. Cell viability was then assessed using light microscopy and the resazurin viability assay on samples UV-cured in air and in argon. The PEG-ylation of fibrinogen was performed in two steps. In the first step, fibrinogen was dissolved in 50mM PBS with 8M urea (protein concentration 7mg/ml) and TCEP HCl was added and then stirred for 2h. After that, PEG-DA 4kDa was added (145:1 PEG-DA: fibrinogen) and reaction was continued overnight. Obtained product was purified and characterized by NMR (Bruker DPX HD-400 MHz). The hybrid was prepared using telechelic macromonomers and PEG-ylated fibrinogen with photoinitiator followed by solvent evaporation. Photocuring was performed in air atmosphere with use of the same UV-light source.
Results and Discussion:
New catalytic systems allowed to obtain telechelic macromonomers at the shortest reaction times for bismuth (7h) and magnesium-titanium catalysts (9h). Rection yield was similar for all of the materials (65-57%). The cell viability study showed that the use of nontoxic catalysts resulted in high cell viability, regardless of gel fraction. PEGylation of fibrinogen has reached high yield of 88%. The use of co-solvents, here ethyl acetate and/or dichloromethane and/or dimethylsulfoxide allowed to prepare amphiphilic hybrids. Their characteristics and adhesive properties will be discussed during the lecture.
Conclusions:
Photocurable macromonomers were synthesized with new catalysts, being non-toxic as revealed by high cell viability. The use of co-solvents allowed to prepare amphiplilic networks from hydrophobic macromonomer and hydrophilic PEGylated fibrinogen with the use of UV light. Obtained hybrid networks showed also high cell viability and elastomeric properties thus manifesting their suitability as patches for soft tissue engineering.
Acknowledgements:
This work has been financially supported by Polish National Science Center (Grant number: UMO-2019/33/B/ST5/01445).
73296353379
"Introduction
The aim of this research was to design and obtain an injectable thermosensitive hydrogel consisting of methylcellulose (MC) and agarose which would serve as a smart scaffold for tissue engineering applications. The MC provides thermal sensitivity, while heated up to c.a. 37 ℃ becoming a physically crosslinked hydrogel. While agarose enhances crosslinking of MC rate and increases its mechanical properties. To evaluate the usefulness of such an injectable thermosensitive hydrogel system from the perspective of tissue engineering applications, injectability studies and biological tests with the use of two cell lines were carried out.
Methodology
In these studies, the injectability of MC/agarose hydrogel was studied using a dynamometer system, in which the maximum force needed for making injections was measured and compared to the literature reports. Additionally, in vitro cellular studies were performed using fibroblasts and mesenchymal stem cells (MSCs). Cellular morphology was analyzed via scanning electron microscopy (SEM) and fluorescence microscopy (FM), while a cytotoxicity test was carried out on extracts using the Presto Blue assay.
Results
The injectability tests showed the maximum force needed for making the injection of MC/agarose hydrogel was less than 30 N which according to Kim et al. [1], is the maximum force required for injection by a human. Biological studies showed proper cell morphology in comparison to control (tissue culture plastic -TCP). Additionally, cytotoxicity tests confirmed the nontoxic character of studied hydrogel systems.
Conclusions
Injectability studies showed the investigated MC/agarose hydrogel systems might be readily injected by humans, proving their injectability. Investigated MC/agarose hydrogels provided a hospitable ECM-mimicking environment enhancing cell spreading, migration, and proliferation. These studies demonstrate the high potential of investigated materials for tissue engineering applications.
Acknowledgments: Financial support was received from Polish National Science Center (NCN), grant number 2018/29/N/ST8/00780.
References:
1. Kim, M et al. Int. J. Biol. Macromol. 109,57-64 (2018)."
94238107637
"Introduction:
Human Mesenchymal stromal cells (hMSC) are appealing candidates for regenerative medicine applications. However, upon implantation, they encounter an ischemic microenvironment depleted of oxygen and nutrients responsible for their massive death post-transplantation, a major roadblock to successful clinical therapies. To date, various approaches have been proposed to address this issue, albeit with limited clinical success. We hereby propose a paradigm shift for enhancing hMSC survival by designing, developing, and testing an enzyme-controlled, nutritive hydrogel with an inbuilt glucose delivery system for the first time. This novel hydrogel is composed of fibrin, wheat starch (a glucose polymer), and amyloglucosidase (AMG), which hydrolyze glucose from starch.
Methodology:
In vitro: Glucose concentration at the core of hydrogels was determined using a custom-made glucose electrode biosensor. hMSC survival was assessed by cytometry after releasing cells from cell-loaded hydrogels exposed at 0.1% oxygen for up to day 14. The chemotactic potential of hMSCs towards hMSC and Human Umbilical Vein Endothelial Cells (HUVEC) was assessed by collecting conditioned Media (CM) from these hMSC-loaded hydrogels and evaluating migration in Boyden chambers. Moreover, chemotactic and angiogenic cytokines in CM were quantified using Luminex®.
In vivo: fluorescent-labelled AMG leakage from cell-free fibrin hydrogels was monitored using Xenogen live imaging until day 14 after ectopic implantation in nude mice. Luciferase-labelled hMSC survival within both fibrin/starch/AMG and fibrin hydrogels was assessed in an ectopic nude mice model by bioluminescence imaging until day 14. New blood vessel formation in the hydrogel vicinity was determined by µCT scanner, using a radiopaque agent Microfil® perfused within blood vessels at day 7 and 14.
Results:
In vitro fibrin/starch/AMG hydrogels released glucose at physiological concentrations and exhibited a 95 fold increase in hMSC survival compared to fibrin hydrogels after 14 days. CM collected from hMSC loaded fibrin/starch/AMG hydrogels showed (i) a 9- and a 4-fold increase in chemotactic potential towards hMSCs and HUVECs and (ii) a statistically significant rise in most but not all chemotactic and angiogenic cytokines compared to hMSC loaded fibrin hydrogels. In vivo glucose concentration within cell-free fibrin/starch/AMG hydrogels was within physiological ranges at days 7 and 14. Fluorescence monitoring revealed that AMG had completely disappeared within 7 days. hMSCs viability (measured by bioluminescent signal intensity compared to day 1) was 76.4% within fibrin/starch/AMG hydrogels and 22.1% within fibrin hydrogels at day 7. The hMSCs viability decreased drastically between days 7 and 14, corroborating the AMG time course. Last but not least, the formation of new blood vessels in the hydrogel vicinity exhibited a 4-fold increase when using fibrin/starch/AMG hydrogels compared to fibrin hydrogels at day 21.
Conclusion:
We hereby establish the proof of concept that a fibrin/starch/AMG hydrogel provides glucose to hMSCs and maintain their viability and angioinductive potential in vitro and in vivo. AMG sustained delivery is required to extend the survival time of the transplanted hMSCs."
62825411166
"Introduction. Despite being the youngest branch of regenerative medicine, neural tissue engineering has rapidly developed, with numerous advances close to clinical translation. One of the most researched areas are tissue engineering and regenerative medicine approaches for brain repair after ischaemic stroke, with more than 70 preclinical studies testing therapeutic combinations of biomaterials, stem cells, and/or therapeutic proteins. Many of these studies have administered the regenerative therapies at the target site, in the stroke cavity resulted following the clearance of dead tissue in the chronic phase of stroke. This localised administration favours tissue repair by overcoming the blood brain barrier, which drastically limits drug accessibility into the brain. Most studies assess behavioural and histological outcomes. Here, we propose a more specific approach to assess the success of a combination therapy based on the use of an injectable synthetic peptide hydrogel and two multifunctional therapeutic proteins for inducing recovery following ischaemic stroke.
Methodology. We propose outcome evaluation at three main levels: behaviour functional (through sickness, wellbeing, and memory behavioural tests), functional vascular, and histological level. The behaviour functional level assesses brain function, representing the ultimate goal of regenerative therapies. In this regard, sickness, wellbeing, sensory-motor, and memory tests are performed. The functional vascular level assesses the cerebral blood flow in potentially regenerated areas through magnetic resonance imaging (MRI) arterial spin labelling (ASL). The histological level assesses markers of interest for the main repair processes which should be targeted by novel therapies: reduction of inflammation, immune-modulation, angiogenesis, and neurogenesis.
Results. Here, we have assessed the effect on functional recovery of a regenerative construct using wellbeing and sickness behaviour tests like the burrowing and nest building tests, and the sensorimotor neuroscore test. Functional vascular assessment through MRI ASL can discern cerebral blood flow differences after stroke, differences which minimise at 28 days post stroke. Histological assessment of brain inflammation was done through scoring of brain immune cells, Iba1+ (ionising calcium binding adaptor molecule 1) microglia, into discrete activation states. Relevant for tissue repair, measurement of glial scar thickness was made by GFAP+ (glial fibrillary acidic protein) integrated density quantification. Assessment of angiogenesis, the most targeted repair process in stroke, was done by measuring the ratio between endoglin, marker of new endothelial cells, and PECAM-1, an endothelial cell adhesion molecule. Finally, neurogenesis is assessed by integrated density of doublecortin positive cells infiltrated into the infarct and ratio of ipsilateral to contralateral doublecortin integrated density in the neurogenic niche-containing brain ventricles.
Conclusions. Our approach proposes the stroke outcome assessment in pre-clinical models at three different levels, behaviour functional, vascular functional, and histological functional. These are instrumental in evaluating the success of regenerative therapies for brain recovery after stroke, before further progression into clinical practice."
94238162648
"Adhesive biomaterials have been studied by the scientific community in an attempt to surpass the current disadvantages of sutures and staples in surgery, such as the challenging implementation in some tissues and the risk of infection. The developed bioadhesives have been used as: i) glues for maintaining biological tissues together after laceration; ii) tissue sealants for preventing the leakage of body fluids, and; iii) hemostatic agents for helping generate the blood clot[1]. Despite the potential of this area, whose market size was valued at USD 1.8 billion in 2015 and is projected to grow 9.7% annually[1], existent adhesive materials approved by the Food and Drug Administration (FDA), still present several limitations. Some are not biocompatible or their degradation produces cytotoxic by-products, others lack bulk strength and bioactive properties, and some have proinflammatory potential[2].
Regarding wet-adhesion, mussel-inspired bioadhesives have gained attention, mimicking the mussel´s strong underwater adhesion, using catechol groups in the compound’s structures[3]. Tannic acid (TA), a plant-derived polyphenol, is a safe and low-cost source of catechol/pyrogallol groups. It allows polymeric crosslinking through hydrogen and ionic bonding, or hydrophobic interactions, improving biomaterials adhesiveness and mechanical performance, while endowing it with anti-microbial, anti-inflammatory and antioxidant properties[4]. Hence, by combining laminarin (LAM-OH) or pullulan (PUL-OH), two natural origin polisaccharides with TA, bioinspired adhesive biomaterials for biomedical applications were produced.
A library of bioadhesives was fabricated by combining PUL-OH and LAM-OH with TA in several concentrations, having the best formulations been chosen by lap shear test performance. Then, LAM-OH and PUL-OH were functionalized with methacrylic groups, having the modification of the polymers backbone (LAM-MET and PUL-MET) been successfully confirmed by 1H-NMR spectroscopy and FTIR. In the previous best formulations, the natural polysaccharides were substituted by LAM-MET and PUL-MET, respectively, and the bioadhesives presented adhesion to wet porcine skin, contrary to some already commercialized cyanoacrylate adhesives. Rheological and biological properties were also evaluated. Therefore, the present bioadhesives show good perspectives for being implemented as soft tissue bioadhesives.
References
[1] Z. Ma, G. Bao, J. Li, Z. Ma, G. Bao, and J. Li, “Multifaceted Design and Emerging Applications of Tissue Adhesives,” Adv. Mater., vol. 33, no. 24, p. 2007663, Jun. 2021.
[2] G. M. Taboada et al., “Overcoming the translational barriers of tissue adhesives,” Nat. Rev. Mater. 2020 54, vol. 5, no. 4, pp. 310–329, Feb. 2020.
[3] H. Lee, N. F. Scherer, and P. B. Messersmith, “Single-molecule mechanics of mussel adhesion,” Proc. Natl. Acad. Sci. U. S. A., vol. 103, no. 35, pp. 12999–13003, 2006.
[4] K. Kim et al., “TAPE: A Medical Adhesive Inspired by a Ubiquitous Compound in Plants,” Adv. Funct. Mater., vol. 25, no. 16, pp. 2402–2410, Apr. 2015."
31412743105
"Introduction. Breast cancer is considered nowadays the most common cause of death for female population. Since traditional treatments (e.g., chemotherapy, radiotherapy, surgery) showed several drawbacks [1], Drug Delivery Systems (DDS) recently gained interest due to the possibility to release therapeutic agents locally and controllably in targeted sites. Herein, we aimed to develop stable drug-loaded alginate microspheres (MS) encapsulating a natural anti-tumoral drug, curcumin, to be released in the tumor site [2]. Unloaded and drug-loaded MS were investigated by a morphological, chemo-physical, and biological characterization.
Materials and Methods. Sodium alginate (3% w/V) was dissolved in distilled water and alginate MS were obtained by extrusion dripping, using a coaxial needle (dint = 26G, dext = 20G), connected to a compressor which allowed a laminar air flow (P = 0.5 bar). Alginate droplets were chemically crosslinked in a CaCl2 (450, 900 mM) bath, and MS were then collected. In vitro stability tests were carried out on curcumin-loaded (0.3% w/V) and unloaded MS formulations, in neutral (pH = 7.4) and acidic (pH = 5.3) environment. Then, curcumin-loaded alginate MS were investigated in terms of encapsulation efficiency (EE%) and drug release. Lastly, in vitro biological tests were performed to investigate the effect of curcumin-loaded alginate MS on MCF-7 tumoral cells, previously 2D-seeded.
Results and Discussion. Extrusion parameters were selected through MS morphological analysis (threshold: Ø < 1.5 mm, circularity > 0.6) [3] [4]: A3-C450-0.5, A3-C900-0.5. In vitro tests stability exhibited no significant differences (p > 0.05) between alginate MS formulations in the same environment. This finding suggested that alginate MS reached the maximum swelling when crosslinked in 450 mM CaCl2. Differences (p < 0.05) were noticed comparing swelling kinetic in the two environments: degradation rate appeared faster (130 h) at pH 7.4 than at pH 5.3 (1008 h). EE% resulted higher (p < 0.05) for A3-C450-0.5-Cur than for A3-C900-0.5-Cur. As regards, alginate instantaneous crosslinking allowed for a higher drug release from MS, diminishing EE% [5]. Furthermore, drug release tests did not show significant differences (p > 0.05) between two CaCl2 concentration. Otherwise, the environment allowed for a lower drug release in acidic environment for both the formulations. In vitro biological tests showed that MCF-7 tumoral cells exhibited an increasing metabolic activity when cultured in contact with drug-unloaded alginate MS, as expected. Cell metabolism decreased when MCF-7 cells were cultured in presence of curcumin-loaded alginate MS.
Conclusions. Optimized alginate MS were tested as DDS in our work to develop a stable and controllable anti-tumoral therapy for breast cancer treatment. As regards, in vitro biological tests showed DDS efficiency in terms of drug release, which resulted time prolonged and specific. As regards, MCF-7 tumoral cells cultured on TCPS decreased their cell metabolism once in presence with curcumin-loaded MS.
1 American Cancer Society, American Cancer Society (2019)
2 Sookkasem, A. et al., RSC Advances, 8753-8756 (2015)
3 Lee, B. B. et al., Chemical Engineering and Technology, 1627-1642 (2013)
4 Lin, S. F. et al., PLOS ONE (2016)
5 Rastogi, R. et al., International Journal of Pharmaceutics, 71-77 (2007)"
41883642006
Introduction
Neurodegenerative diseases (NDs) are a group of chronic disorders (e.g. Alzheimer’s disease, Parkinson’s disease and multiple sclerosis -MS) characterized by progressive neurological dysfunction. Despite different neuronal populations can be affected, NDs share major clinical manifestations, namely motor impairment, cognitive disability and/or dementia. Effective treatments do not exist, but stem cell therapies emerged as treatment modalities with potential to cure NDs. However, despite high initial expectations, their clinical use is still limited. To overcome their crucial limitations, such as poor cell survival and low penetration into the central nervous system (CNS), we designed a hydrogel to deliver bone marrow mesenchymal stem cells (BMSCs) intrathecally or intracerebroventricularly.
Methodology
The hydrogel physically crosslinked with liposomes was based on biomolecules (phospholipids and hyaluronic acid-HA) naturally present in the CNS. The characterization comprised the determination of size, polydispersity index (PDI), surface charge and temperature transition of large unilamellar liposomes (LUVs) and their distribution in the HA matrix as well as the assessment of the gel’s thermal and rheological behaviors. Hydrogel cytocompatibility was assessed using BMSCs isolated from healthy rats. To determine biocompatibility and efficacy two rat strains were used, namely Wistar Han rats and Lewis rats, respectively. This choice was based on their ideal use for testing the formulations’ safety and therapeutic efficacy in experimental autoimmune encephalomyelitis (EAE). The assessment of the hydrogel’s in vivo compatibility was performed through its direct injection into the rat’s ventricular space. Moreover, a fluorescent-labeled hydrogel was used to investigate its brain distribution. To determine the efficacy of the developed formulation, containing a significantly lower number of cells than previously reported, the daily body weight, clinical score, and neuropathology levels were assessed in EAE rat models.
Results
LUVs presented a homogeneous (PDI=0.088+0.022) size of 115.7+3.5 nm and a significant negative surface charge (-33.4+3.7 mV). Hydrogels with LUVs displayed a rougher surface than the glycosaminoglycan hydrogel. The shift to a lower value of the temperature of the endothermic peak of HA also confirmed the presence of liposomes. Moreover, liposomes increased the elastic and viscous moduli of the HA matrix as well as the viscosity of the formulation. The encapsulation of BMSCs in the 3D matrix demonstrated they were able to adhere to, survive and proliferate within the hydrogels to a higher extent than in 2D cultures. In vivo studies confirmed hydrogel safety. Moreover, the hydrogel diffused into the corpus callosum, which is ideal for NDs treatment, as the damage of this white matter structure is responsible for important neuronal deficits. The BMSCs-laden hydrogel significantly decreased the maximum mean clinical score and average mean clinical score when compared with the control group of EAE and eliminated the relapse.
Conclusions
The developed formulation was more efficacious in reducing disease severity and maximum clinical score in EAE rats than cells suspensions, demonstrating the added value of cell incorporation in the hydrogel. Therefore, the engineering of stem cells therapies using this natural carrier can result in efficacious treatments for MS and related debilitating conditions.
Acknowledgements: FCT-Cells4_IDs-PTDC/BTM-SAL/28882/2017, IF/003472015, FROnTHERA-NORTE-01-0145-FEDER-000023 and NORTE2020 Structured Project
94238135166
"INTRODUCTION
The regenerative effect of Platelet Rich Plasma on skin and other tissue lesions is well known. If, on the one hand, research aims to optimize PRP standardized protocols, on the other hand, it aims to identify substrates as vehicles for the platelet content release to the lesion site. For the latter purpose, hyaluronic acid (HA) is proposed thanks to its viscoelastic and biological properties and biocompatibility. The aim of this study was the set-up and characterization of an “off the shelf” freeze-dried and injectable device based on HA that entraps PRP in a stable matrix sustaining the platelet growth factor release.
METHODOLOGY
High MW HA (1400 KDa – HA-HMW) and Low MW HA (56, 90, 200 KDa – HA-LMW) were used in combination with PRP at the platelet concentration of 2.5x106 plts/ul in the ratio 1:1. The Rheology of selected HAs with defined hydrodynamic parameters was analyzed at the concentration used for PRP mixing and lyophilization. After regeneration, the resulting lyophilized mixtures HA/PRP were tested for in vitro cell proliferation and scratch assays on human primary fibroblasts. The biological activity of freeze-dried HA-LMW/PRP formulations were also tested during storage at different temperatures (25°C, 4°C and -20°C) up to 6 months.
RESULTS
In a first set of experiments, HA-HMW/PRP was evaluated for its biological activity, showing that the freeze-dried and regenerated HA-PRP combination supported human dermal fibroblast proliferation in a comparable way to PRP alone. Although the biological properties of the HA-HMW/PRP were maintained, the formulation needed almost half an hour for full regeneration and quite a strong pressure to be extruded by a 21-gauge needle.
To overcome these limitations, hyaluronans of low molecular weight were selected after a specific hydrodynamic (SEC-TDA) analysis, namely HA 56 KDa, HA 90 KDa, HA 200 KDa. All formulations obtained by the combination of these HA with PRP induced cell proliferation. For a clinical application of an “off the shelf” lyophilized product it is mandatory to preserve the PRP activity along time. We already reported that the long-term storage of the freeze-dried PRP was associated to a progressive biological activity loss. In this work, the HA/PRP formulation were tested to evaluate the possible stabilization by HA at different temperatures and length of storage.
All formulations induced cell proliferation comparable to PRP alone at the different tested temperatures, but, interestingly, the 56 KDa HA/PRP formulation, after 6 months of storage at 25°C, showed significant preservation of the proliferation activity compared to PRP alone, suggesting a protective effect of HA versus the PRP bioactive factors. In addition, some of the low molecular weight HA-PRP formulation showed, at the same storage condition, a superior healing rate in a scratch assay followed by time lapse video microscopy.
CONCLUSIONS
In conclusion, we developed a lyophilized HA-based/PRP device, that may improve bioadhesive properties of the sole PRP also improving on site delivering (e.g. wound treatment). These formulations proved to release platelet factors preserving their biological activity over time. HA/PRP allows the development of promising products, for topical and intra-articular applications."
83767224367
In this presentation the latest advances in the field of biomaterials for tendon repair will be presented. Current technologies put forward for engineering tendon tissue will be discussed. The focus of the presentation will be on the latest developments in electrospun based scaffolds exhibiting suitable time dependant mechanical properties, nanoscale (fibrous) topography and the ability to deliver locally growth factors. Emerging developments based on tailored combinations of synthetic and natural polymers incorporating nanovectors for local growth factor delivery, related to the EU project P4FiT, will be reported and discussed.
83871202586
"Epithelial-to-mesenchymal transition (EMT) is a key event in embryo development and post-natal life in which epithelial cells undergo to a transdifferentiation into mesenchymal cells by acquiring a mobile state. This cell transitioning process recognizes the activation of signaling pathways which occur under controlled environments in response to factors controlling stem cell epigenetic reprogramming, self-renewal and differentiation (1). The investigation of EMT processes controlling tissue patterning and organization as well as disregulating healing processes leading to fibrosis, as for tendinopathies, has been addressed for enabling the advancement of regenerative medicine strategies based on the control of EMT-mediated events in generating a favorable local environmental of stem cell-host tissue dialogue as well as of repairing feedback loop between extracellular matrix (ECM) and progenitor/host cells (4).
In order to verify the role exerted by EMT mediated decision during tenogenesis an epithelial stem cell source derived from the amnion were used to analyse the mechanisms underlying the recovery of tendon microarchitecture and function in relation to cell phenotype. To this aim validated in vitro protocols have been used in order to control the phenotype status of AECs before transplantation by obtaining three subset of cells, starting from a unique genome makeup: epithelial (eAECs), mesenchymal (mAECs) (2) and tendon-like (tdAECs) cells (5). The results of this research demonstrate that eAECs and tdAECs are the most suitable phenotypes to use in order to accelerate the process of regeneration in experimental injured tendons. The healing advantage obtained using these two AECs’ phenotypes is however obtained through two different underlying mechanisms. Epithelial AEC were able to positively influence the process of tendon healing mainly through the modulation of the host tissue immune environment, targeting a potential shift from pro-inflammatory and pro-fibrotic to pro-regenerative cellular responses, which probably led to a reduced infiltration of inflammatory cells responsible of the ordered deposition of ECM components. On the other hand tdAECs transplantation played the major role in accelerating the deposition and organization of the ECM without strongly influencing the host immune system. Thus, the ability to control the cell phenotype by reproducing the epithelial-mesenchyme-tenodifferentiation stepwise process, allowed us to comprehensively determine how to drive the regenerative process by supporting a favorable stem cell-host tissue cross-talk. Studies that probe the mechanisms underlying the relationship between EMT and EMT/ECM interactions thus represent the next steps forward in elucidating strategies for reducing fibrosis.
References
1. Wilson M. M. et al. Trends in Cancer 2020;
2. Canciello A et al. Sci Rep, 2017;
3. Barboni B et al. J Tissue Eng Regen Med, 2018;
4. Russo V et al. Cells 2020;
5. Barboni B et al. PLOS ONE 2012
Acknowledgments: This research is funded by H2020-MSCA-ITN-EJD-P4 FIT grant agreement No 955685."
94355103066
The poor healing ability of tendons as well as the limitations of currently used therapies have motivated tissue engineering (TE) strategies to develop living tendon substitutes. At the same time, the significant lack of knowledge on tendon homeostasis and disease mechanism trigger our interest to focus on the development of adequate 3D tissue models that can provide important insights for developing better and innovative therapies. Our team has been exploring the development of cell-laden 3D magnetically responsive systems that recapitulate key features of the native tissue and that can be remotely actuated both during in vitro culture and/or upon in vivo implantation, through the application of external magnetic stimuli. We are exploring conventional and innovative tools such as multimaterial 3D bioprinting to design magnetic responsive systems mimicking specific aspects of tendon tissue architecture, composition and biomechanical properties, which, combined with adequate stem cells, shall render appropriate behavioural instructions to stimulate the regeneration of tendon tissue. We have demonstrated that the magnetic stimulus of different intensities/frequencies can trigger tenonegic differentiation od hASCs and/or modulate inflammatory response of various cell types. Simultaneously, the 3D cell-laden magnetic system are also being as sophisticated 3D tissue models to unravel mechanisms behind tendon homeostasis and repair that shall support the base knowledge to establish rational design criteria for the biofabrication of living tendon substitutes offering the prospect of tendon regeneration as opposed to simple tissue repair.
Acknowledgments: Authors thank Hospital de Guimarães for tissue samples; FCT for project MagTT PTDC/CTM-CTM/29930/2017 and HORIZON 2020 for ERC CoG MagTendon (772817) and Twinning Project Achilles (810850)
"Introduction: MicroRNAs are short, non-coding RNA sequences with the ability to inhibit the expression of a target mRNA at the transcriptional level. MiRNAs are involved in the regulation and modulation of both regenerative and degenerative processes, playing crucial regulatory roles in tissue healing and regeneration. This particular feature makes this family of molecules a very interesting niche to explore in pursuit of novel therapeutic tools in the fields of tissue engineering and regenerative medicine. Among the many tissues comprised in the musculoskeletal system, the tendon-to-bone enthesis is notoriously difficult to treat due to the heterogeneity of its composition. Upon injury, the fibrocartilaginous transition between the tendon and the bony ends of the enthesis usually doesn’t regenerate. Furthermore, the occurring fibrotic process typically yields a scar tissue with poor mechanical properties prone to recurrent rupture. With this study, we aimed to investigate the early expression patterns of fibrosis-related miRNAs in an injured enthesis to select the ultimate miRNA candidate(s) to be used as a therapeutic tool to aid in the treatment of entheses defects.
Methodology: A longitudinal defect was created at the patellar enthesis of adult rats. Explants were collected at one (n=6) and 10 days (n=6) after the injury. Tissue samples of the contralateral (healthy) side were used as control. MiRNA expression was assessed by a miScript qPCR array specific for fibrosis (Qiagen) containing a total of 86 miRNAs. Fourteen potentially enthesis-injury/regeneration-related miRNAs resulted de-regulated after the injury compared to the expression in the native tissue. The expression of these miRNAs was then validated in each separate sample. Target prediction was carried out by Ingenuity Pathway Analysis (IPA-Qiagen). The expression of the predicted mRNA targets was investigated by qPCR in each sample. Additionally, protein expression levels of the collagens type I, II, III, and X were investigated by western blot analysis.
Results: We observed that enthesis-injury/regeneration-related miRNAs showed a three- to five-fold down-regulation after one day of the injury and two- to 14-fold up-regulation after 10 days. Further IPA analysis predicted potential mRNA targets relevant for enthesis development and healing for seven of the fourteen de-regulated miRNAs. These were miR-16, -17, -100, -124, -133a, -155 and -182. Furthermore, the predicted mRNA targets included Col2a1, Runx2, Egfr1, Smad2, and Smad3. The mRNA expression pattern confirmed their regulation according to the up- or down-regulation of their respective targeting miRNA. Furthermore, at the protein level, the collagens type I and II resulted down-regulated directly after the injury and up-regulated after 10 days while collagens III and X showed the opposite pattern of expression.
Conclusions: These preliminary results bring new insights on the role of miRNAs in the early healing phases of the enthesis upon injury. Moreover, miRNA expression can be modulated by using mimics or antagomirs. Therefore, the mimics or antagomir of these de-regulated miRNAs could be used as time-sensitive, therapeutic tools to enhance or inhibit the miRNA modulatory effect over their mRNA target to aid in the regeneration process of the injured enthesis."
83767238706
Tendon/ligament injuries are a relevant clinical problem in modern society. Although these tissues can selfheal when a lesion occurs, the complete functional recovery is difficult to achieve due to their low cellularity and vascularity. Moreover, reconstruction strategies have a non-negligible failure rate [1]. Failures often occur at the enthesis, the tendon/ligament-bone insertion [2]. This junction is a heterotypic tissue characterized by a graded structure from soft (ligament) to hard (bone) tissues with a heterogeneous distribution of cell types, matrix components and architecture [3]. In this work, a multimaterial and multiscale approach was developed, to fabricate scaffolds mimicking the complexity of these tissues, exploiting the combination of electrospinning and 3D printing technologies. Firstly, commercial, medical grade, and bioresorbable polymers both natural (porcine gelatin and gelatin methacryloyl(GelMA)) and synthetic (poly(l-lactic acid)(PLLA), poly(lactic-co-glycolic acid)(PLGA), polycaprolactone (PCL)) were systematically investigated to select the most valuable candidate. On supports made by solvent casting, we analyzed cell viability, proliferation, and gene expression of bone marrowderived mesenchymal stem cells (BM-MSCs). Among the tested materials, PLGA and PCL displayed the best ability to promote the proliferation of BM-MSCs. Further studies highlighted the ability of PLGA and PCL to promote, respectively, the tenogenic and osteogenic differentiation of BM-MSCs. Subsequently, a scaffold fabrication protocol was developed. For the region that interacts with bone, PCL grid-shaped scaffolds were 3D printed by fused deposition modelling (FDM) technology [4]. The ability of
these constructs to promote the BM-MSCs osteogenic differentiation was validated by confocal microscopy imaging. The tendon-like aligned fiber network was replicated by electrospinning PLGA fibers collected on a rotating drum collector. Electrospun structures were mechanically tested, and the fiber alignment was evaluated as function of drum revolutions per minute. Viability and proliferation tests highlighted the possibility to use these electrospun structures as scaffold for tendon/ligament engineering. The enthesis was replicated by directly extruding PCL onto PLGA electrospun films. A fine tuning of the extrusion parameters allowed the PCL deposition onto the electrospun PLGA mates without affecting the fibers integrity, as highlighted by scanning electron microscopy analysis. Scaffolds presenting the three different regions were, then, fabricated and the strength of the interface between 3D printed and electrospun structures was evaluated performing tensile tests. Finally, following this fabrication protocol bidimensional and 3D-dimensional anterior cruciate ligament (ACL) scaffolds, presenting the osteotendinous junction, were fabricated.
References
[1] Jason T. Shearn et al., Musculoskelet Neuronal Interact. 2011 Jun; 11(2): 163–173.
[2] Zhao S. et al., Colloids Surf., B Biointerfaces (2017): 157, 407.
[3] Hammoudi T M. et al., Biomaterials for Tissue Engineering Applications, Springer pp 307–41.
[4] G.Criscenti et al., Biofabrication, vol. 8, no. 1, p. 15009, 2016
62825415687
"Introduction: Tendinopathies are one of the most common musculoskeletal conditions. Unsatisfactory healing has a significant impact on the life of patients and imposes a remarkable socioeconomic burden. The recovery from tendon injuries is slow and requires extensive rehabilitation. The resulting scar tissue lacks the mechanical integrity of the original tissue, and therefore complete recovery is rarely achieved1 2. Several biological therapeutics have been proposed so far, such as the delivery of growth factors, stem cells and recently the application of gene therapy3, but limited success has been achieved. In this project, lipid-polymer hybrid nanoparticles (LPNs) are proposed for the co-loading of two biological drugs with the aim to achieve tendon regeneration. Interleukin-4 (Il-4) is an anti-inflammatory cytokine extensively used in macrophage polarization towards the anti-inflammatory M2 phenotype. Co-loading of IL-4 with an siRNA against one of the genes involved in fibrosis could potentially render a dual therapeutic effect: immunomodulation and fibrosis prevention 4,5.
Methodology: LPNs, consisting of a PLGA core and a lipid shell, were prepared using a newly developed method based on nanoprecipitation using a glass-capillary microfluidics technique. Empty LPNs were also analyzed by transmission electron microscopy (TEM) doing negative staining with phosphotungstic acid (PTA). The toxicity of the empty particles was assessed in RAW 264.7 cells with a luminescent CellTiter Glo® assay. The loading of a model siRNA (i.e., eGFP siRNA) in the lipid shell was quantified using the fluorescent Ribogreen assay. The loading of IL-4 in the PLGA core was determined using an ELISA assay. The transfection efficiency of the siRNA-loaded LPNs, measured as eGFP expression inhibition, was evaluated in RAW 264.7 cells expressing eGFP by flow cytometry. The ability of the IL-4-loaded LPNs to polarize macrophages was assessed in the same cell line by quantification of an increased expression of markers of M2 macrophages as compared to M1.
Results: Homogenous PLGA cores with sizes smaller than 300 nm were obtained by a glass-capillary microfluidics technique upon optimization of the process and formulation parameters. LPNs were prepared by two different microfluidics methods involving one and two steps, respectively, rendering homogenous particles of approximately 350 nm. The TEM images confirmed the results obtained by DLS and unraveled a spherical shape for the PLGA cores and a spherical/elongated shape for the hybrid NPs. LPNs were loaded with eGFP siRNA in the lipid shell and IL-4 in the PLGA core and a lack of toxicity was proved up to concentrations of 200 µg/mL.
Conclusion: This work demonstrates the potential of hybrid nanoparticles to load biological drugs with different physicochemical properties and therapeutic effects, allowing for the development of novel nanosystems with dual effects. The developed platform could potentially be used in tendinopathies to promote scarless tissue repair and recovery of the biomechanical function of the tissue."
41883611855
Modular tissue-engineering approaches provide a promising strategy for building complex living structures from the bottom-up, through the co-assembly of microscale tissue units. Using biofabrication tools, multiple modular units of parenchymal, stromal, and vascular tissues can be rationally combined to recreate structurally/functionally different compartments of human organs. Microtissue units present a high surface area, which facilitates the diffusion/mobility of oxygen, molecules, and cells through interstitial gaps, affording a useful tool to generate densely cellularized 3D constructs. In this talk, we outline different approaches to engineering vascularized microtissues and describe some of their applications in the fields of regenerative medicine and in vitro modeling.
31451703204
TBA
"Background and Aims: Cholangiocarcinoma (CCA) is a highly aggressive tumor which arises from the biliary duct epithelium. Currently available models fail to recapitulate the full complexity of CCA, particularly the desmoplastic environment and the interplay between cancer cells and the extracellular matrix (ECM). We aimed to create an improved 3D in vitro model by combining patient-derived CCA organoids (CCAOs) with native CCA tumor and liver ECM, obtained by decellularization, to study the role of tumor cells in desmoplasia and ECM remodelling.
Method: Patient-derived CCA matrix (CCA-M) and tumor-free liver matrix (TFL-M) were obtained by decellularization of tumor and tumor-free liver biopsies from CCA patients. The decellularized scaffolds were biochemically and mechanically assessed using nanoindentation and rheology. Subsequently, CCA-M and TFL-M were recellularized with CCAOs. Tumor cell behavior of CCAOs in CCA-M, TFL-M was studied on a transcriptome level with bulk RNA-sequencing, and protein level with immunocytochemical stainings and Stable Isotope Labeling by Amino acids in Cell culture (SILAC)-based mass spectrometry. Cell viability measurements were taken for quantifying CCAO response to chemotherapeutics. Standard culture conditions of CCAOs in basement membrane extract (BME) were used as control.
Results: Decellularization of CCA tumor and liver tissue resulted in effective removal of cells while preserving ECM structure and retaining important characteristics of the tissue origin, including stiffness, collagen content, and the presence of desmoplasia, typically associated with CCA, in the tumor ECM. When culturing CCAOs in CCA-M, the expression profile of differentially expressed genes much more resembled the transcriptome of primary CCA tumor tissue in vivo compared to TFL-M (correlation coefficient (CC) CCA-M 0.83±0.03 vs CC TFL-M 0.70±0.03, p = 0.004) and BME (CC CCA-M 0.88±0.04 vs CC BME 0.63±0.06, p < 0.0001). This was accompanied by a significant difference in cell viability in response to exposure to gemcitabine, which is the standard of care treatment for CCA patients (mean viability at 100uM CCA-M 0.86 vs TFL-M 0.64, p = 0.018). These results provide evidence that the desmoplastic extracellular environment in CCA plays an important role in chemoresistance. Moreover, CCA-M induced specific extracellular matrix protein production in CCAOs, such as fibronectin 1 (FN1), which is related to desmoplasia and decreased patient survival. In TFL-M, lacking desmoplasia, CCAOs initiated a desmoplastic reaction directly through increased production of multiple collagen types (e.g. COL1A1, COL1A2, COL6A1, COL6A3).
Conclusion: This study demonstrates that combining tumor organoids and decellularized matrix provides a complex in vitro tumor model that can recapitulate key components of CCA biology, including transcriptome profiles, drug responses, and ECM remodeling activity. The increased production of ECM proteins, primarily collagens, indicates that epithelial tumor cells are able to contribute to their own desmoplastic environment. Complementing organoid-based culture models with tumor decellularized matrix is applicable to a variety of tumors and could result in overall better recapitulation of tumor behavior in vivo."
31412708526
Inner ear disorders (e.g., hearing loss) are common, but it is difficult to develop a therapeutic drug and to find a specific mechanism because of a lack of the research platforms. Inner ear organoids (IEO) are perceiving as an innovative research platform to reproduce the complex inner ear systems and to solve the previous problems. To improve uniformity and reproducibility of IEO, we develop a microengineered system that can collect cells and form aggregates rapidly. Using microengineered system, we can easily control the size of aggregates by the initial cell number and find the optimal condition to develop mature IEO. Compared with the traditional approach to develop IEO (i.e., IEO grown on U well plate; U-IEO), it is possible to develop IEO with the mass production and more reproducible shape. In addition, IEO grown on microengineered system (M-IEO) have an improved functions including the formation of mature kinocilia and the electrophysiological function than U-IEO. Thus, we conclude M-IEO may have great potential to overcome the limitations of the traditional approach, and it may be used as an advanced platform for inner ear disorder.
94238154186
"Introduction: An abundant and important receptor that regulates numerous ECM-cell interactions is the integrin receptor. To illustrate, integrins influence the cells’ polarity by controlling the apical-basal orientation. Kidney epithelial cells and intestinal organoids cultured in suspension and thus in the absence of matrix, invert the direction of their polarization, with an apical membrane facing outwards. These observations raise the question whether direct control over the integrins via the matrix’ stiffness and degree of bioactivity could possibly control the orientation of polarization, thereby directing epithelial morphogenesis and controlling organoid behavior.
Therefore, we propose synthetic, modular supramolecular hydrogels based on the ureido-pyrimidinone (UPy) motif, which use directional, non-covalent interactions. These assemblies are eminently suitable to study cell-material interactions, because herein we have full and independent control over several different properties, like stiffness and ligand concentration, allowing that the effects of such microenvironment components can be assessed individually. The UPy hydrogels consist of 3 molecules: monofunctional (M), bifunctional (B) and bioactive (RGD) UPys. Herein, the M UPys can form one-dimensional fibers, while the B UPys could act as a crosslinker between the M UPys to create a network with adjustable mechanical properties, by changing the M/B UPy ratio or by varying the hydrogel’s concentration. And finally, RGD UPys could be mixed in as integrin-binding ligands.
Here, we aim to investigate the influence of stiffness on renal epithelial cell and intestinal organoid polarization in 3D using supramolecular hydrogels.
Methodology: UPy supramolecular hydrogels were prepared in a fixed molecular ratio of 80 M to 1 B molecules at the desired concentration (wt%). For kidney cell studies in 3D, 0.5 mM of RGD UPy was incorporated into the hydrogel as part of the M molecules. Rheological experiments were measured at 1 rad/s and 1% strain.
Results: Rheology showed that the mechanical properties of the UPy hydrogels could be varied from ~ 0.1 kPa to 1 kPa to 2 kPa for the 0.6, 1.25 and 2.5 wt% gels, respectively.
Madin-Darby Canine Kidney (MDCKs) were then encapsulated in 3D in UPy hydrogels of different weight percentages and corresponding stiffness. After 10 days, cell encapsulation resulted in cyst formation with apical-basal polarization inside all supramolecular hydrogels. Three different structures were formed, ranging from cell aggregates with inverted polarity to well-polarized cysts with a lumen inside. Quantification of the frequencies of the different morphologies in the different conditions showed that cell aggregates were predominantly formed in the 0.6 wt% gels, whereas the cysts with hollow lumens were mostly observed in the 2.5 wt% gels. This observation indicates that the cell-cell interactions dominate when less hydrogel, and thus less mechanical support, is present.
Conclusions: We showed that the cell-cell interactions dominate in the 0.6 wt% gels, whereas well-polarized cysts are formed in the higher, 2.5 wt% hydrogels. To further investigate the origin of the difference in organization, we will test the influence of ligand concentration. Currently, we are also investigating the influence of mechanical support (i.e. the wt% of UPy gels) on growth and polarity of intestinal organoids."
20941849689
"Background and Aims:
Biliary complications that may arise after liver transplantation, such as non-anastomotic strictures and diffuse bile leakage, are challenging and complex. Ischemia-related cell death and impaired regeneration of damaged biliary epithelium is known to be involved in causing these complications. Intrahepatic cholangiocyte organoids (ICO) allow for the expansion and study of cholangiocyte-like cells, but access to the lumen of the organoids is limited and can only be studied after disrupting the 3D structure. There are currently no in vitro models mimicking the circumstances as exposure of bile ducts to warm ischemia time or cold storage, and therefore we aimed to establish a microfluidic bile-duct-on-chip (BDOC) platform for studying the effect of these conditions on biliary epithelium in vitro
Method:
ICO were initiated from human liver biopsies (N=5) obtained during liver transplant procedures. Three-channel BDOC (dimensions; length 1cm, width and height 500µm) were prepared by casting polydimethylsiloxane (PDMS) into a mold. Subsequently, plasma treated PDMS chips were bonded to glass slides. The BDOC channels were filled with solubilized decellularized human liver extracellular matrix and collagen type I pre-gel. Viscous finger patterning procedures were used to create channels inside these hydrogels. ICO-derived cells (50∙103 cells/channel) were introduced into the channels and ICO expansion medium was added to the reservoirs. The BDOC were incubated for up to 21 days. Growth of epithelial cells was monitored using confocal microscopy and histology.
Results:
ICO-derived cells populated the entire surface of the channels with a single layer of cells within 7 days after seeding. Whole mount confocal imaging revealed that cells were columnar in shape and morphologically looked like biliary epithelium. Zonula Occludens-1 (ZO-1) staining showed cholangiocyte-like polarization of cells in honey comb patterns. The cells express the cholangiocyte markers cytokeratin 7 and 19 on gene and protein level. Moreover, presence of glycocalyx components, such as fucosyl and sialic was confirmed based on histochemistry. This indicates that ICO form functional barriers.
Conclusion:
The results show that microfluidic approaches combined with cholangiocyte-like (KRT 7 and 19-positive) cells from ICO can be used to create healthy small diameter intrahepatic bile duct structures in vitro. This BDOC can thus serve as a platform to study epithelial damage and regeneration during warm and cold ischemic conditions in more detail in vitro. The platform can also be used to study the onset and progression of biliary diseases. <!--[if gte mso 9]><xml>
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Name=""List Continue 3""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true""
Name=""List Continue 4""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true""
Name=""List Continue 5""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true""
Name=""Message Header""/>
<w:LsdException Locked=""false"" Priority=""11"" QFormat=""true"" Name=""Subtitle""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true""
Name=""Salutation""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true""
Name=""Date""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true""
Name=""Body Text First Indent""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true""
Name=""Body Text First Indent 2""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true""
Name=""Note Heading""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true""
Name=""Body Text 2""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true""
Name=""Body Text 3""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true""
Name=""Body Text Indent 2""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true""
Name=""Body Text Indent 3""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true""
Name=""Block Text""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true""
Name=""Hyperlink""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true""
Name=""FollowedHyperlink""/>
<w:LsdException Locked=""false"" Priority=""22"" QFormat=""true"" Name=""Strong""/>
<w:LsdException Locked=""false"" Priority=""20"" QFormat=""true"" Name=""Emphasis""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true""
Name=""Document Map""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true""
Name=""Plain Text""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true""
Name=""E-mail Signature""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true""
Name=""HTML Top of Form""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true""
Name=""HTML Bottom of Form""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true""
Name=""Normal (Web)""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true""
Name=""HTML Acronym""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true""
Name=""HTML Address""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true""
Name=""HTML Cite""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true""
Name=""HTML Code""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true""
Name=""HTML Definition""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true""
Name=""HTML Keyboard""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true""
Name=""HTML Preformatted""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true""
Name=""HTML Sample""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true""
Name=""HTML Typewriter""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true""
Name=""HTML Variable""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true""
Name=""Normal Table""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true""
Name=""annotation subject""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true""
Name=""No List""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true""
Name=""Outline List 1""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true""
Name=""Outline List 2""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true""
Name=""Outline List 3""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true""
Name=""Table Simple 1""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true""
Name=""Table Simple 2""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true""
Name=""Table Simple 3""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true""
Name=""Table Classic 1""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true""
Name=""Table Classic 2""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true""
Name=""Table Classic 3""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true""
Name=""Table Classic 4""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true""
Name=""Table Colorful 1""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true""
Name=""Table Colorful 2""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true""
Name=""Table Colorful 3""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true""
Name=""Table Columns 1""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true""
Name=""Table Columns 2""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true""
Name=""Table Columns 3""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true""
Name=""Table Columns 4""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true""
Name=""Table Columns 5""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true""
Name=""Table Grid 1""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true""
Name=""Table Grid 2""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true""
Name=""Table Grid 3""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true""
Name=""Table Grid 4""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true""
Name=""Table Grid 5""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true""
Name=""Table Grid 6""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true""
Name=""Table Grid 7""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true""
Name=""Table Grid 8""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true""
Name=""Table List 1""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true""
Name=""Table List 2""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true""
Name=""Table List 3""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true""
Name=""Table List 4""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true""
Name=""Table List 5""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true""
Name=""Table List 6""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true""
Name=""Table List 7""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true""
Name=""Table List 8""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true""
Name=""Table 3D effects 1""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true""
Name=""Table 3D effects 2""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true""
Name=""Table 3D effects 3""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true""
Name=""Table Contemporary""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true""
Name=""Table Elegant""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true""
Name=""Table Professional""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true""
Name=""Table Subtle 1""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true""
Name=""Table Subtle 2""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true""
Name=""Table Web 1""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true""
Name=""Table Web 2""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true""
Name=""Table Web 3""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true""
Name=""Balloon Text""/>
<w:LsdException Locked=""false"" Priority=""39"" Name=""Table Grid""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" UnhideWhenUsed=""true""
Name=""Table Theme""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" Name=""Placeholder Text""/>
<w:LsdException Locked=""false"" Priority=""1"" QFormat=""true"" Name=""No Spacing""/>
<w:LsdException Locked=""false"" Priority=""60"" Name=""Light Shading""/>
<w:LsdException Locked=""false"" Priority=""61"" Name=""Light List""/>
<w:LsdException Locked=""false"" Priority=""62"" Name=""Light Grid""/>
<w:LsdException Locked=""false"" Priority=""63"" Name=""Medium Shading 1""/>
<w:LsdException Locked=""false"" Priority=""64"" Name=""Medium Shading 2""/>
<w:LsdException Locked=""false"" Priority=""65"" Name=""Medium List 1""/>
<w:LsdException Locked=""false"" Priority=""66"" Name=""Medium List 2""/>
<w:LsdException Locked=""false"" Priority=""67"" Name=""Medium Grid 1""/>
<w:LsdException Locked=""false"" Priority=""68"" Name=""Medium Grid 2""/>
<w:LsdException Locked=""false"" Priority=""69"" Name=""Medium Grid 3""/>
<w:LsdException Locked=""false"" Priority=""70"" Name=""Dark List""/>
<w:LsdException Locked=""false"" Priority=""71"" Name=""Colorful Shading""/>
<w:LsdException Locked=""false"" Priority=""72"" Name=""Colorful List""/>
<w:LsdException Locked=""false"" Priority=""73"" Name=""Colorful Grid""/>
<w:LsdException Locked=""false"" Priority=""60"" Name=""Light Shading Accent 1""/>
<w:LsdException Locked=""false"" Priority=""61"" Name=""Light List Accent 1""/>
<w:LsdException Locked=""false"" Priority=""62"" Name=""Light Grid Accent 1""/>
<w:LsdException Locked=""false"" Priority=""63"" Name=""Medium Shading 1 Accent 1""/>
<w:LsdException Locked=""false"" Priority=""64"" Name=""Medium Shading 2 Accent 1""/>
<w:LsdException Locked=""false"" Priority=""65"" Name=""Medium List 1 Accent 1""/>
<w:LsdException Locked=""false"" SemiHidden=""true"" Name=""Revision""/>
<w:LsdException Locked=""false"" Priority=""34"" QFormat=""true""
Name=""List Paragraph""/>
<w:LsdException Locked=""false"" Priority=""29"" QFormat=""true"" Name=""Quote""/>
<w:LsdException Locked=""false"" Priority=""30"" QFormat=""true""
Name=""Intense Quote""/>
<w:LsdException Locked=""false"" Priority=""66"" Name=""Medium List 2 Accent 1""/>
<w:LsdException Locked=""false"" Priority=""67"" Name=""Medium Grid 1 Accent 1""/>
<w:LsdException Locked=""false"" Priority=""68"" Name=""Medium Grid 2 Accent 1""/>
<w:LsdException Locked=""false"" Priority=""69"" Name=""Medium Grid 3 Accent 1""/>
<w:LsdException Locked=""false"" Priority=""70"" Name=""Dark List Accent 1""/>
<w:LsdException Locked=""false"" Priority=""71"" Name=""Colorful Shading Accent 1""/>
<w:LsdException Locked=""false"" Priority=""72"" Name=""Colorful List Accent 1""/>
<w:LsdException Locked=""false"" Priority=""73"" Name=""Colorful Grid Accent 1""/>
<w:LsdException Locked=""false"" Priority=""60"" Name=""Light Shading Accent 2""/>
<w:LsdException Locked=""false"" Priority=""61"" Name=""Light List Accent 2""/>
<w:LsdException Locked=""false"" Priority=""62"" Name=""Light Grid Accent 2""/>
<w:LsdException Locked=""false"" Priority=""63"" Name=""Medium Shading 1 Accent 2""/>
<w:LsdException Locked=""false"" Priority=""64"" Name=""Medium Shading 2 Accent 2""/>
<w:LsdException Locked=""false"" Priority=""65"" Name=""Medium List 1 Accent 2""/>
<w:LsdException Locked=""false"" Priority=""66"" Name=""Medium List 2 Accent 2""/>
<w:LsdException Locked=""false"" Priority=""67"" Name=""Medium Grid 1 Accent 2""/>
<w:LsdException Locked=""false"" Priority=""68"" Name=""Medium Grid 2 Accent 2""/>
<w:LsdException Locked=""false"" Priority=""69"" Name=""Medium Grid 3 Accent 2""/>
<w:LsdException Locked=""false"" Priority=""70"" Name=""Dark List Accent 2""/>
<w:LsdException Locked=""false"" Priority=""71"" Name=""Colorful Shading Accent 2""/>
<w:LsdException Locked=""false"" Priority=""72"" Name=""Colorful List Accent 2""/>
<w:LsdException Locked=""false"" Priority=""73"" Name=""Colorful Grid Accent 2""/>
<w:LsdException Locked=""false"" Priority=""60"" Name=""Light Shading Accent 3""/>
<w:LsdException Locked=""false"" Priority=""61"" Name=""Light List Accent 3""/>
<w:LsdException Locked=""false"" Priority=""62"" Name=""Light Grid Accent 3""/>
<w:LsdException Locked=""false"" Priority=""63"" Name=""Medium Shading 1 Accent 3""/>
<w:LsdException Locked=""false"" Priority=""64"" Name=""Medium Shading 2 Accent 3""/>
<w:LsdException Locked=""false"" Priority=""65"" Name=""Medium List 1 Accent 3""/>
<w:LsdException Locked=""false"" Priority=""66"" Name=""Medium List 2 Accent 3""/>
<w:LsdException Locked=""false"" Priority=""67"" Name=""Medium Grid 1 Accent 3""/>
<w:LsdException Locked=""false"" Priority=""68"" Name=""Medium Grid 2 Accent 3""/>
<w:LsdException Locked=""false"" Priority=""69"" Name=""Medium Grid 3 Accent 3""/>
<w:LsdException Locked=""false"" Priority=""70"" Name=""Dark List Accent 3""/>
<w:LsdException Locked=""false"" Priority=""71"" Name=""Colorful Shading Accent 3""/>
<w:LsdException Locked=""false"" Priority=""72"" Name=""Colorful List Accent 3""/>
<w:LsdException Locked=""false"" Priority=""73"" Name=""Colorful Grid Accent 3""/>
<w:LsdException Locked=""false"" Priority=""60"" Name=""Light Shading Accent 4""/>
<w:LsdException Locked=""false"" Priority=""61"" Name=""Light List Accent 4""/>
<w:LsdException Locked=""false"" Priority=""62"" Name=""Light Grid Accent 4""/>
<w:LsdException Locked=""false"" Priority=""63"" Name=""Medium Shading 1 Accent 4""/>
<w:LsdException Locked=""false"" Priority=""64"" Name=""Medium Shading 2 Accent 4""/>
<w:LsdException Locked=""false"" Priority=""65"" Name=""Medium List 1 Accent 4""/>
<w:LsdException Locked=""false"" Priority=""66"" Name=""Medium List 2 Accent 4""/>
<w:LsdException Locked=""false"" Priority=""67"" Name=""Medium Grid 1 Accent 4""/>
<w:LsdException Locked=""false"" Priority=""68"" Name=""Medium Grid 2 Accent 4""/>
<w:LsdException Locked=""false"" Priority=""69"" Name=""Medium Grid 3 Accent 4""/>
<w:LsdException Locked=""false"" Priority=""70"" Name=""Dark List Accent 4""/>
<w:LsdException Locked=""false"" Priority=""71"" Name=""Colorful Shading Accent 4""/>
<w:LsdException Locked=""false"" Priority=""72"" Name=""Colorful List Accent 4""/>
<w:LsdException Locked=""false"" Priority=""73"" Name=""Colorful Grid Accent 4""/>
<w:LsdException Locked=""false"" Priority=""60"" Name=""Light Shading Accent 5""/>
<w:LsdException Locked=""false"" Priority=""61"" Name=""Light List Accent 5""/>
<w:LsdException Locked=""false"" Priority=""62"" Name=""Light Grid Accent 5""/>
<w:LsdException Locked=""false"" Priority=""63"" Name=""Medium Shading 1 Accent 5""/>
<w:LsdException Locked=""false"" Priority=""64"" Name=""Medium Shading 2 Accent 5""/>
<w:LsdException Locked=""false"" Priority=""65"" Name=""Medium List 1 Accent 5""/>
<w:LsdException Locked=""false"" Priority=""66"" Name=""Medium List 2 Accent 5""/>
<w:LsdException Locked=""false"" Priority=""67"" Name=""Medium Grid 1 Accent 5""/>
<w:LsdException Locked=""false"" Priority=""68"" Name=""Medium Grid 2 Accent 5""/>
<w:LsdException Locked=""false"" Priority=""69"" Name=""Medium Grid 3 Accent 5""/>
<w:LsdException Locked=""false"" Priority=""70"" Name=""Dark List Accent 5""/>
<w:LsdException Locked=""false"" Priority=""71"" Name=""Colorful Shading Accent 5""/>
<w:LsdException Locked=""false"" Priority=""72"" Name=""Colorful List Accent 5""/>
<w:LsdException Locked=""false"" Priority=""73"" Name=""Colorful Grid Accent 5""/>
<w:LsdException Locked=""false"" Priority=""60"" Name=""Light Shading Accent 6""/>
<w:LsdException Locked=""false"" Priority=""61"" Name=""Light List Accent 6""/>
<w:LsdException Locked=""false"" Priority=""62"" Name=""Light Grid Accent 6""/>
<w:LsdException Locked=""false"" Priority=""63"" Name=""Medium Shading 1 Accent 6""/>
<w:LsdException Locked=""false"" Priority=""64"" Name=""Medium Shading 2 Accent 6""/>
<w:LsdException Locked=""false"" Priority=""65"" Name=""Medium List 1 Accent 6""/>
<w:LsdException Locked=""false"" Priority=""66"" Name=""Medium List 2 Accent 6""/>
<w:LsdException Locked=""false"" Priority=""67"" Name=""Medium Grid 1 Accent 6""/>
<w:LsdException Locked=""false"" Priority=""68"" Name=""Medium Grid 2 Accent 6""/>
<w:LsdException Locked=""false"" Priority=""69"" Name=""Medium Grid 3 Accent 6""/>
<w:LsdException Locked=""false"" Priority=""70"" Name=""Dark List Accent 6""/>
<w:LsdException Locked=""false"" Priority=""71"" Name=""Colorful Shading Accent 6""/>
<w:LsdException Locked=""false"" Priority=""72"" Name=""Colorful List Accent 6""/>
<w:LsdException Locked=""false"" Priority=""73"" Name=""Colorful Grid Accent 6""/>
<w:LsdException Locked=""false"" Priority=""19"" QFormat=""true""
Name=""Subtle Emphasis""/>
<w:LsdException Locked=""false"" Priority=""21"" QFormat=""true""
Name=""Intense Emphasis""/>
<w:LsdException Locked=""false"" Priority=""31"" QFormat=""true""
Name=""Subtle Reference""/>
<w:LsdException Locked=""false"" Priority=""32"" QFormat=""true""
Name=""Intense Reference""/>
<w:LsdException Locked=""false"" Priority=""33"" QFormat=""true"" Name=""Book Title""/>
<w:LsdException Locked=""false"" Priority=""37"" SemiHidden=""true""
UnhideWhenUsed=""true"" Name=""Bibliography""/>
<w:LsdException Locked=""false"" Priority=""39"" SemiHidden=""true""
UnhideWhenUsed=""true"" QFormat=""true"" Name=""TOC Heading""/>
<w:LsdException Locked=""false"" Priority=""41"" Name=""Plain Table 1""/>
<w:LsdException Locked=""false"" Priority=""42"" Name=""Plain Table 2""/>
<w:LsdException Locked=""false"" Priority=""43"" Name=""Plain Table 3""/>
<w:LsdException Locked=""false"" Priority=""44"" Name=""Plain Table 4""/>
<w:LsdException Locked=""false"" Priority=""45"" Name=""Plain Table 5""/>
<w:LsdException Locked=""false"" Priority=""40"" Name=""Grid Table Light""/>
<w:LsdException Locked=""false"" Priority=""46"" Name=""Grid Table 1 Light""/>
<w:LsdException Locked=""false"" Priority=""47"" Name=""Grid Table 2""/>
<w:LsdException Locked=""false"" Priority=""48"" Name=""Grid Table 3""/>
<w:LsdException Locked=""false"" Priority=""49"" Name=""Grid Table 4""/>
<w:LsdException Locked=""false"" Priority=""50"" Name=""Grid Table 5 Dark""/>
<w:LsdException Locked=""false"" Priority=""51"" Name=""Grid Table 6 Colorful""/>
<w:LsdException Locked=""false"" Priority=""52"" Name=""Grid Table 7 Colorful""/>
<w:LsdException Locked=""false"" Priority=""46""
Name=""Grid Table 1 Light Accent 1""/>
<w:LsdException Locked=""false"" Priority=""47"" Name=""Grid Table 2 Accent 1""/>
<w:LsdException Locked=""false"" Priority=""48"" Name=""Grid Table 3 Accent 1""/>
<w:LsdException Locked=""false"" Priority=""49"" Name=""Grid Table 4 Accent 1""/>
<w:LsdException Locked=""false"" Priority=""50"" Name=""Grid Table 5 Dark Accent 1""/>
<w:LsdException Locked=""false"" Priority=""51""
Name=""Grid Table 6 Colorful Accent 1""/>
<w:LsdException Locked=""false"" Priority=""52""
Name=""Grid Table 7 Colorful Accent 1""/>
<w:LsdException Locked=""false"" Priority=""46""
Name=""Grid Table 1 Light Accent 2""/>
<w:LsdException Locked=""false"" Priority=""47"" Name=""Grid Table 2 Accent 2""/>
<w:LsdException Locked=""false"" Priority=""48"" Name=""Grid Table 3 Accent 2""/>
<w:LsdException Locked=""false"" Priority=""49"" Name=""Grid Table 4 Accent 2""/>
<w:LsdException Locked=""false"" Priority=""50"" Name=""Grid Table 5 Dark Accent 2""/>
<w:LsdException Locked=""false"" Priority=""51""
Name=""Grid Table 6 Colorful Accent 2""/>
<w:LsdException Locked=""false"" Priority=""52""
Name=""Grid Table 7 Colorful Accent 2""/>
<w:LsdException Locked=""false"" Priority=""46""
Name=""Grid Table 1 Light Accent 3""/>
<w:LsdException Locked=""false"" Priority=""47"" Name=""Grid Table 2 Accent 3""/>
<w:LsdException Locked=""false"" Priority=""48"" Name=""Grid Table 3 Accent 3""/>
<w:LsdException Locked=""false"" Priority=""49"" Name=""Grid Table 4 Accent 3""/>
<w:LsdException Locked=""false"" Priority=""50"" Name=""Grid Table 5 Dark Accent 3""/>
<w:LsdException Locked=""false"" Priority=""51""
Name=""Grid Table 6 Colorful Accent 3""/>
<w:LsdException Locked=""false"" Priority=""52""
Name=""Grid Table 7 Colorful Accent 3""/>
<w:LsdException Locked=""false"" Priority=""46""
Name=""Grid Table 1 Light Accent 4""/>
<w:LsdException Locked=""false"" Priority=""47"" Name=""Grid Table 2 Accent 4""/>
<w:LsdException Locked=""false"" Priority=""48"" Name=""Grid Table 3 Accent 4""/>
<w:LsdException Locked=""false"" Priority=""49"" Name=""Grid Table 4 Accent 4""/>
<w:LsdException Locked=""false"" Priority=""50"" Name=""Grid Table 5 Dark Accent 4""/>
<w:LsdException Locked=""false"" Priority=""51""
Name=""Grid Table 6 Colorful Accent 4""/>
<w:LsdException Locked=""false"" Priority=""52""
Name=""Grid Table 7 Colorful Accent 4""/>
<w:LsdException Locked=""false"" Priority=""46""
Name=""Grid Table 1 Light Accent 5""/>
<w:LsdException Locked=""false"" Priority=""47"" Name=""Grid Table 2 Accent 5""/>
<w:LsdException Locked=""false"" Priority=""48"" Name=""Grid Table 3 Accent 5""/>
<w:LsdException Locked=""false"" Priority=""49"" Name=""Grid Table 4 Accent 5""/>
<w:LsdException Locked=""false"" Priority=""50"" Name=""Grid Table 5 Dark Accent 5""/>
<w:LsdException Locked=""false"" Priority=""51""
Name=""Grid Table 6 Colorful Accent 5""/>
<w:LsdException Locked=""false"" Priority=""52""
Name=""Grid Table 7 Colorful Accent 5""/>
<w:LsdException Locked=""false"" Priority=""46""
Name=""Grid Table 1 Light Accent 6""/>
<w:LsdException Locked=""false"" Priority=""47"" Name=""Grid Table 2 Accent 6""/>
<w:LsdException Locked=""false"" Priority=""48"" Name=""Grid Table 3 Accent 6""/>
<w:LsdException Locked=""false"" Priority=""49"" Name=""Grid Table 4 Accent 6""/>
<w:LsdException Locked=""false"" Priority=""50"" Name=""Grid Table 5 Dark Accent 6""/>
<w:LsdException Locked=""false"" Priority=""51""
Name=""Grid Table 6 Colorful Accent 6""/>
<w:LsdException Locked=""false"" Priority=""52""
Name=""Grid Table 7 Colorful Accent 6""/>
<w:LsdException Locked=""false"" Priority=""46"" Name=""List Table 1 Light""/>
<w:LsdException Locked=""false"" Priority=""47"" Name=""List Table 2""/>
<w:LsdException Locked=""false"" Priority=""48"" Name=""List Table 3""/>
<w:LsdException Locked=""false"" Priority=""49"" Name=""List Table 4""/>
<w:LsdException Locked=""false"" Priority=""50"" Name=""List Table 5 Dark""/>
<w:LsdException Locked=""false"" Priority=""51"" Name=""List Table 6 Colorful""/>
<w:LsdException Locked=""false"" Priority=""52"" Name=""List Table 7 Colorful""/>
<w:LsdException Locked=""false"" Priority=""46""
Name=""List Table 1 Light Accent 1""/>
<w:LsdException Locked=""false"" Priority=""47"" Name=""List Table 2 Accent 1""/>
<w:LsdException Locked=""false"" Priority=""48"" Name=""List Table 3 Accent 1""/>
<w:LsdException Locked=""false"" Priority=""49"" Name=""List Table 4 Accent 1""/>
<w:LsdException Locked=""false"" Priority=""50"" Name=""List Table 5 Dark Accent 1""/>
<w:LsdException Locked=""false"" Priority=""51""
Name=""List Table 6 Colorful Accent 1""/>
<w:LsdException Locked=""false"" Priority=""52""
Name=""List Table 7 Colorful Accent 1""/>
<w:LsdException Locked=""false"" Priority"
41883605555
"Possible biomarkers to predict the risk of healing delays are of huge clinical interest since 10% of fracture patients progress to delayed or non-union of fractures [1]. During endochondral ossification, which takes place in mechanically unstable regions with higher risk for delayed fracture healing, the bone regenerates through chondrogenic differentiation leading to a cartilage tissue before being remodeled into bone.
MicroRNAs (miRNA) are small, non-coding RNAs known to be involved in cell regulation [2] and play potential role in differentiation of human bone marrow derived mesenchymal stromal cells (hBM-MSCs). This work aims to identify miRNA differentially expressed during early differentiation of hBM-MSCs to be used as predictive biomarkers for non-union fracture healing.
hBM-MSCs were seeded and cultured for 28 days either in chondropermissive medium (CP) as negative control (DMEM 4.5 g/l glucose, 1% Pen/Strep, 1% ITS-x, 1% non-essential amino acids, 50 µg/ml ascorbic acid, 100 nM dexamethasone) or in chondrogenic medium (CH) as positive control (CP + 10 ng/ml TGF-β1) (N=4). Medium samples and pellets were collected on day 3, 7, 14, 21 and 28. Next, a multiaxial loading bioreactor is used for mechanically induced chondrogenesis of hBM-MSCs seeded in fibrin-polyurethane scaffolds, and three groups are compared (N=4): 1) scaffolds cultured in CP-medium (negative control), 2) scaffolds cultured in CH-medium (positive control), 3) scaffolds cultured in CP-medium + mechanical loading. To validate the differentiation of MSCs under chondrogenic conditions, histology with Safranin O/Fast Green staining was performed, as well as TGF-β1 ELISA and GAG analysis on medium and scaffold samples.
RNA was isolated from the pellets and scaffolds, followed by RT-qPCR to quantify miRNA and gene expression of chondrogenic marker genes, such as ACAN, SOX9, and COL2A1 during differentiation. As investigated in previous studies, miR-193a-5p is regulated chondrogenic differentiation of hBM-MSCs [3] and miR-193a-5p can be involved in regulation of TGF-β pathway [4]. We hypothesized that this miRNA has a regulatory role in chondrogenic differentiation, driven by TGF-β1 or during mechanically induced chondrogenesis.
Results miR-193a-5p are significantly downregulated in the scaffolds and appears downregulated in pellets cultured under chondrogenic conditions, confirming previous results [3]. Levels of miR-193a-5p during mechanically-induced chondrogenesis more closely resemble those in CP conditions. Since it has been reported that miR-193a-5p can be involved in bone metabolism by inhibiting the TGF-β pathway [4], we are investigating the correlation between levels of active TGF-β1 produced by mechanical stimulation with the miRNA expression at early days of differentiation processes. If there is a direct correlation, this miRNA may function as predictive markers for MSC differentiation and can be further validated for its role during endochondral ossification.
[1] Wildemann et al. 2021. doi:10.1038/s41572-021-00289-8.
[2] Lai 2002. doi:10.1038/ng865.
[3] Della Bella et al. 2020. doi:10.3390/cells9020398.
[4] Pu et al. 2016. doi:10.1007/s10585-016-9783-0."
31412754957
The onset and progression of aging-associated pathologies is paralleled by continuous local extracellular matrix (ECM) remodelling. This process serves as a compensatory strategy for tissues to cope with the altered conditions.
The modifications in the nanostructure and mechanics of cardiac ECM are driven by the activation of cardiac fibroblasts and impair cardiac cell function to progressively lead to organ failure. In turn, cardiomyocytes respond to the ensuing biomechanical stress by re-expressing fetal contractile proteins, by transcriptional and post-transcriptional processes, such as alternative splicing.
Our group demonstrated that the aberrant activation of mechanosensitive Yes Associated Protein (YAP) alters the assembly of focal adhesions in response to mechanical stress. Additionally, we contributed knowledge on YAP regulation during the acquisition of cardiac phenotype by adult and pluripotent stem cells, and found that its hyperactivation in patient-derived cardiac fibroblasts promotes ECM pathological remodelling, thus favoring the fibrotic process and fueling heart failure.
Lately our experimental data highlighted how the pathological remodeling of ECM in the failing heart directly affects the expression and function of RNA binding proteins in cardiomyocytes. This discovery demonstrated that mechanical stress can effectively rewire the alternative splicing of numerous genes involved in cardiomyocyte contractility, calcium handling and mechanosensing.
These studies allowed us to describe different layers of intracellular mechanosensing responsible for finely tuning the expression of splicing variants of important cardiac genes in response to pathological mechanical turmoil.
"Following ECM pathological remodelling, the aberrant activation of the mechanosensing apparatus contributes to the establishment and progression of age-related pathologies, like those affecting the cardiovascular system, and cancer. The mechanical turmoil associated with ECM remodeling is known to determine opposite consequences in rather different cell types, like cardiac and tumor cells: while cardiomyocytes in the heart are induced to hypertrophy by mechanical stress, cancer cells tend to proliferate and disseminate following similar stimuli.
Our group recently demonstrated how ECM pathological remodeling determines changes in the assembly of focal adhesions through the mechanical control of Hippo pathway downstream effector Yes Associated Protein (YAP) in breast cancer cells. The stained activation of this co-transcriptional activator has been independently associated to the growth and dissemination of many different tumor types.
Our group also described how YAP aberrant activation following pathological cardiac ECM remodelling plays a pivotal role in heart failure, by contributing to cardiac fibroblast activation and contractility.
In the context of the failing heart, we identified RNA binding proteins which are mechanosensitive and confer mRNA metabolism sensitivity to cardiac ECM mechanical turmoil and impinging on YAP alternative splicing. Finally, by employing bioengineered tools allowing the tight control of cell-matrix interaction and pluripotent stem cells, we also highlighted that mechanosensing is controlled in a stage-specific fashion and contributes critically to phenotype specification.
In conclusion, through a vast array of cellular models and bioengineered tools, we highlighted the pivotal role of mechanical cues in cell function and disease. "
41883636846
"Objectives
The damaged nervous system leads to the impairment of the motor, sensory and autonomic functions, since the central nervous system (CNS) regeneration is very limited. Failure of axon regeneration in the CNS is partly due to the inhibitory environment, and partly due to the intrinsic loss of regenerative ability with neuronal maturation. A key molecule for promoting migration and growth is membrane-associated PIP3, which is produced by PI3Kδ. In mature neurons many axon growth molecules are excluded from axons, and PI3Kδ expression enables anterograde transport of developmentally restricted molecules, such as the integrins, which has been shown to promote growth. The regenerating axons must overcome nonpermissive extracellular matrix of the glial scar, in which, among others, tenascin-C is upregulated after spinal cord injury and contributes to the inhibitory environment around the lesion site. The migration-inducing tenascin-binding integrin is alpha9beta1, which is expressed in the embryonic nervous system but downregulated in adults and not upregulated after injury.
Methods
In our studies we focus on regeneration of sensory axons and corticospinal tract axons (CST) using two different approaches based on gene therapy. Sensory regeneration was achieved in animals with dorsal column crush lesion using AAV based viral vector delivery of the integrin α9 and kindlin 1 genes to the dorsal root ganglia (DRG). We addressed two different levels of SCI, C4 lesion with DRG C6 and C7 injections for forelimb sensory restoration and T10 lesion with DRG L4 and L5 injections for hindlimb sensory restoration. To regenerate CST, we performed C4 dorsal lesion and injected the right motor cortex at 4 sites concurrently with viral vector mixture of PI3KCD and GFP. Functional outcome was assessed with battery of behavioral tests (tape removal, von Frey, Plantar test, grip strength test, ladder walking and staircase). Regenerating axons were identified by immunostaining.
Results
Significant improvement was observed in Von Frey test for mechanical perception and Hargreaves test for thermal sensation in α9 kindlin 1 treated animals with both, cervical and thoracic lesions when compared to controls. Tape removal test was improved only in treated animals with T10 lesion. Positive behavioural outcome was confirmed by counting axons from α9 and kindlin 1 group above the lesion and c fos staining showing the connectivity of newly grown axons in the treated groups. Significant improvement was detected in paw reaching test and grip strength test in PI3KCD treated animals compared to controls. Expression of PI3Kδ by cortical neurons elicited growth of CST axons at least 1.3 cm below lesion 12 weeks after SCI.
Conclusion
The AAV-mediated gene therapy leads to robust sensory and CST axon regeneration after SCI proved by behavioural tests and immunohistochemical staining.
Supported by: NEURORECON CZ.02.1.01/0.0/0.0/15_003/0000419"
20941864267
"Cardiovascular diseases (CVD) remain as the leading cause of death worldwide, and there is an increasing focus on developing physiologically relevant in vitro cardiovascular tissue models suitable for studying personalized medicine and pre-clinical tests. While recent technologies provide some insight into how human CVDs can be modelled in vitro, they may not always give a comprehensive overview of the complexity of the human heart due to their limits in cellular heterogeneity and physiological complexity[1][2][3]. Furthermore, animal models may not always faithfully reflect the features that are unique to human biology and disease [4].
We have optimized a scaffold-free protocol to generate multicellular, beating, self-organized and functional human cardiac organoids (hCOs) derived in vitro from induced pluripotent stem cells (iPSCs). The hCOs contain multiple cell types of the heart, are viable for more than 50 days, and show functional beating response without external stimuli. We have shown that hCOs have improved cardiac specification, survival and maturation compared to standard 2D monolayer cardiac differentiation. Furthermore, the hCOs has shown functional beating response to cardioactive drugs, and could be used to model chemotherapy-induced cardiotoxicity. Our study demonstrates that culture dimensionality and time are important for survival, differentiation, and maturation of in vitro cardiac tissue models in long term, and could enable further possibilities in translational research in cardiovascular biology.
References:
[1] S. Cho, C. Lee, M. A. Skylar-Scott, S. C. Heilshorn, and J. C. Wu, “Reconstructing the heart using iPSCs: Engineering strategies and applications,” J. Mol. Cell. Cardiol., vol. 157, pp. 56–65, Aug. 2021.
[2] J. Kim, B. K. Koo, and J. A. Knoblich, “Human organoids: model systems for human biology and medicine,” Nat. Rev. Mol. Cell Biol., vol. 21, no. 10, pp. 571–584, Oct. 2020.
[3] M. A. Lancaster and J. A. Knoblich, “Organogenesis in a dish: Modeling development and disease using organoid technologies,” Science (80-. )., vol. 345, no. 6194, pp. 1247125–1247125, Jul. 2014.
[4] S. B. Gorzalczany and A. G. Rodriguez Basso, “Strategies to apply 3Rs in preclinical testing,” Pharmacol. Res. Perspect., vol. 9, no. 5, Oct. 2021."
83767252566
"Title: Electrospun silica nanofibres as multifunctional substrate for drug delivery and tissue regeneration
Introduction: Over the last two decades, electrospun nanofibres were demonstrated to be interesting material applicable in regenerative medicine and drug delivery, possessing number of unique properties including high specific surface area, high porosity and small pore size. Properties such as chemical and mechanical stability, biocompatibility and degradation kinetics then depend on chemical composition, crosslinking or possibly functionalization. While polymer nanofibres may exhibit serious disadvantages including swelling in moist environment and during degradation or low surface functionality or limited bioactivity, inorganic nanofibres represent a family of nanofibres unlimited by these factors and potent for medical applications. Silica nanofibres, being member of this family, combine traditional properties of nanofibres based on their structure and advantages of inorganic bioactive material. The aim of this paper is to outline properties and performance of silica nanofibres as biocompatible, biodegradable, and easy to modify high performance material for regenerative medicine and drug delivery.
Methodology: Silica nanofibres were prepared by sol-gel method and needle-less electrospinning, which led to formation of nanofibrous matrix of 5 – 30 g/m2 and mean fibre diameter 180 – 850 nm depending on the spinning conditions. Biocompatibility was tested in vitro on several model cell lines including 3T3-A31 fibroblasts, Hacat keratinocytes, Vero cells and HepG2 hepatocyte-like cells in compliance to ISO 10993-5. Biodegradation of silica nanofibres was evaluated in vitro under simulated conditions (37 °C, SBF). Silica release into the SBF was measured using ICP-MS. Impact of degradation on the surface morphology was evaluated by electron microscopy (SEM). Surface availability for functionalization and its impact on relevant properties was tested by APTES aminosilane silanization.
Results: Silica nanofibres, obtained by electrospinning, were confirmed to promote multitissue biocompatibility in vitro. Fast degradation under simulated conditions in vitro was observed with surface erosion appearing after 24 hours in simulated body fluid (SBF) with limited swelling and sustained integrity of the nanofibrous matrix. Silica released upon degradation in form of orthosilicic acid, was confirmed to have a beneficial impact on cellular proliferation in vitro, which is known effect provided silica nanomaterials in general. Successful grafting of –NH2 amino group by silanization of surface silanol group was confirmed without relevant impact on the fibre morphology or integrity. Positive impact of the silanization process on biocompatibility was verified.
Conclusion: The electrospun silica nanofibres were confirmed as biocompatible and bioactive nanomaterial with capacity to promote tissues regeneration through its degradation products. Moreover, the unique degradation mechanism reminiscent of Stöber silica degradation mechanism was revealed. The high specific surface available for surface functionalization, can be applied in drug and bioactive molecules delivery in wound healing and tissue regeneration."
52354553526
Organoids, 3D multicellular aggregates, provides excellent possibilities to recapitulate pato/physiological properties of human tissues or organs. Methodological obstacles lie in uniform formation, differentiation and long-term cultivation. Preparation and maintenance of organoids can be labor-intensive, medium exchange is usually discontinuous, and individual organoids are highly heterogenous in size, morphology, and cellular composition. Microfluidics help to overcome some of these disadvantages. Due to automation and proper design of chips, microfluidic systems are capable of continual medium flow in space and time, which allows to create better controlled microenvironments for cells.
In this work, we present designs of microfluidic system for uniform formation of organoids with low divergence of size and shape. System enables a parallel perfusion culture of large amount of cell spheroids as well as long term cultivation and cell differentiation. Upon in silico simulations, we optimized conditions for long-term cultures in chip. To improve their nutrient support, we also examined approaches for their vascularization.
This work was supported by the European Regional Development Fund - project INBIO (No. CZ.02.1.01/0.0/0.0/16_026/0008451), project from Masaryk University (MUNI/IGA/1297/2021), Czech Science Foundation GA21-06524S and Ministry of Health of the Czech Republic (NU21-08-00561).
Intravital microscopy (IVM) has revolutionized our understanding of single-cell behavior in complex tissues by enabling real-time observation of molecular and cellular processes in their natural environment. In preclinical research, IVM has emerged as a standard tool for mechanistic studies of therapy response and the rational design of new treatment strategies. For understanding immune function in the tumor microenvironment, higher harmonic generation, a label-free multiphoton imaging technique, provides tissue context and reveals guiding structures which steer immune cell migration and modulate T cell efficacy. The talk will highlight the role of IVM for understanding cellular interactions with implanted materials and immune cell behavior in tumors within tissue-engineered bone.
In this study, fluorescence lifetime imaging of NAD(P)H-based cellular autofluorescence is applied as a non-invasive modality to classify two contrasting states of human macrophages by proxy of their governing metabolic state. Macrophages were obtained from human blood-circulating monocytes, polarised using established treatments, and metabolically challenged using small molecules to validate their responding metabolic actions in extracellular acidification and oxygen consumption. Fluorescence lifetime imaging microscopy (FLIM) quantified variations in NAD(P)H-derived fluorescent lifetimes in large field-of-view images of individual polarised macrophages also challenged, in real-time with small molecule perturbations of metabolism during imaging. We uncover FLIM parameters that are pronounced under the action of carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) which strongly stratifies the phenotype of polarised human macrophages. This stratification and parameters emanating from a FLIM approach, served as the basis for machine learning models. Applying a random forest model, identified three strongly governing FLIM parameters, achieving a ROC AUC value of 0.944 when classifying human macrophages. Visualisation of the data show a clear classification of IFNγ-M1 and IL-4-M2 macrophages in response to real-time imaging when treated with FCCP. The excellent performance of machine learning models, applied on the data extracted from the non-invasive technique, underlines further the efficiency of this workflow. This workflow can be easily adopted to non-invasively characterise macrophage polarisation in in vivo models and in vitro multicellular organoid models to study foreign body interactions, biomaterial assessment, pharmaceutical research and screening and clinical applications such as disease diagnosis. Precise regulation of macrophage activation state is key to understanding disease control, tissue homeostasis and implant response, with this regulation shown to be directly related with macrophage intracellular metabolism. Therefore, impaired macrophage metabolism results in impaired function such as the case of diabetes, the foreign body response to biomaterials, obesity or cancer.
73296360669
"Introduction
Multicellular spheroids can be a powerful model mimicking the physiological environment of the tissue in a microscale format. They are often seen as ‘microscale-bioreactors’ providing an appropriate environment for cell differentiation and stem and cancer cell niche, which makes them important for physiological studies and biofabrication. However, this advantage becomes a problem when it comes to the question of characterization / standardization of spheroids produced in different labs, by different methods or even cell passage number1. The gradients of cell proliferation, oxygenation, cell death and other biomarkers are thus largely unexplored and ignored.
Methodology
Here we describe a nanosensor-based analysis of live spheroid oxygenation helping to routinely estimate O2 gradients on a conventional fluorescence microscope. Described method helps spheroid phenotype characterization (size, relative hypoxia), informing on their metabolism and viability. We optimized generation of spheroids loaded with ratiometric red (650 nm reference) / near infrared- (760 nm O2-sensing channel) emitting O2-sensing biocompatible nanoparticles. Presented visualization of oxygenation can be combined with multiparametric analysis in available ‘blue’ and ‘green’ channels such as cell death staining or advanced imaging modalities such as FLIM and PLIM2.
Results
Presented approach was tested in different experiments with homo- and heterocellular (human dental pulp stem hDPSC with endothelial HUVEC cells) spheroids produced from stem and cancer cells: (1) we performed long-term monitoring of individual spheroids oxygenation for more than 14 days; (2) we detected changes in oxygenation upon adding mitochondrial uncouplers / inhibitors; (3) we performed ‘endpoint’ multiparameter analysis of oxygenation coupled with labeling cell death by SYTOX Green. To standardize the analysis of oxygenation in spheroids we looked at oxygenation at spheroid core and periphery, value of O2 gradient and their ‘steepness’.
We found that in contrast to hDPSC spheroids, ‘addition’ of HUVEC cells to spheroids provided higher oxygenation and significantly steeper gradient. Heterocellular spheroids were also statistically larger, suggesting that their oxygenation was caused by cell composition-related differences in bioenergetics agreeing with the known data on HUVEC and hDPSC metabolism.
To illustrate the applicability of the approach for biofabrication we compared O2 gradients in hDPSC spheroids before and on a day 1 after bioprinting in GelMA. Bioprinted hDPSC spheroids had significant changes in periphery which affected the range and steepness of their periphery-to-core O2 gradients. The dead cell staining was more profound in bioprints.
Conclusions
We demonstrated that spheroid oxygenation reflects the bioenergetic state and viability of cells in 3D, allowing application of ratiometric oxygenation analysis for standardization of spheroid phenotype. Usage of ratiometic analysis versus phosphorescence lifetime calculation enables for more ‘cost-efficient’ O2 gradients studies with almost all types of conventional fluorescence microscopes. The method is compatible with multi-parameter physiological measurements (e.g., cell death, proliferation, and cell composition) and downstream assays (immunofluorescence, FACS etc.), and long-term monitoring, essential for bioprinted constructs containing spheroids.
References
1 Peirsman, A. et al., Nature methods. 18, 1294-1303 (2021).
2 Dmitriev, R. I. et al., Biomaterials. 34, 9307-9317 (2013)."
41883616084
Introduction
Advanced in vitro models (e.g., organoids) are three-dimensional (3D) constructs usually generated from cells with a degree of stemness. This, accompanied by the multiplicity of parameters which condition organoid growth and morphology, results in constructs of different shapes and size and thereby functional properties. To date, a quantitative framework for robust measurement and modelling of organoid growth processes in relation to morphological features is still lacking and urgently required. In silico methods integrate physical data and biochemical models using computational tools, and are a powerful support for tissue engineering. They are particularly relevant for studying organoids: the multiplicity of parameters which condition their growth and morphology can be explored in virtual models, facilitating experimental design, and enabling prediction and extrapolation of behaviour and function. Here we describe a framework for quantifying organoid morphometry with imaging tools and mapping shape and size to virtual organoids generated through evolutionary algorithms.
Methodology
Multi tissue hepatic organoids and spheroids are generated using standard protocols. They are imaged at multi-scale by means of an integrated approach involving light-sheet, confocal and super-resolution microscopy. Thus, we can resolve objects spanning from few millimetres (e.g., construct shape) down to microns (e.g., cells) to tens of nanometers (e.g., mitochondria, tight junctions). Ad hoc image processing algorithms and routines are developed for dealing with the acquired datasets. In particular, algorithms based on the intensity distributions of background and foreground, described locally within the dataseta, are exploited for identifying and isolate single cells and/or subcellular constructs.
Evolutionary algorithms based on the optimization of a cost function which incorporates resource uptake, surface energy and cooperative metabolic effort are used to generate virtual organoids within a range of masses.
Results
Quantitative descriptors to characterize construct and cell shape, cell arrangement in the 3D space as well as cell-cell and cell-substrate interactions are identified. Evolutionary algorithms are honed by matching imaging data with the virtual organoids. We are thus able to identify how resource assimilation and physical phenomena affect organoid formation and growth.
Conclusions
Our framework enables the characterisation of structural features of 3D constructs, which in turn may give insights on their functionality. In addition, the outputs are used for implementing more accurate computational models that take into account the real shape of the constructs and the real arrangement of the cells. This will serve to quantitatively assess to what extent organoids are similar to their in vivo counterparts, and thus define strategies for improving reproducibility and viability. Integrating experimental and modelling approaches is key for designing constructs with translational value and hence useful for robust in vitro to in vivo extrapolation, paving the way towards predictive and precision medicine and reducing animal tests. Currently the models are deterministic, future efforts will be dedicated to incorporating fluctuations as an inevitable and ubiquitous feature of any functional biological system.
62825437764
Introduction: Lasers have been used for years in the field of cancer therapy. Nanosecond pulsed lasers, in particular, have been used in the generation of reactive oxygen species via plasma for the triggering of immunogenic cell death[1][2], for the delivery of biomolecules intracellularly via optoporation[3] and for tumor resection[4]. Notwithstanding this large range of uses, the full impact of nanosecond pulsed lasers on cellular mechanisms is not fully understood. Their effects on epithelial to mesenchymal transition (EMT), for instance, are unknown. While being a key mechanism in embryogenesis and wound healing, EMT has also been associated to tumor progression, invasion and resistance through the action of players like the transforming growth factor beta (TGF-β) family.
Here, the putative effects of nanosecond pulsed laser ablation on the expression of known EMT players in melanoma spheroids models are studied.
Methodology: Multicellular spheroids comprising the human melanoma cell line VMM-15 and human dermal fibroblasts (hDFbs) were produced while using monoculture spheroids of each cellular type as controls. Cellular aggregation was allowed to occur over 7 days, at which time partial ablation of the spheroids using a nanosecond 355nm laser was performed. Ablated spheroids were collected after allowed to recover for 3 hours or 3 days. Several key properties of the ablated spheroids where subject to evaluation. Morphology recovery was assessed using time-lapse microscopy. Gene and protein expression were analyzed by Real-Time PCR, Western Blot and immunohistochemistry.
Results: Ablated spheroids displayed variable angular openings of the wound surface, with greater values being verified for the multicellular spheroids. Laser ablation triggered an up-regulation of the gene expression of EMT mediator TGFβ1 across all conditions, which was also verified for its receptor TBFβ-R1. Canonical pathway transducer pSMAD2/3 presented a higher protein expression post-ablation. Additionally, increased gene expression was verified for PLOD2 which correlated with the increase of Col1 protein confirmed through western-blot. Further analysis of EMT players showed a decrease in the expression of epithelial marker E-Cadherin after ablation recovery, while an increase in the expression of mesenchymal marker N-Cadherin and ZEB1 was observed when compared to control. Gene expression of stem cell master regulators SOX2, OCT4 and NANOG was shown to be overexpressed in melanoma spheroids after laser ablation.
Conclusions: It was demonstrated that laser ablation triggers a phenotypical shift from an epithelial state into a mesenchymal state in melanoma cell spheroids mediated by the expression of TGF-β1. This was further reflected in the increased expression of key stem cell regulators linked to stemness. These results strongly suggest that nanosecond pulsed laser ablation is capable of promoting EMT in determined conditions.
[1] A. Lin, B. et al., Int. J. Mol. Sci. 2017, 18, 966.
[2] H. T. M. Nguyen et al., Nanoscale 2021, 13, 3644.
[3] P. Gupta et al., Analyst 2021, 146, 4756.
[4] J. Hornef et al., Sci. Rep. 2020, 10, 5122.
Acknowledgments: FCT/MCTES through the grants SFRH/BD/119756/2016 and IF/00347/2015 and EU Horizon 2020 research and innovation programme under the ERC grant CapBed (805411).
41883615759
In this talk, author's decades-long research on inducing bone from adipose stem cell (ASCs) will be introduced, including 1) the exploration of osteogenic potentioal of ASCs versus bone marrow stem cell (BMSCs), 2) investigation of small molecules, peptides, gene transfer to enhance osteogesis, 3) synergistic effect of coculture of ASCs and BMSvs in osteogenesis and angiogenesis, 4) mining of de novo factors that promote the survival and bone induction from poorly osteogegenic ASC clones.
62903403666
This lecture will show how biomaterials and components of the extracellular matrix, i.e. structural proteins and growth factors, affect the osteogenic potential of human adipose-derived mesenchymal stromal cells (ASC). Examples of bone formation by various human adipose derived cells-based engineered matrix/tissue, via either intramembranous or endochondral ossification will be presented. The lecture will also present the development of an advanced therapy medicinal product (ATMP) based on an intraoperative use of the stromal vascular fraction (SVF) of human adipose, containing mesenchymal and endothelial cells, to support bone repair with tissue harvest, cell isolation, seeding onto scaffolding material and implantation within 3-4 hours. A translation of this concept into a first-in-man clinical trial, demonstrating safety, feasibility and providing proof-of-principle of the biological functionality (i.e., bone formation) of the implanted graft will be presented. Another clinical case based on the use of such ATMP for mandibular bone regeneration will be shown. More recent and future research directions will be discussed.
94355104697
Objectives
The glucocorticoid receptor (GR) is a nuclear receptor that controls critical biological processes by regulating the transcription of specific genes. GR transcriptional activity is modulated by a series of ligand and coenzymes, where a ligand can act as an agonist or antagonist. GR agonists, such as the glucocorticoids dexamethasone (DEX) and prednisolone, are widely prescribed to patients with inflammatory and autoimmune diseases. DEX is also used to induce osteogenic differentiation in vitro. Recently, we highlighted that DEX induces changes in osteogenic differentiation of human mesenchymal stromal cells by inhibiting the transcription factor SRY-box transcription factor 9 (SOX9) and upregulating the peroxisome proliferator activated receptor γ (PPARG) [1]. SOX9 is fundamental in the control of chondrogenesis, but also in osteogenesis by acting as a dominant negative of RUNX2. There are still many processes to be clarified during cell fate determination, such as the interplay between the key transcription factors. The main objective pursued by this work is to shed light on the interaction between GR and SOX9 in presence and in absence of DEX at an atomic level of resolution using molecular dynamics (MD) simulations. The outcome of this work could help the understanding of possible molecular interactions between GR and SOX9 and their role on the determination of cell fate. Moreover, the impact of DEX on the previously mentioned macro molecular interactions will be fully clarified.
Methods
Classical MD has been used to perform a systematic investigation of a series of docked pose between GR, with and without DEX, and SOX9. HDOCK web server was used to obtain the initial docked configuration, considering as docking target for SOX9 the GR’s dimerization and coenzyme moieties. All the SOX9-GR initial docked configuration were simulated in presence and in absence of DEX complexed with GR. The AMBER99SB-ILDN force field and TIP3P water molecules was used for defining system topology.
Results
The results showed that DEX has an influence on the binding behavior between SOX9 and GR. The SOX9 protein docked within the GR’s coenzyme moiety showed a different binding behavior depending on the presence or absence of DEX bound to the GR’s ligand binding domain. It is worth mentioning that when DEX is absent from the GR, SOX9 has the capability to strongly interact with the GR’s coenzyme domain. Contrariwise, when DEX is bound, SOX9 has impaired capability to interact with the GR’s coenzyme domain. Finally, no significant difference has been observed in the simulations of SOX9 docked the GR’s dimerization domain, with and without DEX complexed to GR.
Conclusions
This work sheds light on the modulator effect carried out by DEX on the interaction between GR and SOX9. The fruitful information extracted from this study may help the understanding at molecular level of the interaction between the nuclear receptor GR and SOX9 in the presence or absence of DEX, which can prompt a better understanding of the entire osteogenic differentiation pathway of human mesenchymal stromal cells.
[1] Della Bella E. et al. (2021) Int J Mol Sci 22 (9):4785.
83767229706
The use of mesenchymal stem cells (MSCs) for bone regeneration is a promising alternative to conventional bone grafts. Because local metabolic alterations seem to be critical for bone regeneration, metabolomics (through cell extracts and culture media) may unveil novel information on MSCs osteogenic differentiation,1,2 allowing their behaviour to be understood and potentially guided towards improved osteogenic lineage commitment (e.g. through specific media tailoring).3,4 However, only a few reports have monitored osteogenesis (mass spectrometry approaches predominating compared to nuclear magnetic resonance (NMR) spectroscopy), with scarce information interconecting intracellular and extracellular metabolic alterations.2,5 Here, NMR untargeted metabolomics is applied to monitor endo- and exometabolome adaptations of human adipose tissue-derived MSCs throughout 21 days of osteogenic differentiation. Endometabolome results revealed significant fluctuations in the metabolism of amino acids, energy-related compounds, lipids, nucleotides, and metabolic players in protective anti-oxidative mechanisms. Furthermore, exometabolome data highlighted alanine, glutamate, glycerol and citrate as important secretome components. Different metabolic stages are suggested, supported by putative biochemical explanations, and other important issues (such as inter-donor variability and aging effect) are discussed. Overall, this work has shown the great potential of NMR metabolomics to characterize the dynamic metabolism of MSC osteogenenic differentiation, ultimately enabling the potential discovery of universal biomarkers of osteogenic differentiation efficacy, with potential translation to in vivo clinical practice.
Acknowledgements: We acknowledge the Portuguese Foundation for Science and Technology (FCT) for co-funding the BIOIMPLANT project (PTDC/BTM-ORG/28835/2017) through the COMPETE2020 program and European Union fund FEDER (POCI-01-0145-FEDER-028835); CICECO-Aveiro Institute of Materials project (UIDB/50011/2020 & UIDP/50011/2020), financed by national funds through the FCT/MEC and when appropriate co-financed by FEDER under the PT2020. DSB acknowledges the Sociedade Portuguesa de Química and FCT for her PhD grant SFRH/BD/150655/2020. The NMR spectrometer used in this work is part of the National NMR Network, partially supported by Infrastructure Project Nº 022161 (co-financed by FEDER through COMPETE 2020, POCI and PORL and FCT through PIDDAC).
20941805655
Mesenchymal stem cells (MSCs) are in the frontline of tissue engineering and regenerative medicine because they have indefinite self-renewal potential, and also exhibit great multilineage differentiation into a variety of tissues such as such as bone, cartilage, and adipose1. In the field of bone tissue engineering, the osteogenesis of MSCs is a promising therapeutic target, and controlling their differentiation is of critical importance for improving traumatic bone healing and therapy for genetic bone diseases2-4. Although metabolomics has already contributed to some extent understanding of the osteogenic mechanism of MSCs5, studies on the role of lipids metabolism are still scarce6.
In this study, we monitored the lipid profile of human adipose-derived MSCs as a function of osteogenic differentiation times, using nuclear magnetic resonance (NMR) untargeted lipidomics strategy applied to MSCs. Samples were collected at different time points during 2D culturing in both control (undifferentiated) and in osteogenesis-inductive media. Changes in the lipid composition of human adipose-derived MSCs were interpreted in detail based on multivariate and univariate statistical approaches. Our 1H-NMR strategy detected various groups of lipids with statistically relevant changes throughout the whole 21-day period of osteogenic differentiation. These comprised phosphatidylcholine, sphingomyelin, phosphatidylethanolamine, plasmalogen, total fatty acids, polyunsaturated fatty acids and unsaturated fatty acids, as well as total, free and esterified cholesterol. These results may unveil potential osteogenesis-related lipid biomarkers, which are essential to provide a comprehensive picture of the MSCs differentiation metabolism.
Acknowledgements: We acknowledge the Portuguese Foundation for Science and Technology (FCT) for co-funding the BIOIMPLANT project (PTDC/BTM-ORG/28835/2017) through the COMPETE2020 program and European Union fund FEDER (POCI-01-0145-FEDER-028835); CICECO-Aveiro Institute of Materials project (UIDB/50011/2020 & UIDP/50011/2020), financed by national funds through the FCT/MEC and when appropriate co-financed by FEDER under the PT2020. DSB acknowledges the Sociedade Portuguesa de Química and FCT for her PhD grant SFRH/BD/150655/2020. The NMR spectrometer used in this work is part of the National NMR Network, partially supported by Infrastructure Project Nº 022161 (co-financed by FEDER through COMPETE 2020, POCI and PORL and FCT through PIDDAC).
52354509208
Introduction: Lack of sufficient vascularization to support cell viability, growth and function in scaffold guided tissue regeneration (SGTR) is a prevalent challenge facing tissue engineering today. Matching axial vascularization is a regenerative therapeutic approach which incorporates the benefits of flap-based techniques for neo-vascularization to further aid tissue regeneration. Methodology: An ovine critical-size tibial defect model (Medium defect volume=9.5 cm3) was undertaken in eight sheep to evaluate the novel tissue engineering approach involving a 3D-printed medical-grade ε-polycaprolactone b-tricalcium phosphate (mPCL-TCP) scaffold with a cortico-periosteal flap (CPF). Biomechanical, radiological, histological and immunohistochemical analysis confirmed functional bone regeneration comparable to the clinical gold standard control (autologous bone graft) and was superior to a scaffold control group (mPCL-TCP only). A pilot study was performed on two sheep where the defect volume was doubled to 19 cm3 (X-Large defect volume) to represent the most challenging clinical situation. Results: Positive results of both the M and XL defect volume study supported clinical translation. A 27-year-old adult male underwent reconstruction of a 36 cm near-total intercalary defect of the tibia secondary to osteomyelitis using this original scaffold guided bone regeneration concept. Bone regeneration was confirmed postoperatively both radiologically and histologically with complete independent weight bearing achieved within 24 months. Conclusion: This article represents the widely advocated and seldomly accomplished concept of “bench-to-bedside” research. The presented original scaffold-guided treatment concept to reconstruct major bone loss in load-bearing limbs will have significant implications for reconstructive surgery and regenerative medicine more generally.
41883658008
Impaired tendon function leads to pain and restricted movement of the joints. This impairment can be due to the degeneration of the tendon and, in the worst-case, due to rupture. Conservative and surgical treatments are available, however treatment duration can be long and the failure rate is high. In order to develop new treatment strategies, a better understanding of the processes underlying tendon alterations as well as the factors influencing them is necessary. This talk will give insights into the process of tendon degeneration and biology obtained from human tissue samples and animal studies.
73387302328
"Tendon disorders and injuries are one of the most common musculoskeletal disorders. Our knowledge of the causes and underlying mechanisms for the development of tendinopathies still remain fragmentary. Recent evidence has clearly implicated the presence of immune cells during early tendinopathy and we are beginning to better understand the origin and properties of these cells. Recently, we described tissue-resident cells fulfilling macrophage- or monocyte-related functions in healthy tendons, most likely serving as sentinels which are activated upon tendon tissue injury or pathological stress.
Various intrinsic and extrinsic risk factors have been identified and next to mechanical overuse, other known predisposing factors include rheumatoid arthritis, diabetes, obesity or smoking, all of which elicit or are accompanied by a mild systemic inflammation. We could show that not only local inflammation can affect tendon quality, but the mere presence of a low-grade, allergy-induced systemic inflammation is sufficient to induce structural alterations in tendons and impair tissue function.
In this presentation I will give an overview on the recent advancements in defining the key role of immune-mediated mechanisms in tendon disease."
94355101524
"Introduction
Tendon injuries occur commonly in human and equine athletes. Post-injury, the healing response is inadequate leading to increased deposition of scar-tissue and high re-injury rates. This has motivated the development of novel treatments which promote superior tissue regeneration. Particular interest has surrounded the use of bone-marrow mesenchymal stromal cells (BM-MSCs), with clinical investigations showing promising results1. Nevertheless, how BM-MSCs encourage tendon healing is unclear. Tendon injuries invoke an inflammatory response, and although moderate levels of inflammation are required to initiate tendon repair, evidence suggests inadequate resolution of inflammation contributes to fibrotic healing2. Previously, IL-1β was evidenced to exhibit negative effects on equine tenocytes, yet these consequences could be rescued by exogenous IL-1 receptor antagonist protein (IL1Ra)3. In contrast, embryonic stem cell (ESC) derived tenocytes appeared to be protected from the adverse effects of IL-1β, making them the ideal model to investigate tendinopathy further.
Methodology
Three biological replicates of equine adult tenocytes and ESC-tenocytes were cultured with IFN-γ (100 ng/ml), TNFα (10 ng/ml) and IL-1β (1 nM) and/or IL1Ra (100 ng/ml) prior to gene expression analysis and immunocytochemistry to determine inflammatory pathway activation. A 3-D culture model was used to determine the effects of IFN-γ, TNFα and IL-1β on collagen gel contraction by adult or ESC-tenocytes over 14 days. Adult tenocytes were also stimulated with IFN-γ, TNFα and IL-1β alone or in co-culture with BM-MSCs or BM-MSC conditioned media and the effects on signal induction, gene expression and 3-D collagen gel contraction measured.
Results
Stimulation of adult tenocytes with IFN-γ, TNFα and IL-1β resulted in significant changes in tendon-associated gene expression. Furthermore, these cytokines significantly inhibited 3-D collagen gel contraction by adult tenocytes. Immunocytochemistry demonstrated this combination of cytokines activated NF-κB, but not STAT1, JNK or P38 MAPK in adult tenocytes. These adverse effects could not be rescued by IL1Ra or factors produced by BM-MSCs. Conversely, ESC-tenocytes appear to be protected from IL-1β, TNFα and IFN-γ; generating tendon-like constructs indistinguishable from controls and demonstrating little to no changes to gene expression. Additionally, inflammatory stimulation failed to activate key inflammatory signalling pathways in ESC-tenocytes.
Conclusion
We demonstrate IL-1β, TNFα and IFN-γ work synergistically to induce greater detrimental consequences for adult tendon function than when used individually. Moreover, these adverse effects cannot be rescued by direct suppression of IL-1β. However, ESC-tenocytes appear to be protected from inflammatory stimulation, exhibiting minimal effects on gene expression and no activation of NF-κB, suggesting an association between undesired cellular activities in tenocytes and NF-κB signalling. Our results suggest BM-MSCs are unable to protect adult tenocytes from the adverse effects of inflammation. Understanding the mechanisms by which NF-κB signalling is blocked in ESC-tenocytes, and how BM-MSCs facilitate healing, may enable us to identify novel interventions for tendon injuries which minimise scar-tissue formation, resulting in diminished re-injury rates and improved quality of life.
References
"
83767205346
"Despite the high incidence of tendon injuries globally, an optimal treatment strategy has yet to be defined1. A key challenge for tendon repair is the alignment of the repaired matrix into orientations which provide maximal mechanical strength2, 3. Using oriented implants for tissue growth combined with either exogenous or endogenous stem cells may provide a solution. Previous research has shown how oriented fiber-like structures within 3D scaffolds can provide a framework for organized extracellular matrix deposition4. In this paper, we present our data on the remote magnetic alignment of collagen hydrogels which facilitates long-term collagen orientation. Magnetic nanoparticles (MNPs) at varying concentrations and size can be contained within collagen hydrogels. In the presence of an external magnetic field, gelation is initiated by incubation at 37oC for 30-minutes. Our data shows how, in response to the magnetic field lines, MNPs align and form string-like structures orientating 90o from the applied magnetic field from our device. This can be visualized through light and fluorescence microscopy and persists 21-days post application of magnetic field. Confocal microscopy demonstrates anisotropic macroscale structure of MNP-laden collagen gels subjected to a magnetic field, compared to gels without MNP dosing. Matrix fibrillation was compared between non- and biofunctionalized MNP hydrogels, and different gels dosed with varying MNP concentrations. Human adipose stem cells (hASCs) seeded within the magnetically-aligned gels were seen to align in parallel to MNP and collagen orientation 7-days post application of magnetic field. hASCs seeded in isotropic gels were randomly organized. To summarize, we have developed a convenient, non-invasive protocol to control collagen I hydrogel architecture. Through the presence or absence of MNP dosing and a magnetic field, collagen can be remotely aligned or randomly organized, respectively, in situ. This can be considered as an innovative approach particularly useful in tissue engineering or organ-on-a-chip applications for remotely controlling collagen matrix organization. In this way cellular constructs analogous to healthy and diseased tendon can be engineered ex vivo for regenerative therapies.
"
41883627366
"A well-balanced shift from pro- to anti-inflammatory processes during healing appears essential for successful regeneration and may prevent from chronic diseases or tissue damage. Resolution of inflammation is an active process, which is regulated by specialized pro-resolving mediators (SPMs) such as lipoxins, annexins, and resolvins. The contribution of these mediators to the severity of rotator cuff (RC) tendinopathy has previously been shown. Due to the close localization of RC tendons to the subacromial bursa, we hypothesize that bursae may store pro-resolving mediators to regulate tendon regeneration. We aimed to detect differences at increasing stages of RC tendinopathy in order to identify mediators that may regulate its deterioration.
We investigated bursae from healthy patients (n=13-16) versus patients with partial (n=13-17) or full-thickness RC tears (n=18-32). Bursa tissues were harvested for immunofluorescence staining, gene expression analysis and release of SPMs (Annexin A1, Lipoxin A4, Resolvin D1/D2) and/or their receptors (FPR2, ChemR23, GPR18). To understand regulatory mechanisms of SPM signaling in bursae, bursa-derived cells from patients with full-thickness RC tears (n=6) were subjected to physiological (2%) or pathological (8%) straining under uniaxial cyclic loading for 4 hours/day on 3 consecutive days. After stimulation, cells were analyzed by flow cytometry for SPM receptors (FPR2, ChemR23), fibroblast markers (CD90, CD105), adhesion molecules (CD54, CD106), human leucocyte antigens (HLA-DR, HLA-ABC) and proliferation marker Ki67. Furthermore, gene expression of SPM related genes (Annexin A1, FPR1, FPR2, ChemR23, GFP18), Col I, matrix degrading enzymes and inhibitors (MMP1, -2, TIMP1), and pro-inflammatory cytokines (IL6, IL1b) were analyzed.
Multiplex fluorescence staining revealed FPR2+ and ChemR23+ cells in perivascular, but also in fibrous or fatty bursa tissue. Single or double positive FPR2/ChemR23 cells were identified partially as CD45+ leukocytes but also as CD45- cells (e.g. endothelial cells, fibroblasts). Annexin A1 gene expression showed a trend towards an increase in bursae of partial RC tears compared to full-thickness RC tears (p=0.076), whereas SPM receptor gene expression was not significantly affected. In tissue culture, bursae of partial RC tears showed increased Annexin A1 and Resolvin D1 release compared to bursae of intact controls (p=0.029, p=0.007, respectively). Resolvin D1 release was also increased in bursae of full-thickness RC tears compared to intact controls (p=0.008). Pathological, but not physiological mechanical straining resulted in significantly increased MMP1 and ChemR23 gene expression compared to unstimulated controls (p=0.002, p=0.040). On surface marker level, no significant regulations were observed after mechanical stimulation of bursa-derived cells.
The study shows for the first time SPM signaling mediators in subacromial bursa tissue with differences depending on the severity of RC tears. The increase in Annexin A1 and Resolvin D1 particularly in early tendinopathy (partial RC tear) may indicate that these bursae are trying to balance the pro-inflammatory response at this stage. Pathological mechanical straining induced ECM remodeling, due to strongly increased MMP1 gene expression and might be an initiator of a pro-resolving response, as ChemR23 gene expression was up-regulated. In summary, the results provide first evidence that the subacromial bursa is involved in pro-resolving processes in RC tendinopathy."
20941868049
INTRODUCTION
Tendon tissues host different cell populations that play important roles in their physiology and pathophysiology. A hallmark of tendon injuries and diseases is the persistent inflammatory response that can self-amplify and lead to chronicity. The inflammatory phase of tendinopathy is characterized by increased vascularization and influx of immune cells (mast cells, macrophages, T cells) at the healing site. A better understanding of this complex multicellular crosstalk and environmental cues is critical for decoding the healing mechanisms of tendon injuries and to find new therapeutic options. However, the current lack of representative in vitro models of tendinopathy is a major barrier to the progress of this field. The aim of this work is to establish a multicellular organotypic 3D model recreating key signaling hallmarks of that immune response, in particular for the study of the interactions between stromal tenocytes and the circulating T cells in tendon vasculature under healthy and diseased conditions.
METHODOLOGY
To recreate the anisotropic fibrillar architecture of tendon ECM and induce cell alignment in 3D within the chip, we produced magnetically responsive microfibers (MNF@PCL). Microfibes were prepared by cryo-sectioning electrospun PCL meshes incorporated with iron oxide nanoparticle. A three channeled microfluidic chip was used as platform to build the model, where human tendon derived cells (hTDCs) were encapsulated in the central channel in either transglutaminase crosslinked Gelatin or Platelet Lysate (PL) hydrogels along with MNF@PCL. For in-situ alignment of MNF@PCL, the chip was placed under a uniform magnetic field created by two parallel magnets. Hydrogel formation allowed to fix the fiber pattern after removal of the magnetic forces. Microvascular cells were co-cultured in the side channel to recreate the open vasculature of the extrinsic tendon compartment, where T cells can be subsequently circulated for evaluating their interactions with stromal tenocytes.
RESULTS
Analysis of 3D hTDCs cytoskeleton organization within hydrogel matrices showed that the topographical cues created by the microfiber alignment strongly dictates the cell's aspect ratio and orientation. The synergy between the PL matrix bioactivity and magnetically aligned MNF@PCL revealed to be the most effective strategy for inducing cell anisotropic organization within central compartment and maintenance of a tenogenic phenotype. The microvascular cells co-cultured in the side channels organized into compartmentalized tubular monolayer with open lumen. We are currently assessing the effects stemming from the crosstalk between tendon and vascular cells on genes and proteins related with ECM, tenogenic markers and inflammatory signaling pathways. This physiomimetic system is also being explored to study the effects of hTDCs on the behavior of circulating T cells (migration and activation), as well as the impact of these crosstalk mechanisms on the stromal compartment.
CONCLUSION
In this work, we propose a compartmentalized tendon-on-chip model able to recapitulate ex-vivo some of the characteristic microstructural features of healthy and diseased (fibrotic) tendon stroma interfacing with the vasculature of the extrinsic tendon compartment which is capable of supporting circulating immune cells. This physiomimetic system is being leveraged for better understanding not only the mechanisms of tendinopathy, but also of tendon tissue regeneration and repair.
31412741288
The natural extracellular matrix (ECM) is a highly dynamic, supramolecular structure composed of various bioactive molecules held together by specific interactions. The ECM directly interacts with cells and dictates cell behavior to a large extend. Our goal is to synthetically mimic this intricate natural system using supramolecular materials based on hydrogen bonding units. The dynamics of the supramolecular system is shown to be important in the presentation of bioactive epitopes to cells. By design, highly dynamic supramolecular fibrous assemblies decorated with cell adhesive peptide motifs were made and studied in solution. It was shown that these soluble fibrous structures interact with the cell surface, and that the dynamics of bioactive presentation is dependent on the method of supramolecular incorporation. Transient networks and hydrogels composed of similar molecules were shown to have slowed down dynamics, compared to the particles in solution. These hydrogels, when formulated in the right way, were able to enhance cell viability and adhesion. When highly robust solid materials were made using the same supramolecular motif, cell adhesion and migration could be tuned. However, the ECM displays a plethora of bioactive peptide signals. Therefore, a high throughput screening approach was taken using a design of experiments set up, to investigate a synthetic library of peptides supramolecularly incorporated as additives in the base material. It was found that several sequences and/or combinations outperformed others, showing the importance of the high throughput screening approach. Our proposal is that both the dynamics and presentation of bioactive sequences determine cell behavior. In this way we aim to make steps towards the design of a synthetic ECM analogue.
41935605049
Human induced pluripotent stem cell (hiPSC)-derived kidney organoids have prospective applications ranging from basic disease modelling to personalised medicine, however, there remains a requirement to refine the biophysical and biochemical parameters that govern kidney organoid formation. Here we describe the differention and maturation of hiPSC-derived kidney organoids within fully synthetic self-assembling peptide hydrogels (SAPHs) of variable stiffness (storage modulus, G′). The resulting organoids contained complex structures comparable to those differentiated within the animal-derived matrix, Matrigel. Single-cell RNA sequencing (scRNA-seq) was then used to compare organoids matured within SAPHs to those grown within Matrigel or at the air-liquid interface. A total of 13,179 cells were analysed, revealing 14 distinct clusters. Notably, differentiation within a higher G’ SAPH generated podocytes with more mature gene expression profiles. Additionally, maturation within a 3D microenvironment significantly reduced the derivation of off-target cell types, which are a known limitation of current kidney organoid protocols. Finally, we show that these organoids can be used to faithfully model pathogenic processes; by integrating single cell gene expression and epigenome profiling, we identified de novo ACTA2+ve /POSTN+ve cell clusters in kidney organoids treated with TGFbeta, characterised by increased SMAD3-dependent cis chromatin accessibility and the expression of several genes associated with fibroblast activation in patients with Diabetic Kidney Disease. This work demonstrates the utility of synthetic peptide-based hydrogels with a defined stiffness, as a minimally complex microenvironment for the modelling of renal fibrosis.
31451707839
"Introduction
Patients requiring soft tissue reconstruction caused by defects or pathology may require biomaterials that provide a void volume for subsequent vascularization and new tissue formation, as autografts are not always a viable option. Here supramolecular hydrogels represent promising candidates due to their 3D structure being similar to the native extracellular matrix and their cell entrapment capability. Over recent years, guanosine (Guo)-based hydrogels have increasingly emerged, in which the nucleoside self-assembles into ordered structures (G4-quadruplex) and ultimately into nanofibrillar networks by the π-π stacking of G-quartets and coordination of central K+ ions. While such hydrogels typically exhibit a short lifetime, the use of boronic acid (BA) significantly enhances their stability. The aim of the present study was to combine this technology with 3D bioprinting to produce binary cell-laden hydrogels consisting of Guo and guanosine 5-monophosphate (GMP) stabilized by BA and K+, and to optimize printability and the survival of the entrapped cells. Such a system would then allow to tailor the biomaterial to the respective soft tissue defect and thus improve tissue reconstruction.
Experimental Methods
Various compositions of Guo (10-120 mM), GMP (10-120 mM), BA (10-60 mM) and KOH (10-60 mM) were mixed at 80 °C, slowly cooled down, and assessed for gelation by inversion test. Printability was subsequently evaluated by a semi-quantitative filament collapse and fusion test. The very best hydrogel composition was then immersed in a hyperbranched poly(ethylenimine) (PEI) solution (5 mgmL-1, 15min) and characterized by scanning electron microscopy (SEM) and rheological studies (strain sweep, dynamic step-strain sweep and peak-hold assay). Furthermore, nutrient permeability (FITC-Dextran) and hydrogel stability (immersion in complete medium at 37 °C) were determined. Finally, rat mesenchymal cells (rMCs) were entrapped in the hydrogels and studied for 21 days after printing, including cell viability and morphology assessment by confocal laser scanning microscopy (CLSM), and monitoring of the adipogenic differentiation (Oil-red O solution).
Results
From 49 hydrogel compositions that passed the inversion test, 15 exhibited suitable 3D printing properties. To improve long-term stability, hydrogels were subsequently treated with PEI, and the best composition was analyzed by SEM, showing nanofibrillar structures evident of successful G4-quadruplex formation. Furthermore, rheological analysis revealed good printing and thixotropic properties, while successful diffusion of FITC-dextran molecules (70, 500 and 2000 kDa) into the hydrogel confirmed that nutrients of various sizes may diffuse through the scaffold. Finally, a cell viability of 85% after 21 days was observed but with exclusively rounded morphology. However, lipid droplets were identified after 7 days, indicating cell functionality and successful differentiation under adipogenic conditions.
Conclusions
Our results demonstrate that printed Guo/GMP hydrogels exhibit extensive nanofibrillar networks and good printability and thixotropic properties. Stability was verified for 21 days in medium, and embedded cells showed good survival despite rounded morphology. Under adipogenic conditions, lipid droplets were observed, witnessing successful differentiation and functionality of the entrapped cells. Due to the demonstrated bioprintability, our Guo/GMP hydrogels may hold great potential for the reconstruction of soft tissues."
83767209244
"After decades of cutting-edge academic work, novel electrospun biomaterials are finally starting to enter clinics and patients worldwide. The process of taking an electrospun material from the lab, to the clinic, and finally to market is long and difficult. It requires the collaboration of many professionals over the course of years. But what it creates are world-class, ground-breaking medical devices which treat previously untreatable conditions, and improve the lives of patients.
This presentation will give a thorough overview of those devices containing electrospun materials which are currently available on the market, as well as those in various stages of clinical trials. We will explain how we, The Electrospinning Company Ltd, work with our clients to take exciting concepts born out of academia and private research, and guide them through proof-of-concept, design development, design control, production development and finally production. This presentation will also explain how pre-clinical and clinical trials fit into our framework, and how this all culminates in a final product release.
At The Electrospinning Company we can tailor our materials to achieve bespoke characteristics suitable for specific therapeutic indications and target tissues. By using nano and micro fibre structures, we can tightly control mechanical strength, flexibility, resorption time and architecture. Critically, we focus on producing these materials on an industrial scale, with the methodology, consistency, and quality necessary for medical applications. We refine ideas into standardised, repeatable processes to produce materials which can confidently be implanted into people.
Additionally, this presentation will give a detailed account of how The Electrospinning Company can take promising ideas and turn them into profitable products. Case studies of advanced materials developed for ophthalmological, dura mater repair, musculoskeletal, and cardiovascular applications will illustrate the process, showing how our scientists and engineers overcome one challenge after another on the long road to client and regulatory approval. The case studies will focus on specific technical and scientific challenges that had to be overcome, for devices in the clinic or about to enter the clinic."
41883648366
"INTRODUCTION: The use of biomaterials inside the body always entails the risk of infection. This risk might even be higher in in situ tissue engineering applications. Since the porous scaffold materials can form a niche for invading bacteria, the intended in situ production of novel tissue may be severely compromised by infection. Therefore, we aim to develop a new polymeric supramolecular scaffold material, exerting two important functions: preventing microbial adhesion and thereby preventing biofilm formation, and inducing endogenous (eukaryotic) cells to regenerate the body.
METHODOLOGY: In our research, supramolecular contact-killing materials based on antimicrobial peptides (AMP) are developed. A special class of supramolecular biomaterials are based on fourfold hydrogen bonding 2-ureido-4[1H]-pyrimidinone (UPy) moieties. The supramolecular base material consists of an UPy end-functionalization polycaprolactone (i.e. PCLdiUPy). These UPy-materials can be functionalized with bioactive compounds, either via a modular approach in which the UPy-base material is mixed with UPy-modified additives1, or via a post-modification strategy to specifically functionalize the surface of the biomaterial using click chemistry2. The antimicrobial activity is introduced via UPy-functionalized AMPs, using SAAP-148, a synthetic derivative of LL-373. The regenerative activity is introduced via an UPy-functionalized heparin binding peptide (UPy-HBP). The peptides were synthesized by manual Fmoc-based solid phase peptide synthesis. Solid polymer films were prepared by drop-casting PCLdiUPy with UPy-SAAP-148 or UPy-TC84 on glass coverslips. The antimicrobial activity of the UPy-AMPs in solution and when incorporated in the drop-casted samples was evaluated against Escherichia coli ESBL and Staphylococcus aureus JAR060131 and LUH14616 (MRSA) and Acinetobacter baumannii RUH875 using the LC99.9 (i.e. the lowest concentration killing at least 99.9% of the inoculum) and the JISZ2801 surface antimicrobial assay, respectively. Moreover, the cytotoxicity of these AMPs was tested against human dermal fibroblasts.
RESULTS: Coupling of the UPy-linker to SAAP-148 did not influence its antimicrobial activity in solution. For the solid drop-casted materials, incorporation of 5 mol% UPy-SAAP-148 is sufficient for killing all 4 bacterial strains tested. This indicates that the peptide remains active after immobilization in the materials. Unfortunately, TC84 loses its antimicrobial activity upon UPy-coupling, both in solution and as a solid. QCM-D adsorption studies revealed that heparin adsorbed to spin coated material films of PCLdiUPy with 5 mol% UPy-HBP mixed via the modular strategy.
Current studies focus on characterization of the UPy-SAAP-148/TC84 and multifunctional biomaterial with XPS, AFM, WCA, zeta potential and leakage experiments to investigate the material properties. Moreover, we assess the in vivo efficacy of dip-coated titanium implants with 5% UPy-SAAP-148 in the experimental biomaterial-associated infection mouse model.
CONCLUSIONS: In conclusion, this modular approach will enable a stable but dynamic incorporation of AMPs, and control of cell adhesion by using cell-adhesive peptides. Ultimately, we aim to use such materials for in situ infection-free tissue engineering.
REFERENCES: 1. Dankers, P.Y.W. et al., Nat. Mater. 4 (7), 568-574 (2005), 2. Goor, O.J.G.M. et al., Adv. Mater. 29 (5), 1604652 (2017), 3. de Breij A. & Riool, M. et al., Sci. Transl. Med. 10 (423), eaan4044 (2018)."
20941815547
"INTRODUCTION
Current treatments for oesophageal cancer and oesophageal atresia, that involves the repair of the entire thickness of the oesophagus, present various complications and challenges due to the lack of functional oesophageal replacement tissue. Through the combination of cells, scaffolds and biologically active molecules, tissue engineering presents an innovative approach to develop constructs that can mimic the multi-layered architecture of the oesophagus. This study analyses the response of primary oesophageal epithelial cells and fibroblasts on Manchester BIOGEL’s various self-assembling peptide hydrogels (PeptiGels), and primary oesophageal smooth muscle cells’ response on hydrogel-coated polycaprolactone (PCL) scaffolds with aligned fibres to determine a suitable hydrogel and PCL scaffold combination to tissue engineer a simplified oesophagus, consisting of epithelial, submucosal and muscle layers.
METHODOLOGY
2D culture of human oesophageal epithelial cells on PeptiGels was performed using a cell density of 3,000 cells/mm2; and cell viability assay, metabolic assay and immunohistochemical analysis were performed up to day 21. 3D culture of human oesophageal fibroblasts within PeptiGels was performed using a cell density of 100,000 cells/100µL volume of hydrogel; and cell viability assay, proliferation assay and immunohistochemical analysis were performed up to day 21. Aligned electrospun PCL fibres were coated with PeptiGel Alpha4_RGD_GFOGER before human oesophageal smooth muscle cells were seeded onto the PeptiGel-coated PCL scaffolds at 40,000 cells/cm2. Cell viability assay, metabolic assay and immunochemical analysis were performed up to day 21.
RESULTS
Viable cells, increase in metabolic activity and increased cell proliferation at greater timepoints were recorded for epithelial cells cultured in 2D and fibroblasts cultured in 3D for all PeptiGels up to day 21. Immunohistoanalysis showed positive expression of ZO-1 tight junction protein and involucrin markers by epithelial cells seeded on all PeptiGels up to day 14, and positive expression of Collagen I and Collagen III proteins, and Keratinocyte Growth Factor by fibroblasts seeded into all PeptiGels up to day 14. Analysis of results at this stage indicated that a positively charged hydrogel with RGD and GFOGER motifs, i.e., PeptiGel Alpha4_RGD_GFOGER, to the be most suitable out of all hydrogels for 2D and 3D culture of epithelial cells and fibroblasts respectively. PeptiGel Alpha4_RGD_GFOGER was chosen to pre-coat aligned PCL fibres for smooth muscle cell culture.
Viable cells and increase in metabolic activity at greater timepoints were recorded for smooth muscle cells cultured on PeptiGel-coated PCL scaffolds up to day 21 and positive expression of smooth muscle alpha-actin marker by smooth muscle cells seeded on PeptiGel-coated PCL scaffolds were recorded up to day 21.
CONCLUSIONS
The results collectively indicate that employing a combination of synthetic peptide hydrogels and hydrogel-coated aligned PCL fibres to tissue engineer the multi-layered structure of the oesophageal tissue is a viable option."
52354517559
"Introduction
Delayed or severed tissue regeneration is often caused by dysfunctional immune system.[1] One solution to tackle this issue is to design immunomodulatory materials that support tissue regeneration by priming immune system to a pro-regenerative state.[2] For example, it is known that high molecular weight (>1000 kDa) hyaluronic acid (HA) can polarize macrophages to an M2 pro-regenerative phenotype, whereas low molecular weight HA drives pro-inflammatory M1 polarization.[3] Understanding the rules for designing functional materials that incorporate immunomodulatory effects, biocompatibility and allow stable long-term polarization of macrophages is therefore of high interest in multiple tissue engineering (TE) scenarios, notably for 3D printing TE.
Methodology
Here, to answer the new demands, we designed a selection of two-component hydrogels built from self-assembling β-sheet forming peptides[4] and immunomodulatory tyramine-modified HA (THA)[5], that can be processed by 3D micro-extrusion printing. A selection of peptide sequences was based on the alternation of hydrophobic and hydrophilic amino acids: XYXZXYXZ (X: hydrophobic residue: phenylalanine or tyrosine, Y/Z: hydrophilic residue e.g.: lysine or glutamic acid), stemming from the known parental FEFKFEFK sequence and it subsequent modifications.[4] All parental peptides self-assemble into semi-flexible networks and hydrogels, as derived from oscillatory rheology measurements and contain high β-sheet content, as measured by FTIR. 280 kDa and 1640 kDa THA were synthesized as previously described.[4] The successful THA synthesis was confirmed using 1H-NMR and degree of modification was calculated from UV absorption.
Results
A parametric study was carried out to verify the effect of rational peptide sequence modification on final physico-chemical and biological properties of composite hydrogels. Self-assembly, and rheological properties can be controlled by the choice of primary peptide sequence, fabrication technique and final crosslinking mechanisms including enzymatic (HRP, H2O2) and visible green light crosslinking using Eosin. These hydrogels are characterised by shear-thinning behaviour and rapid recovery allowing extrusion-based fabrication of both simple (lines, grids) and more complex shapes retaining post-printing fidelity.
Conclusions
The versatile crosslinking mechanisms allow post-crosslinking structure stabilization with longer-term degradation, deeming them a modular and versatile inks platform to endow with multiple biological cues for TE and immunomodulation.
ACKNOWLEDGEMENTS: This work was supported by the European Union’s Horizon 2020 (H2020-MSCA-IF-2019) research and innovation programme under the Marie Skłodowska-Curie grant agreement 893099 — ImmunoBioInks.
REFERENCES:
[1] B. Shan, X. Wang, Y. Wu, C. Xu, Z. Xia, J. Dai, M. Shao, F. Zhao, S. He, L. Yang, M. Zhang, F. Nan, J. Li, J. Liu, J. Liu, W. Jia, Y. Qiu, B. Song, J.-D. J. Han, L. Rui, S.-Z. Duan, Y. Liu, Nature Immunology 2017, 18, 519.
[2] C. M. Walsh, J. K. Wychowaniec, D. F. Brougham, D. Dooley, Pharmacology & Therapeutics 2021, 108043.
[3] J. E. Rayahin, J. S. Buhrman, Y. Zhang, T. J. Koh, R. A. Gemeinhart, ACS Biomaterials Science & Engineering 2015, 1, 481.
[4] J. K. Wychowaniec, A. M. Smith, C. Ligorio, O. O. Mykhaylyk, A. F. Miller, A. Saiani, Biomacromolecules 2020, 21, 2285.
[5] C. Loebel, S. E. Szczesny, B. D. Cosgrove, M. Alini, M. Zenobi-Wong, R. L. Mauck, D. Eglin, Biomacromolecules 2017, 18, 855."
20941807266
Nanotechnology is now found in almost every aspect in life, from the liposomes that carry COVID-19 vaccines to coatings placed on floors to reduce wear. Over the past 20 years, the use of nanotechnology in medicine has grown from the unknown to now significantly helping to prevent, diagnosis, and treat numerous diseases. This is true for regenerative medicine and tissue engineering as well. Specifically, nanotechnology has been used to create biologically-inspired nanotextures to promote tissue formation, limit infection, and decrease inflammation. It has also been used to create materials whose shape and function can be changed after being implanted into the body. Such materials can be used to straighten the curves of spines for patient suffering from scoliosis, close sphincters in the body such as from the stomach to intestines, increase pressure on juxtaposed bones to promote bone growth, control drug delivery, and so much more. This invited talk will dsicuss all of the promising applications of nanotechnology in tissue engineering, in particular emphasizing in vivo studies and what is needed for the incorporation of nanotechnology into tissue engineering to continue to grow and meet the high demands of medicine in the future.
83871202439
Synthetic hydroxyapatite is therapeutically used as bone graft substitute, bone filler, or as coatings to support attachment of bone to metal implants. Here I will present some data on how our group have used hydroxyapatite nanoparticles in combination with various polymers and fabrication techniques to support bone cell differentiation and matrix formation in both static and mechanically stimulated culture conditions. However, the slow degradation rate of hydroxyapatite compromises its osteogenic activities so recently we have collaborated with industrial partners to create a multisubstituted HAP (sHAP) with Magnesium and Strontium. We used a continuous flow method and a Design of Experiments approach to optimise the method and amounts of magnesium and strontium in the hydroxyapatite to increase its solubility, osteogenic integrity and bioactivity. The powders were tested using immortalised human mesenchymal stem cells Y201 in serum-free media and shown to support osteogenesis with no cytotoxicity at any dose tested. A particular need for improved orthopaedic devices is in spine repair where 50% of spinal fusion surgeries need revision partly due to poor osseointegration. Therefore we employed our substituted hydroxyapatite in two spine repair devices 1) Spinal fusion cages: as a coating on a titanium oxide base on a PEEK spinal cage 2) Bone graft substitute: as a particulate phase within a sHAP polycaprolactone composite use as an ink to print an insert for spinal cages which can be used in place of bone graft. Materials containing sHAP were tested for their ability to support osteogenesis and their potential to accelerate the fusion process in spine repair.
31451706027
"Introduction
Efficient regeneration of different tissue types requires solutions that are specifically tailored to meet certain criteria. This is particularly true for the group of hard-to-regenerate tissues, such as cardiac, neural or chondral, which are known to have a low self-regeneration potential. The cells of these tissues often require certain factors to induce their division, differentiation and maturation. These would include usage of scaffolds with: specific surface properties (chemical composition, presence of certain motifs), presence of some sort of bioactive compound (various types of biomolecules), electro-donor properties, electrical conductivity, surface morphology, mechanical properties [1, 2]. Positive therapeutic effect can be further boosted by introducing additional exogenous stimuli, such as electrical, magnetic, or mechanical stimulation [3, 4]. Certainly, all of the positive outcomes can be boosted when all of the above-mentioned cues are combined.
Despite controversies regarding their safe biomedical applications, carbon nanotubes (CNTs) of well- defined properties have been proven be biocompatible, creating interesting modificators for the fabrication of multifunctional scaffolds with new or improved properties. Electrical conductivity, ability to bind and controllably release bioactive compounds, or introducing the stimuli-responsiveness are just some examples of these properties.
Methodology
In this study, surface functionalization of CNTs have been employed to grant them with cytocompatibility and bioactive properties. Next, the CNTs were used as matrix and surface modifiers. Chemical composition, electrical and mechanical properties of the as-obtained scaffolds were evaluated and the materials were tested for their effect on cells, antibacterial, and anticancer properties.
Results & conclusion
In the course of this study, electrically conductive and cytocompatible materials based on CNTs were fabricated. Altered electro-donor, physichochemical and electrical properties yielded surfaces of bactericidal and anti-cancer properties that at the same time were able to enhance the cellular adhesion, growth and proliferation.
Acknowledgements
This study was supported by the National Science Centre, Poland, under grants nos. UMO-2017/24/C/ST8/00400 and 2020/37/B/ST5/03451.
Citations:
31412769528
"Introduction
In situ tissue-engineered vascular grafts (TEVGs) based on resorbable synthetic scaffolds have the potential to overcome the limitations of prosthetic graft replacement and provide off-the-shelf availability. Despite massive efforts in investigating new materials, up to date no TEVG is clinically available. One of the most important challenges for successful regeneration is to balance the scaffold degradation and tissue formation. There is an increasing demand for tools to monitor these parameters simultaneously and at a spatial resolution, as most of the currently applied methods can either access polymer degradation or tissue regeneration.
Methodology
Raman microspectroscopy allows to characterize a sample based on molecule-specific spectral fingerprints, which enables label-free evaluation and imaging of a sample. This study investigated the potential of Raman microspectroscopy as an in situ tool to monitor degradation kinetics and mechanisms of supramolecular polymers which are applied as degradable scaffolds in in situ tissue engineering. Raman imaging was applied on in vitro degraded polymers, investigating different polymer materials, subjected to oxidative and enzymatically induced degradation. Furthermore, the method was transferred to analyze in vivo degradation of tissue-engineered carotid grafts after 6 and 12 months in a sheep model.
Results
Multivariate data analysis allowed to trace degradation and to compare the data from in vitro and in vivo degradation, indicating similar molecular observations in spectral signatures between implants and oxidative in vitro degradation. In vivo degradation appeared to be dominated by oxidative pathways. Furthermore, collagen remodeling at the implant interface was monitored simultaneously to the assessment of polymer degradation.
Conclusion
Our results demonstrate the sensitivity of Raman microspectroscopy and imaging to determine degradation stages and the assigned molecular changes non-destructively, encouraging future exploration of this techniques as a quality assessment tool to monitor in situ tissue engineering."
31412719397
Introduction: Allograft vasculopathy is an aggressive form of accelerated atherosclerosis that manifests uniquely in transplanted hearts, lungs, and kidneys. Activated blood vessel endothelial cells (ECs) stimulate alloreactive CD4+ and CD8+ T-lymphocytes to result in sustained inflammation. Transition metal carbides, MXenes, are an emerging class of nanomaterials that have recently been shown to have unique immunomodulatory properties. Here, we present the development and application of novel tantalum carbide MXene (Ta4C3Tx) quantum dots for in vivo immunomodulation and prevention of allograft vasculopathy.
Methodology: To infer mechanisms and to ensure reproducibility of results, detailed physicochemical characterization of Ta4C3Tx MXene quantum dots was performed using scanning/transmission electron microscopy, x-ray diffraction, and x-ray photoelectron spectroscopy. In vitro studies were carried out using co-cultures of human umbilical vein endothelial cells (HUVECs) with allogeneic peripheral blood mononuclear cells, and immunomodulatory function was assessed using flow cytometry. Mechanisms for immunomodulation was ascertained using quantitative real-time PCR. A rat aortic transplantation and allograft vasculopathy model was used for in vivo validation of safety and immunomodulatory function.
Results: A facile hydrofluoric acid-free protocol was rationally designed to synthesize a zero-dimensional MXene quantum dot (MQD) material. These MQDs were surface modified with high amounts of different functional groups. The average diameter of single particles was found to be about 3.5 nm. Using the in vitro co-culture system, we found that Ta4C3Tx MQDs interact with activated human ECs to reduce activation and pro-inflammatory Th1 polarization of allogeneic CD4+ lymphocytes. Furthermore, we showed that treatment with MQDs significantly increased endothelial surface expression of the T-cell co-inhibitory molecule PD-L1 and decreased expression of the costimulatory molecule CD86. Finally, when applied in vivo¸ our data suggested that treatment with MQDs could significantly reduce lymphocyte infiltration and preserve medial smooth muscle cell integrity within transplanted vessel segments.
Conclusion: These findings offer the promise of next generation Ta4C3Tx MQDs as a smart material for treatment of allograft vasculopathy and other inflammatory diseases. This research also opens the door to development of rationally designed Ta4C3Tx MXene quantum dot technologies for other immune-sensitive regenerative medicine applications.
Keywords: MXene, Immunomodulation, Nanomaterials, Regenerative Medicine
41883625484
Introduction
Spinal cord injury (SCI) is a devastating condition that disrupts both sensory and motor function, with very limited prospects of functional recovery. Electrical stimulation (ES) has become a common clinical remedy to lessen the impact of SCI-induced pain after injury. However, regeneration-focused lesion site electrostimulation has not had clinical translation yet, despite promising evidence of directing axonal growth and encouraging cord repair. Furthermore, neural interfaces still face challenges over long implantation times due to delamination, insufficient water barrier properties and inflammatory responses. MXenes, a novel class of 2D electroconductive layered materials, possess unique properties for developing ES systems that can wrap around the injured cord to deliver charge safely and efficiently.
Here we show the aerosol jet 3D printing (AJP) of a neural interface cuff with highly conductive MXene (Ti3C2TX) electrodes, protected and insulated by a polytetrafluoroethylene (PTFE) structure.
Methodology
MXene films were produced using doctor blade, vacuum-assisted and AJP-printing to assess the effect of fabrication methods on their physical properties and biocompatibility. Conductivity, hydrophilicity and mechanical properties from the films were evaluated.
To assess biocompatibility, NSC-34 mouse motor neurons were seeded on the MXene films to study the morphology influence onto the cells over 3 days and their morphology, proliferation and metabolic rate were studied.
PTFE substrate was spin-coated with a commercial ink followed by 3D printing of the MXene circuit using an Optomec AJP-300 3D printing system. Then, the circuit was passivated and protected with another layer of PTFE by 3D printing with the same system, and then sintered at 360⁰C under non-oxidizing argon atmosphere to create a solid structure.
Results
Results showed that all MXene films were biocompatible, supporting neuron cell viability via similar proliferation and metabolism rate to tissue culture polystyrene controls. Also, neurons grown on AJP-printed films displayed enhanced neuronal neurite outgrowth and cell morphology, possibly, due to their enhanced conductivity (∼12000 S/cm) and higher hydrophilicity (35⁰) in comparison to filtered and doctor blade films. Top and cross-section SEM images of the device showed conformal deposition of MXenes onto a printed PTFE substrate, facilitated by the surfactant-induced hydrophilicity of the PTFE commercial ink. After sintering at 360⁰C, the PTFE nanoparticles coalesced, effectively bounding the printed MXenes onto its otherwise hydrophobic surface. During the thermal treatment, the surfactant and remaining moisture from the PTFE ink evaporated, switching the behaviour of the PTFE surface from hydrophilic (41⁰) to hydrophobic (125⁰) (p<0.0001).
Conclusion
Direct adhesion of 3D printed MXenes after PTFE sintering postulates as a facile method to construct bespoke neural electrode implants for stimulation of the injured spinal cord, while limiting abiotic and biotic faults due to the excellent biocompatibility and pliability of the device.
References
Zamhuri et al. BioMed. Eng. Online, 20:1-24, 2021
Secor et al. Flexible and Printed Electr., 3:1-12, 2018
41883634455
Introduction:
Lack of a cornea’s donor is still a huge problem in ophthalmology leaving over 98% of people waiting for the transplantation. As the solution, bioengineered scaffolds, mimicking the fibrous structure of the corneal stroma are proposed. To obtain a full regeneration, such substrates should provide both cornea cells repopulation and optical transparency. In the native cornea, the hierarchical microstructure of collagen fibrils is responsible for maintaining those properties. Studies have proved that both primary morphology (organization of the fibres) and secondary morphology (fibres nanotopography) have a huge impact on the phenotype of the cells.
In our study, we investigated the impact of different primary (aligned and random fibres) and secondary microstructure (shish-kebab, beads-on-strings) of the electrospun PCL nonwovens on the viability of different cells (macrophages, keratinocytes, fibroblasts) to check whether they show a preference for a specific type of surface. We also examined the correlation between the morphology of the substrate and the optical transparency of the scaffold.
Methodology
The nonwoven was obtained by the electrospinning method based on polycaprolactone (PCL). To obtain the aligned fibres the rotating drum collector was used. Coaxial electrospinning was used to prepare beads-on-strings fibres using different concentrations of PCL solution in a shell/core part. To obtain shish kebab morphology, the neat fibres were modified by directional crystallization in PCL solution. The morphology of the samples was observed under scanning electron microscopy. The influence of the obtained fibres morphology on the physicochemical properties of the membrane (wettability, surface energy) was also investigated. The light transmission through the scaffold was examined by UV-VIS analysis. A biocompatibility test was performed by contacting the cells with the fibrous scaffold after 3 and 7 days.
Results
The SEM observations showed the presence of the randomly oriented and aligned nano- and submicron fibres. All of the obtained fibres have unimodal size distribution in a range 400 – 1000nm. The directional crystallization enabled achieving fibres with lamellae parts, characteristic of the shish-kebab morphology. The use of coaxial electrospinning enabled to obtain bead-on-strings fibres with a reduced core diameter (in a range of 200 – 700nm). Independently on the modification all of the samples exhibited hydrophobic properties (contact angle ~130°). The research also showed a slight difference in translucency between random oriented and aligned fibres, however, the best light transmission was recorded for beads-on-strings fibres. All of the samples exhibited high viability of all cells types independently of the orientation nor morphology of the fibres. None of the tested scaffolds showed a cytotoxic effect.
Conclusion(s)
Electrospun nanofibers with different microstructures were successfully obtained and demonstrate biological and optical properties that indicate the strong potential as extracellular matrix – mimicking substrates for cornea regeneration.
Acknowledgement
This work was supported by the subsidy of the Ministry of Education and Science for the AGH University of Science and Technology in Kraków No 16.16.160.557.
References
1. Gain, P. et al. JAMA Ophthalmol. 134, 167–173 (2016) 2. Kennedy, K. M. et al. Acta Biomater. 50, 41–55 (2017) 3. Zaarour, B. et al. ChemistrySelect 5, 1335–1348 (2020)
83767273539
Biomanufacturing cells and tissues from human pluripotent stem cells (hPSCs) typically strives to guide differentiation through developmentally relevant pathways in a well-defined, dynamic bioreactor environment. While great strides have been made in differentiating hPSCs to many somatic cell types, robust biomanufacturing remains a roadblock to clinical progress of hPSC-derived cell and tissue therapies. In particular, scaling manufacturing to meet clinical needs, reducing cost, improving cell phenotypes, and improving process robustness are critical challenges. hPSC-derived cardiomyocytes have tremendous potential to restore cardiac function to heart failure patients. However, these cells suffer from poor survival and functional integration in preclinical models of heart disease. We have developed protocols to differentiate hPSCs to endothelial cells and cardiac fibroblasts, and demonstrated that inclusion of these cells during cardiomyocyte biomanufacturing accelerates acquisition of maturation phenotypes such as morphology, sarcomere protein expression, and calcium handling in the cardiomyocytes. Importantly, these heterotypic cell interactions must be provided to cardiac progenitor cells, allowing the cell types to co-differentiate. To reduce costs and improve scale of cardiomyocyte biomanufacturing, we have transitioned 2D cardiomyocyte differentiation to 3D, reducing cost by approximately 85% and permitting manufacturing of greater than one trillion cardiomyocytes in a 300 mL spinner flask bioreactor. To improve biomanufacturing process robustness, we have performed a multi-omic characterization of differentiating cardiomyocytes and utilized unbiased data analytics to identify genes, proteins, and metabolites that when measured before day 5 predict successful vs. failed batches at day 15, determined by the percentage of cells expressing cardiac troponin T. We envision that these multivariate predictive critical quality attributes can be used to more quickly identify failed batches and eventually lead to closed-loop control strategies to improve biomanufacturing process robustness.
20967801764
"Over 300 million cases of osteoarthritis were reported in 2017, stating one of the most prevalent chronic joint diseases worldwide characterized predominantly by long-term progressive cartilage and subchondral bone degeneration. Conventional therapy approaches utilize pharmacotherapy mostly for pain relief and at end stage disease treated by whole joint replacement surgery to retrieve some mobility and function. Novel Regenerative Medicine (RM) strategies employing Tissue Engineered implants could enable cure, more than care, of such life-constraining disabilities to meet the rising demand for medical interventions due to an ageing world population. Engineering joint tissue implants for the regeneration of the cartilage-bone unit of the joints remains a challenge due to the complex structural organization and functionality of native joint tissue. The use of microtissue/spheroid platforms has enabled differentiation and maturation of cartilage intermediates and gives hope for the engineering of efficient large-scale implants for osteochondral regeneration. However, there is still lack of enabling technologies for scaling of these approaches and robust manufacturing with end-to-end automation of such advanced therapeutic medicinal products (ATMPs).
To allow sufficient scaling, overcome risks of contamination as well as inconsistent product quality in manual production procedures, the automated, GMP-compliant manufacturing platform »JointPromise« is developed. By establishing a robust, large-scale manufacturing process, a reliable microtissue-based product for the treatment of deep osteochondral defects can be generated with suitable productivity. The platform concept is based on the translation of Standard Operating Procedures (SOPs) for microtissue production, harvest and condensation into a sequence of automated process steps. Derived process design criteria and technical specifications result in device requirements for an automated production process. After initiating the conceptualizing stage of the platform design by creating a 2D layout according to the material flow of the translated SOPs, the final arrangement of devices was optimized in the overall 3D CAD model. The resulting production platform model combines all required devices for cell cultivation, microtissue harvest and ATMP production in an overall layout consisting of pipetting units, an incubator, centrifuge, bioprinter and housing for a defined hygienic environment. Following the SOPs, about 28,000 microtissue spheroids can be produced within 21 days of culture out of 1 mL cell suspension per tissue culture plate. To reach the required productivity of around 100 tissue culture plates per implant, the production platform will need to process around 70 L of liquids during seeding and harvest processes and 5 L per cell media change to produce around 2.8M microtissue spheroids in 21 days. The build-up of the »JointPromise« platform is followed by the implementation of the control software COPE (Control Operate Plan Execute, Fraunhofer IPT, Aachen, Germany) for process controlling and monitoring during cell seeding, cultivation and harvest.
This project received funding from the European Union’s Horizon 2020 research and innovation program under grant agreement no 874837."
62825429844
"Introduction: Progenitor cells from the periosteum are major contributors in fracture healing with contribution to the formation of the cartilaginous fracture callus. It was previously demonstrated that microspheroids of human periosteum derived cells, differentiated towards the chondrogenic lineage, could be assembled into scaffold-free constructs that healed murine critical-size long bone defects [1]. However, the stability of such scaffold-free implants can be compromised when scaling-up. In this work, cartilaginous microspheroids were combined with tailored melt electrowritten (MEW) meshes to create an engineered biohybrid callus-like membrane able to induce bone formation via endochondral ossification.
Methods: Human periosteum-derived cells (hPDCs) were seeded in non-adherent microwells (AggreWell™400, STEMCELL Technologies) to form microspheroids with approximately 250 cells. The microspheroids were cultured in a chemically defined chondrogenic media (CM) for 4 days where after they were seeded onto MEW polycaprolactone (PCL) meshes with defined pore size. The PCL meshes were printed with Spraybase® Melt Electrospinning instrument with an average fiber diameter of 10.9 ± 2.3 µm and coated with 0.1% gelatin before seeding. Microspheroids on MEW-PCL meshes were differentiated in CM for an additional 14 days before gene expression analysis or subcutaneous implantation in immunodeficient mice was performed.
Results: Melt electrowritten polycaprolactone (MEW-PCL) meshes were tailored to contain pores with a size (116 ± 28 µm) designed to allow efficient entrapment of microspheroids (180 ± 15 µm in diameter). Four days microspheroids attached and spread over the MEW-PCL mesh. After 14 days in chondrogenic media, gene expression analysis demonstrated up-regulation of chondrogenic (9-fold: SOX9, 140-fold: COL2) and prehypertrophic (71-fold: IHH) gene markers. To assess the bone forming capacity of the “living membranes” (day 14), they were implanted subcutaneously with MEW-PCL meshes only as control. No mineralization was detected in mesh-only explants but bone, bone marrow and mineralized cartilage was detected in all “living membranes”. A total amount of 23 ± 3 % (MV/TV) mineralized tissue was observed. These data demonstrated differentiation towards prehypertrophic chondrocytes in vitro and bone formation in vivo.
Discussion: The high versatility of this biofabrication approach lies in the possibility to tailor the scaffolds to shape and dimensions corresponding to the clinical indication and the individual patient using robust bone forming building blocks. We believe that these strategies will be instrumental in the development of designed living tissues with predictive clinical outcomes.
Acknowledgment: Research funded by BONE INTERREG North West Europe, European Regional Development Fund
Reference: 1. Nilsson Hall, G. et. al., Adv. Sci. 7, 1–16 (2020)."
62825467239
"INTRODUCTION:
In the field of bioprinting, which is growing very rapidly, the bioextrusion technology represents more than 80% of the market. The other technologies (e.g. microvalve or inkjet) are used for their ability to print droplets. Among them, laser-assisted bioprinting (LAB) represents the most advanced technology to achieve high resolution printing and high cell viability [1][2]. Poietis, a leading company in LAB technology, has developed a unique machine combining all bioprinting technologies in a single instrument to take advantage of their complementary performances paving the way toward personalized ATMPs for therapeutic applications [3]. In this context, bioprinting of spheroids is of great interest to produce efficacious tissue constructs for clinical applications [4][5]. The objective of this talk is to present the capabilities of the LAB approach to print spheroids in a reproducible manner while ensuring post-printing structural maintenance of spheroids and cell viability.
RESULTS:
Spheroid LAB printing is possible by raising the laser energy to 25 µJ, the deposited volume to 30 µL and ablated gold surface to 7000 μm². First, a very thin, high speed jet appeared followed by a thicker jet at lower speed which is capable to transport the spheroid onto the receiver surface. These settings resulted in the formation of a jet with a diameter of 200µm and spheroid transfer success of 94%. The ultra-short laser was limited in the size of spheroid printing capability while the nanosecond laser gave a broader range.
Spheroids with different chondrogenic maturation were tested and day 7 ones exhibit the best printing efficiency possibly due to a trade-off in their composition (# cells, ECM) and size. Semi-quantification of viability staining demonstrated that printing using ultra-fast laser resulted in a similar viability as non-printed spheroids. Although the nanosecond laser resulted in a slightly lower cell viability after printing, the broader range of spheroid printability motivated its use for further experiments.
Furthermore, time resolved imaging technique enabled quantification of the jet dynamics to get experimental behavior laws for each condition. Next, numerical simulations contributed with physical interpretation to the experimental data.
DISCUSSION & CONCLUSION:
We provide proof-of-concept for the use of LAB technology for spheroid-based tissue production, providing a reproducible and precise manner of transferring spheroids. A better understanding of the underlying physical processes and specific biological conditions have been obtained through dedicated experiments and simulations.
REFERENCES:
[1] Murphy K & Atala A, 3D bioprinting of tissues and organs, Nature Biotech, V32 N°8 (2014)
[2] Ali M et al, Controlling laser-induced jet formation for bioprinting mesenchymal stem cells with high viability and high resolution, Biofabrication 6 (2014)
[3] Guillemot F et al, Tissue Manufacturing by Bioprinting: Challenges & Opportunities, Cell Gene Therapy Insights; 4(8), 781-790, (2018)
[4] Burdis R & Kelly D, J Biofabrication and Bioprinting Using Cellular Aggregates and Microtissues for the Engineering of Musculoskeletal Tissues, Acta Biomater. (2021)
[5] Nilsson Hall G et al, Cartilaginous spheroid-assembly design considerations for endochondral ossification: towards robotic-driven biomanufacturing. Biofabrication 13 (2021)
ACKNOWLEDGEMENTS:
Joint Promise European Project (RIA, N°H2020-SC1-BHC-2018-2020) / BPI / Nouvelle-Aquitaine Council"
62825464088
"Introduction: For a viable and compliant clinical translation of tissue-engineered products, the adoption of automated technologies has been acknowledged as a prerequisite. Recently, the use of chondrogenic microtissue and organoid assemblies has shown promising results in long-bone defect regeneration through endochondral ossification[1]. Hence, automated biomanufacturing technologies able to culture and handle these tissue building blocks are of great interest. Here, we present an automation strategy through the use of different robotics for (i) media change during differentiation, (ii) plate movement, and (iii) image-based picking of microtissues for enabling spheroid-specific quality control.
Methods: Periosteum derived cells were seeded in a commercial microwell platform (AggrewellTM800, 1000 cells/spheroid or AggrewellTM400, 250 cells/spheroid). They were cultured for 21 days in chondrogenic medium. Media change of microtissues in microwell platforms requires controlled pipetting to avoid microtissue displacement and suspension leading to uncontrolled fusion. We set up a design of experiment (DoE) for aspiration and dispension speed during automated media changes. Then, we investigated the effect of robotic plate handling and automated imaging on spheroid movement for different size spheroids. After 21 days, we created ectopic implants through a controlled fusion of 900 large, or 3600 small spheroids to assess the bone-forming capacity, which is evaluated through µCT and safranin-O histochemistry. Subsequently, the automatic cell-screening and -picking system CellCelector™ was used to select and transfer single spheroids in a controlled manner. Here, spheroids were automatically selected based on the presence of only 1 spheroid/well and/or size via image-based analysis.
Results: The first DoE revealed that dispension speed, rather than aspiration speed, had a significant effect on local spheroid movement during media changes. The second DoE showed that smaller microtissues were more susceptible to movement than larger spheroids. Also, plate handling had a significant impact on overall movement. After 21 days, spheroids from all conditions were able to mineralize ectopically. Finally, using the CellCelectorTM, we were able to pick and place single spheroids without affecting their viability.
Discussion: Bottom-up engineering of skeletal implants requires a vast amount of diffusion-unlimited spheroids as building blocks. Culturing these for multiple weeks to achieve the desired differentiation is a complex process that requires expert personnel to avoid spheroid movement that leads to uncontrolled fusion. To enable scale-up and increase process robustness, we demonstrate the development of an integrated bioprocess for culturing and manipulation of cartilaginous spheroids. We anticipate the progressive substitution of manual operations with automated solutions for the manufacturing of microtissue-based living implants.
Reference: [1] G. Nilsson Hall, L.F. Mendes, C. Gklava, L. Geris, F.P. Luyten, I. Papantoniou, Developmentally Engineered Callus Organoid Bioassemblies Exhibit Predictive In Vivo Long Bone Healing, Adv. Sci. 7 (2020) 1–16. doi:10.1002/advs.201902295."
41883637086
"Cartilage microtissues are promising tissue modules for bottom up biofabrication of implants leading to bone defect regeneration. Most of the protocols for the development of these cartilaginous microtissues have been carried out in static setups, however in order to further achieve higher scales suspension process needs to be investigated. In the present study we explored the impact of dynamic culture in a novel stirred microbioreactor system.
In order to generate cartilaginous microtissues, we first allowed cells to aggregate for 3 days, forming stable microtissues, before inoculating the bioreactors. Moreover a coupled CBM-CFD model was used in order to estimate the magnitude of shear stress on the individual microtissues. Experiments with the miniBR setup illustrated that we could culture cells dynamically in to form of microaggregates for up to 14 days. Furthermore, we observed that spheroids were able to fuse into larger structures , we used two static controls; one where spheroids cultured with the same dimensions as the initial inoculation and a second control where multiple spheroids were fused in to larger sized at the same time point as we inoculated the bioreactor. We observed that viability was not affected ether by the fusion event on (day2) or size of the microtissues while the same applied for culture in the bioreactor. Additionally, we saw a distinct difference between static and dynamic condition with a much lower fraction of EDU+ cells present in the bioreactor condition. This difference could be linked to the commitment of cells towards ECM production rather than proliferation something that is known during chondrogenic differentiation. This can be further evidenced by the histologic sections and Alcian Blue staining. Gene expression values showed a dramatic upregulation of both Indian hedgehog (IHH, 30-fold) and Collagen type X (COLX, 23-fold) well know marker of chondrogenic hypertrophy, for the day7 and day 14 time points for the dynamically cultured microtissues. Moreover Chondromodulin also showed a large upregulation (16-fold) for the last time point. Similarly to the transcriptome level, we saw distinct metabolic profiles between static and dynamic conditions. More specifically, whereas both conditions were characterized by high glucose consumption and lactate production at Day 7 and Day 14, in the bioreactor condition there was significant increase in glucose consumption and lactate production for Day 14 and a similar trend as well at Day 7. Moreover, several amino acids such as proline and aspartate showed significant differences between the conditions.
In this study we explored the impact of bioreactor-cultured microtissues in suspension as compared to statically cultured ones and the influence of shear stress on the acceleration of chondrogenic differentiation towards hypertrophy.Our work provides insights on the effect of the process environment on critical cellular, molecular, and metabolic parameters, and a straightforward strategy for the scalable production of cartilage intermediate microtissues."
20941861537
Therapeutic angiogenesis, i.e. the generation of new vessels by delivery of specific factors, is required both for rapid vascularization of tissue-engineered constructs and to treat ischemic conditions. Vascular Endothelial Growth Factor (VEGF) is the master regulator of angiogenesis. However, uncontrolled expression can lead to aberrant vascular growth, as well as non-vascular side-effects. Major challenges to fully exploit VEGF potency for therapy include the need to control in vivo distribution of growth factor dose and duration of expression. In fact, the therapeutic window of VEGF delivery depends on its amount in the microenvironment around each producing cell rather than on the total dose, since VEGF remains tightly bound to extracellular matrix. On the other hand, short-term expression of less than about 4 weeks leads to unstable vessels, which promptly regress following cessation of the angiogenic stimulus. Here we will briefly overview some key aspects of the biology of VEGF and angiogenesis and discuss their therapeutic implications, with a particular focus on approaches using gene therapy, genetically modified progenitors and extracellular matrix engineering with recombinant factors.
83871202505
"ENGINEERING HIGH DENSITY CAPILLARY-LIKE NETWORKS USING MICROPOROUS ANNEALED PARTICLE TISSUES
M.R. Schot1, C.A. Paggi1, M.L. Becker1, J. Leijten1
Presenting author: Maik Schot, m.r.schot@utwente.nl
1Department of Developmental BioEngineering, TechMed Centre, University of Twente, the Netherlands
INTRODUCTION: The vascular tree is essential for the function and survival of tissues. However, engineering vascular trees within 3D tissues has remained challenging. Current methods to produce channel-like structures in engineered tissues such as 3D printing are able to mimic large vessels, but struggle to produce highly dense capillary networks at high speeds, limiting their translation to clinically sized constructs.1 Microporous annealed particles (MAP) offer an interesting alternative due to their microporous nature, essentially offering an inherent dense microporous network of channels without having to create it.2 Here, we aim to use our recently developed in-air microfluidics (IAMF) technique to produce cell-laden microgels at ultra-high throughput production rates to create microporous annealed particle (MAP) tissues, allowing for the bottom up development of engineered tissues with inherent highly dense capillary sized pore networks.3
METHODOLOGY: Alginate-Tyramine (ATA) was produced by functionalizing alginate with tyramine groups using DMTMM-based coupling. Using IAMF, hepatic cell laden ATA microgels were prepared via ionic crosslinking under oil-free and surfactant-free conditions. ATA microgel slurries were photocrosslinked into MAPs using ruthenium, sodium persulfate and visible light. MAPs were analyzed on viability, micropore size distribution, pore interconnectivity, hydraulic conductivity, perfusability and mechanical properties.
RESULTS: Cell-laden microgels were successfully produced at varying cell concentrations, maintaining viability after MAP formation through visible light crosslinking. Moreover, MAPs were kept in culture for a period of 7 days and encapsulated cells proliferated within MAP constructs. Confocal analysis confirmed a highly dense, porous network within MAPs with the majority of pores having diameters below 40μm. Pores were shown to lead to increased construct perfusability as compared to nanoporous gels and an interconnected porous network was shown by perfusing 1μm fluorescent particles as well as using microCT analysis. Moreover, small molecules are able to easily perfuse in and out of the MAP as compared to traditional nanoporous gels that are hindered by the diffusion limitation.
CONCLUSION: The combined use of IAMF and ATA allows for ultra-high throughput production of cell laden microgels that can be assembled into MAP tissues containing an inherent, highly dense, interconnected capillary-like network. We thus present a novel and highly scalable production platform for the creation of large engineered tissues with inherent capillary-like networks.
REFERENCES
1. Kolesky, David B., et al., Proc. Nat. Acad. Sciences, 113.12, 3179-3184 (2016).
2. Annamalai, R. T. et al., Ann. Biomed. Eng. 47, 1223–1236 (2019).
3. Visser, C. W. et al, Science Advances, 4.1, 1–9 (2018)."
94238147747
"Introduction. Coupling of angiogenesis and osteogenesis is crucial to generate vascularized bone grafts. Semaphorin 3a (Sema3a) regulates osteoblasts and osteoclasts to promote bone synthesis through Neuropilin-1 receptor (NP1). We previously found that: 1) short-term delivery of Vascular Endothelial Growth Factor (VEGF) in osteogenic grafts dose-dependently decreases bone formation by increasing resorption and impairing progenitor differentiation; 2) in skeletal muscle VEGF dose-dependently inhibits endothelial Sema3a expression, impairing recruitment of Neuropilin-1-expressing monocytes (NEM), TGF-b1 production and SMAD2/3 activation. Here we investigated whether: a) VEGF impairs bone formation by inhibiting endogenous Sema3a expression; b) Sema3a treatment could improve both bone formation and vascularization in engineered bone grafts.
Methods. Fibrin matrices were decorated with recombinant VEGF or Sema3a proteins that were engineered with a transglutaminase substrate sequence (TG-VEGF and TG-Sema3a) to allow cross-linking into fibrin hydrogels. Osteogenic grafts were prepared with human bone marrow mesenchymal cells (BMSC) and hydroxyapatite granules in a fibrin hydrogel containing TG-VEGF, TG-Sema3a or both at ratio of 1:1 and implanted ectopically in nude mice. Sema3a blockade was achieved with a specific antibody (anti-NP1A) that prevents Nrp1 binding to Sema3a, but not to VEGF.
Results. 100 mg/ml of TG-VEGF (high dose) caused severe bone loss and significant downregulation of endogenous Sema3a expression. 0.1 µg/ml of TG-VEGF (low dose), instead, preserved both bone formation and Sema3A expression. Loss-of-function experiments showed that blocking Sema3a/NP-1 signaling significantly impaired bone tissue development, increased osteoclasts recruitment and, interestingly, also decreased vascular invasion both in the absence and presence of low TG-VEGF. Further, Sema3a/NP-1 blockade significantly reduced both human progenitor survival and endogenous Sema3a expression, as well as phospho-SMAD 2/3 activation in both human progenitors and host endothelial cells. These data are consistent with a positive feed-back loop between Sema3a and TGF-b1 signaling, as we previously described in skeletal muscle. Conversely, in gain-of-function experiments, TG-Sema3a co-delivery was able to prevent bone loss induced by high TG-VEGF, while preserving efficient vascular growth. Notably, TG-Sema3a alone could increase both the amount of mineralized matrix and vascular invasion in the absence of any TG-VEGF.
Conclusion. These data suggest that Sema3a: 1) is required for intramembranous ossification in osteogenic grafts; and 2) provides a key molecular link coupling angiogenesis and bone formation. These data identify Sema3a as a promising target to generate vascularized bone grafts in a clinical setting."
62825408166
"Introduction
The ultimate goal in vascular tissue engineering is the generation of bioartificial blood vessels that resemble the morphology and function of native vessels as accurately as possible. Previous studies have shown that the tunica intima and tunica media of native blood vessels can be resembled in bioartificial vessels by applying physiological mechanical stimulation using pulsatile perfusion in vitro. However, until today only very few studies have focused on the integration of a functional tunica adventitia including a vascular network known as vasa vasorum, which are of pivotal importance for graft integration and nutrition. For this, it is not only essential to integrate a complex microvascular network in the outer layer of the prosthesis, but also to resemble the physiological longitudinal alignment of capillary vasa vasorum of native vessels. Here, we investigated the effect of physiological in vitro perfusion on the alignment of vasa vasorum capillaries in the tunica adventitia of small-diameter fibrin-based bioartificial blood vessels.
Methodology
Two-layered fibrin vessels were generated in a step-wise molding technique. First, acellular tunica media-equivalents were molded in a cylindrical mold. For this, a high-concentration fibrin matrix was generated using 25 mg/ml human fibrinogen resuspended in Medium 199. Polymerization was initiated by adding thrombin, factor XIII and Calcium chloride. After initial polymerization, compaction of the matrix was performed by centrifugation using a custom-built rotation device. The compacted tunica media-equivalent was then transferred into a second mold and the tunica adventitia-equivalent was molded around it. For cellularization of this layer, red fluorescent protein expressing human umbilical vein endothelial cells (EC) and adipogenous stem cells (ASC) were suspended in a low-concentration fibrin matrix. Consequently, segments were implemented in bioreactors and incubated for 72 h under longitudinal tension of 50% (tension alone), pulsatile perfusion with physiological cyclic stretch of 5% (pulse alone) at a frequency of 60 bpm, or both factors combined (tension+pulse). Control segments were incubated in cell culture tubes without mechanical stimulation (static).
Results
Complex microvascular networks were generated by endothelial cell self-assembly in the tunica adventitia of every group. Both, longitudinal tension and pulsatile perfusion induced physiological longitudinal alignment of vasa vasorum capillaries parallel to the main vessel axis. This effect was even more pronounced when both stimuli were applied simultaneously. Opposed to that, statically incubated controls showed randomly organized capillary networks.
Conclusions
Integration of a pre-vascularized tunica adventitia in bioartificial blood vessels is a promising strategy to facilitate early graft integration and immediate cell nutrition throughout the vessel wall after implantation of bioartificial vascular grafts. With longitudinal tension and cyclic stretch, we identified two mechanical stimuli that influence capillary tube orientation in tunica adventitia equivalents of fibrin-based bioartificial blood vessels in vitro. Thus, mechanical stimulation represents an effective strategy to generate physiologically aligned vasa vasorum capillaries in cardiovascular tissue engineering."
73296308684
"Introduction: Vascularization is crucial for proper implant survival and integration in the body. For an implant to be vascularized in vivo and thereby supplied with oxygen and the needed nutrients, the body’s blood vessels have to invade the implant first. This is a very slow and often insufficient process, especially in thicker tissues, which can result in implant loss. A solution for this are pre-vascularized, fibrin-based implants that can be attached to the blood vessels in vivo, thereby giving a faster and more sufficient blood supply throughout the whole tissue construct enabling implant survival.
Methodology: We here present a bioartificial tissue construct with a fibrin matrix of 5 mg/mL fibrinogen concentration, containing red fluorescent protein-labeled human umbilical vein endothelial cells (RFP-HUVEC) and adipose tissue-derived stem cells (ASC), facilitating capillary-like network formation. Cellularized fibrin gels were either supported by enclosing them in a high-concentration fibrin capsule, or by walls of the custom built flow chamber. Two fibrin-based vascular grafts were integrated on each side of the construct as an in- and outlet for flow. These were interconnected with microchannels penetrating the cellularized matrix. After polymerization of the fibrin matrix, microchannels were endothelialized with RFP-HUVEC as well. Tissue constructs were then perfused for four days at a flow rate of 18 mL/min at 2-3 mmHg, with feeding medium containing VEGF, FGF, Ascorbic Acid, Aprotinin, TPA and reduced serum supplement.
Results: After four days of perfusion, homogenous capillary-like network formation throughout the cellularized fibrin matrix could be observed. Interestingly, cells between endothelialized microchannels started to align during capillary formation. Moreover, a radial alignment of cells around microchannels could be observed, with occasional sprouting of endothelial cells off cellularized microchannels.
Conclusion: By perfusion of low-concentration fibrin gels, vascularization of a tissue construct can be achieved. This offers the opportunity for the generation of various tissue constructs by introducing tissue specific cell types into the construct."
31412728567
"Introduction: The lack of functional vascular networks is one of the most important hurdles in tissue engineering, limiting the obtainment of fully functional organs substitutes. Vascularization promotes efficient nutrients and oxygen supply, representing a key factor for in vivo application [1]. Endothelial cells not only act as the structural building blocks of vascular endothelium, but they also fulfil key functional tasks by interacting with parenchymal cells in a complex cellular interplay. In the context of liver bioengineering, recent works described enhanced hepatic maturation when combining endothelial cells with liver organoids [2,3]. ‘Reset’ vascular endothelial cells (R-VECs) have recently been identified as a promising candidate for graft re-endothelialization by demonstrating good adaptability and efficient colonization capacity in decellularized scaffolds [4]. The present work aims to combine R-VECs endothelial cells and human fetal liver organoids in decellularized ECM scaffold environment, to recapitulate the endothelial – parenchymal cells interplay.
Methodology: Human fetal liver organoids (post conception week, PCW = 5) were generated in Matrigel® 3D culture, after previous cells isolation via tissue disaggregation, and expanded in hepatic organoid expansion medium [5]. R-VECs were cultured on conventional tissue culture flaks supplied by endothelial cells growth medium (EGM2) medium. Mouse livers were cannulated via the portal vein and decellularized via already established detergent-enzymatic treatment [6]. 18 M cells were obtained from enzyme mediated organoid dissociation and were further seeded in decellularized scaffolds via portal vein injection. Repopulated livers were cultured in a custom-designed bioreactor in dynamic conditions provided by a peristaltic pump (flowrate = 3 ml/min) in hepatic organoid expansion medium for 7 days. Then, the same number of R-VECs were seeded with the same seeding technique. Repopulated scaffolds were cultured in the bioreactor with hepatic organoid expansion medium supplied with Oncostatin-M and Dexamethasone [5], and EGM2 in a 1:1 volume ratio. Histological and immunohistochemistry analysis were performed to study scaffold repopulation and the expression of mature hepatic and endothelial markers. qRT-PCR analysis was performed focusing on hepatic maturation markers such as cytochrome 3A4,1A2 (CYP3A4, CYP1A2), hepatocyte nuclear factor 4 alpha (HNF4α) and alpha fetoprotein (AFP).
Results: Mouse livers were successfully decellularized as highlighted by complete translucent appearance. H&E analysis showed efficient scaffold repopulation after 14 - days dynamic bioreactor culture. Immunofluorescence staining revealed the presence of hepatic maturation markers (HNF4α, human albumin and Alpha1-anti-trypsin) in co-presence of endothelial markers (CD31, Von Willebrand factor), highlighting epithelial-endothelial cell interaction and re-arrangement. qRT-PCR results showed enhanced expression of HNF4α, CYP3A4 and CYP1A2 in the 3D dynamic culture compared to the static in vitro control. Accordingly, lower AFP expression was also evidenced with respect to control in vitro culture.
Conclusion: Endothelial cells are involved in key structural and functional tasks that have a pivotal role in building up of functional organ substitutes. In the present work, human fetal liver organoids demonstrated increased phenotypic maturation when cocultured with R-VECs in a 3D organotypic environment. Endothelial cells demonstrated to be key players for the achievement of functional hepatic tissue, boosting fetal stage hepatocytes towards a more mature phenotype."
20941855928
"Introduction
One of the key challenges in the field of tissue engineering is the vascularization of tissue-engineered constructs. Until now, endothelial cells (ECs) derived from human umbilical cord have been the predominant EC type for the engineering of vascularized tissue. However, ECs of different origins display a great heterogeneity, reflecting in tissue- and organ-specific characteristics, which are important for interacting with surrounding cell types. Therefore, the use of skeletal muscle-specific microvascular endothelial cells (SkMVECs) may offer more potential for generating tissue-engineered muscle which mimic better the native muscle structure and physiology. Engineering a vascular network within an engineered tissue can be achieved by co-culturing ECs with myoblasts in a 3D co-culture setting, based on the capacity of ECs to self-assemble and form a vascular network. For this, it may be further beneficial to obtain both cell types from the same biological origin. In this work, we present the isolation of SkMVECs in combination with myoblasts from human skeletal muscle, followed by the investigation of an optimal culture medium for the co-culture of these two cell types. Finally, we demonstrate the application of SkMVECs for generating vascularized bio-artificial muscle.
Methodology
SkMVECs and satellite cell-derived myoblasts were isolated using an in-house developed protocol. Tissue was digested using an automated mechanical and enzymatic tissue dissociation procedure. Isolated single cells were cultured and separated using magnetic-activated cell sorting. Cell characterization was performed based on immunofluorescence staining and flow cytometry. Next, different media compositions varying in type and combination of growth medium and fusion medium were compared. The individual cell types were screened separately for behavior in each media composition. For SkMVECs, the formation of endothelial networks within a fibrin (1 mg/mL) hydrogel was evaluated. For myoblasts, the formation of multinucleated myotubes was assessed by performing a fusion assay. Finally, SkMVECs were applied for engineering co-culture bioartificial muscles as described in (1) using the explored culture media, and visualized using confocal microscopy.
Results
Isolated SkMVECs were found to express the endothelial-specific cell markers vWF and CD31. Isolated satellite cell-derived myoblasts were positive for desmin. The functional characteristics of the two cell types were tested and revealed endothelial network formation of SkMVECs on growth-factor reduced Matrigel, and the formation of multinucleated myotubes by isolated myoblasts. Next, two culture media consisting of a combination of serum-rich medium with a switch to serum-low medium after 3 days, were found to facilitate both a proper myotube and endothelial network formation. In a final step, the determined culture conditions were applied for the 3D co-culture of myoblasts and SkMVECs, and were demonstrated to facilitate the creation of a vascularized bioartificial muscle.
Conclusion
With the developed protocol, SkMVECs can successfully be isolated from human muscle biopsies. In addition, an optimal co-culture medium was identified which further allows the use of SkMVECs to tissue-engineer bioartificial muscles. This paves the way for the follow-up investigation of the vascular properties of SkMVECs and their potential for improving the physiological relevance of muscle tissue constructs.
References
1. Gholobova et al., Methods Mol. Biol., 169-183 (2019)."
83767265448
"Vasculature plays an essential role in skin physiology and its architecture and function are altered in aged and diseased skin. There is thus a need to develop innovative 3D in vitro models with adjustable and amenable vasculature. Several in vitro skin models co-seeding endothelial cells with fibroblasts and keratinocytes have been proposed using scaffolds or bioprinting. However, they all fail in faithfully mimicking native skin microenvironment, including its complex ECM, papillary and reticular specificity or wound-healing associated microenvironment. Indeed, despite cellular remodeling of the scaffold material at the cellular level, exogenous proteins remain as the unique major component thus limiting assessment of the actual dermis microenvironment dynamics.
The aim of this work was to develop a vascularized skin substitute in a dermal microenvironment generated by fibroblasts and displaying plasticity in response to angiogenic factors or physiologic processes. We thus used the scaffold-free approach of cell sheet co-culture to produce the skin microenvironment. Skin primary fibroblast cell sheets co-seeded with endothelial cells or keratinocytes were cultured and superimposed to generate vascularized full-thickness skin substitutes. Using immunofluorescence and transmission electron microscopy, we confirmed the presence of a fully differentiated epidermis and well-structured dermal-epidermal junction. Whole-mount immunofluorescence demonstrated that endothelial cells organized into a dense vascular network throughout the dermis. Capillaries displayed a lumen and were stabilized by a basement membrane and the recruitment of perivascular cells. Modulating Vascular Endothelial Growth Factor (VEGF) concentration in the ng/ml range and time of application differentially affected angiogenesis in our model, resulting in distinct vascular network length and branching. Interestingly, these variations also impacted epidermis differentiation and proliferation. Furthermore, applying a full thickness wound to the skin substitute resulted in wound closure mimicking the time frame and ordered physiological process. In this context, we could follow centripetal revascularization by sprouting angiogenesis from the wound boundaries.
We have thus implemented a novel skin substitute displaying vascular plasticity in response to subtle angiogenic stimuli and wound healing. This model is of interest to mimic physiological and compromised skin conditions involving the vascular component (aging, neuro-inflammatory diseases, etc) and to evaluate the capacity of natural active molecules to restore skin vascular homeostasis."
62825436244
"The development of regenerative therapies for the intervertebral disc (IVD) is of much interest because IVD degeneration is a major cause of low back pain, one of the leading causes of disability worldwide. The most common approaches are to utilize biomaterials, cells, and/or molecular agents, alone or in combination. Some of these approaches are moving toward clinical implementation and have shown promising clinical results. However, actual tissue regeneration has been more elusive.
Tissue engineering and regenerative medicine have often focused on tissue morphology but clinically tissue and organ function may be more important to reduce pain and disability. In terms of the IVD, whose function is mainly biomechanical as a cartilaginous intervertebral joint, this has not always been the focus when designing regenerative approaches. In our and partner laboratories, we have taken a biomimetic approach to develop biomaterials that help to return IVD function. This talk will explain some of these approaches, how they have been evaluated and highlight some major challenges experienced early in their clinical implementation."
83871201684
"Low back pain is the leading cause of morbidity worldwide and yet most therapies fail to target the cause and are purely symptomatic or end stage surgical options. Intervertebral disc degeneration is associated with approximately 40% of low back pain cases and thus a target for potential regeneration. Intervertebral disc degeneration is a catabolic process caused by altered cell behaviour and tissue biomechanics, leading to a harsh environment for potential cell therapies. To generate a successful regeneration strategy for the intervertebral disc this harsh environment must be considered, and therapies assessed within conditions which mimic this degenerate niche.
The utilisation of cells alone for regenerative therapies are unlikely to be successful if the degenerative cascade and mechanical environment are not restored, hence the combination of cells with biomaterials offers advanced therapeutic approaches. Injectable biomaterials which can restore the mechanical properties of the disc, inhibit catabolic processes of disc degeneration whilst delivering a regenerative cell source hold the most promise to halt disc degeneration and enabling regeneration.
Here, the development of novel injectable hydrogel systems which show potential to deliver a three-pronged attack to regeneration of the disc will be discussed. The application of differential cell sources including mesenchymal stromal cells from adipose and bone marrow, notochordal cells and induced pluripotent stem cells will be discussed. Importantly model systems which can recapitulate the degenerate disc environment as testing platforms for potential regenerative approaches for the disc will be introduced."
31451703426
"Introduction
Low back pain due to intervertebral disc (IVD) degeneration is a major health and socioeconomic problem throughout the world. In the young and healthy IVD, large and vacuolated notochordal cells (NCs) are present1. These cells are, in some species (e.g. humans and dogs), replaced by chondrocyte-like nucleus pulposus cells (NPCs) during maturation and ageing2. In previous studies, porcine NC-derived matrix (NCM), containing matrix and biologic factors secreted by NCs, induced regenerative and anti-inflammatory effects in human, canine, and bovine NPCs in vitro and degenerated canine IVDs in vivo3,4. Since the precise mechanism behind NCM remained elusive, we aimed to determine the mode of action of NCM in the degenerative IVD environment.
Methodology
Canine and human NPCs were cultured with and without NCM for 6, 24, and 72 hours in monolayers. After 6, 24, and 72 hours, RT-qPCR was performed on inflammatory markers and after 72 hours, targeted proteomics was performed with DigiWest, a proprietary immunoassay technology which transfers Western Blot to a high-throughput bead-based microarray platform5. Lastly, immunohistochemistry was performed on in vivo canine IVDs treated with NCM for 6 months.
Results
RT-qPCR analysis indicated that NCM induced an initial inflammatory response after 6 hours, since IL-6, IL-8 and COX2 mRNA expression was increased in human and canine NPCs. DigiWest analysis showed that NCM mainly induced changes in the Mitogen-activated protein kinase (MAPK) pathway after 72 hours of treatment, i.e. after the initial pro-inflammatory response. The expression of key proteins downstream the MAPK pathway, such as ERK1/2, JNK, and PKC, was mostly inhibited by NCM, whereas expression of proteins that are known to dephosphorylate MAPK key signaling molecules, DUSP1, 5, and 6, was increased in NCM-treated NPCs. Lastly, also expression of KRT19, a healthy NP marker, was induced by 72 hours of NCM treatment. Confirming the DigiWest results, in vivo canine IVDs treated with an intradiscal NCM injection demonstrated increased KRT19 and DUSP5 immunopositivity compared with controls after 6 months of treatment.
Conclusions
Taken together, these results indicate that NCM induces an initial inflammatory response, but thereafter exerts its prolonged anti-inflammatory effects by influencing the MAPK pathway. The latter leads to reduced expression of inflammatory cytokines after prolonged treatment.
References
1. Bach, F. C. et al. Eur. Cell. Mater. 30, 132–137 (2015).
2. Hunter, C. J., et al. Tissue Eng. 9, 667–677 (2003).
3. Bach, F. C. et al. Oncotarget 9, (2018).
4. de Vries, S., et al. Tissue Eng. A 25, 830–841 (2018).
5. Treindl, F. et al. Nat. Commun. 7, (2016).
The European Union’s Horizon 2020 program (iPSpine; #825925) supported this work."
31412708286
"Introduction
The last decade, efforts have been made in developing more effective diagnostics for low back pain. The focus was addressed towards Modic Changes (MCs), pathological signal intensity changes in the vertebral bone marrow and endplates of the intervertebral disc (IVD), which can be detected on Magnetic Resonance Imaging (MRI)1. In contrast to the human situation, little is known about the prevalence and characteristics of MCs in dogs, that are often used as a model to study intervertebral disc disease and new regenerative therapies2. For this reason, the aim of this study was to examine the prevalence and imaging/histologic characteristics of MCs in dogs.
Methodology
High field 1.5 Tesla MRI images of canine patients with low back pain and/or neurological deficits were retrospectively analysed. Inclusion criteria were the availability of sagittal T1- and T2-weighted turbo spin-echo sequences for the whole lumbar spine and exclusion criteria were (para)spinal neoplasia, resulting in 340 dogs and 2496 spinal segments. Dogs that underwent necropsy on the same day of the MRI were used for histo(patho)logical analysis (modified Boos score3, including endplate morphology, new bone formation, and subchondral bone sclerosis; n=16 dogs, 39 segments). The adjacent vertebral bone was assessed for infiltration of inflammatory cells, neovascularization, fatty infiltration, and the presence of chondroid cells, fibrous tissue, or Schmorl’s nodes. Multivariable logistic regression models were built to test the association between the presence of MCs and explanatory variables.
Results
MCs were most often detected at the lumbosacral junction (L7-S1 in the dog), the majority was MC type 3 (subchondral bone sclerosis). Previous spinal surgery at the investigated level predisposed dogs for the development of MC type 1 (proliferation of fibrovascular (granulation) tissue, oedema in vertebral bone marrow) and 2 (fatty infiltration). As in humans, MCs in dogs were interconvertible over time. The prevalence of MCs appeared positively associated with age and disc protrusion/extrusion. Lasty, histological analysis indicated that IVDs in which MCs were detected showed more histopathological changes in the endplate and vertebral bone than IVDs without MCs. However, the histological changes described in human literature were not detected in the segments with the specific MC types. Instead, mostly chondroid infiltration was encountered in MC types 1 and 3.
Conclusions
As humans, also dogs show MCs, mostly at the lumbosacral junction. However, MCs in dogs exhibit other subchondral bone pathologies than humans, as chondroid proliferation was mostly encountered and little proliferation of fatty, fibrous, or granulation tissue.
References
1. Dudli, S., et al. Eur. Spine J. 25, 3723–3734 (2016).
2. Bach, F. C. et al. BMC Vet. Res. 10, 3 (2014).
3. Bergknut, N. et al. Vet. J. 195, 156–163 (2013).
The European Union’s Horizon 2020 program (iPSpine; #825925) supported this work."
31412709597
"Introduction: Low back pain (LBP) is a leading cause of disability worldwide and intervertebral disc (IVD) degeneration (IVDD) is a major contributor of LBP1. The IVDD is accompanied and often preceded by the replacement of large vacuolated nucleus pulposus cells (NPCs) by non-vacuolated, clustered cells in the notochordal cells (NCs) of the IVD2. The iPSpine project aims to re-populate the degenerated IVD with regenerative iPS-derived NC-like cells (iPS-NLCs). During embryonic development, the determination of cell fates is a result of the combinatorial and concomitant activation of transcription factors. While differentiation of iPS-NLCs can be partially achieved by mRNA transfection of a single gene, such as the notochord-related transcription factor NOTO, such attempts are limited by low and variable differentiation efficiency3. This study aims to achieve optimal notochordal lineage commitment by the concomitant and combinatorial activation of multiple key transcription factors via CRISPR activation (CRISPRa) technology. With CRISPRa, transcription activation complexes are recruited to the endogenous promoters of genes to induce expression.
Methodology: Based on a two-step differentiation protocol, we first established iPS-derived mesodermal progenitor cell by CHIR stimulation to activate the WNT pathway. For notochordal lineage commitment, we focus on NOTO, brachyury (TBXT) and Forkhead Box Protein A2 (FOXA2). Both T and FoxA2 act upstream and are required for the NOTO expression. These three genes were activated at the endogenous gene locus by CRISPRa technology via the synergistic activation mediator (SAM) system, the most efficient dCas9 gene activator4. Gene activation by CRISPRa will be further compared with mRNA transfection to establish the optimal mode of notochordal lineage commitment.
Results: We establish a gene activation and differentiation pipeline in the notochordal cell lineage and show significant activation of all 3 genes by recruitment of CRISPRa to the respective gene promoters in the differentiating iPSCs. Our results indicate better expression levels when transcription is promoted directly via recruitment of transcriptional activation complexes to gene promoters, rather than the introduction of synthetic mRNAs. Time-course analysis of lineage-specific markers shows that combining multiple transcription factors allow for better iPSC-NLC differentiation and commitment toward the notochordal lineage.
Conclusions: We highlight how transcriptional landscapes can be modulated at critical moments of embryonic development to optimize iPSC-NLC differentiation strategies and optimize notochordal lineage commitment.
Funding: iPSpine: Horizon2020(No.825925) and Dutch Arthritis Society (LLP22).
1Clark, S. et al., Lancet. 391(10137):2302 (2018). 2Hunter, C. J. et al., Journal of anatomy. 205(5), 357-362 (2004). 3Colombier, P. et al., Cells. 9(2), 509 (2020). 4Konermann, S. et al., Nature. 517(7536), 583–588 (2015)."
52354506186
"Back pain is often associated with intervertebral disc (IVD) degeneration. Beside surgery, novel treatments relying on stem cell injection have been tested. Unfortunately, the outcomes are disappointing because of cell leakage and incomplete differentiation. Nowadays, a consensus exists on the necessity to encapsulate stem cells within a hydrogel to maintain them in situ and favor their differentiation. As cell behavior depends on biochemical and physical environment, a biomimetic hydrogel would promote IVD regeneration. Nucleus Pulposus is a highly hydrated tissue working as hydraulic shock absorber. Glycosaminoglycans give a high degree of hydration whereas collagen II gives resistance and allows for cell adhesion. With the aim of developing novel biomimetic hydrogels, collagen/hyaluronic acid composites were developed to mimic the structure and the mechanical properties of Nucleus Pulposus. For this purpose, we first studied the impact of the HA content on physical properties. Then, the potential of the different formulations to differentiate mesenchymal stem cells (MSCs) into NP cells was analyzed in detail.
HA functionalized with tyramine groups (HA-Tyr) was mixed with collagen and gelled using Horse Radish Peroxidase and H2O2 at pH 7.4. With a constant collagen concentration (0.4%), the HA-Tyr content was increased up to 2 % to create a platform of Col/HA hydrogels with different properties. The hydrogel structure, the mechanical properties and the degree of hydration were analyzed. Mesenchymal stem cells were encapsulated within the different hydrogel types and cultivated over 28 days. The impact of MSCs on hydrogel stability, metabolic activity and cell morphology were analyzed. Last, the gene expression of Aggrecan, Collagen I and II was quantified by real time PCR.
The physico-chemical study showed the impact of the HA-Tyr content on the hydrogel physical properties. At low HA-Tyr content (less than 0.4 %), the composite behavior was driven by collagen. Hydrogels exhibited a fibrillary network and were characterized by low mechanical properties. From 0.8% HA-Tyr, the mechanical properties and the hydration degree increased to reach those of NP (5kPa) when 2% HA-Tyr was added. Below 0.4% HA-Tyr, encapsulated cells contracted hydrogels after one week in culture. From 0.8%, hydrogels, MSCs did not contract hydrogels and their mechanical properties were stable over the time course of the experiment. With a high HA-Tyr content, cells did not proliferate, suggesting their commitment towards differentiation. At low content, MSCs spread and adopt a fibroblast like morphology. On the opposite, cells encapsulated within hydrogels at high HA-Tyr content were more rounded and resemble NP cells. The gene expression quantification showed that MSCs orientated towards a NP cell phenotype. When 2% HA-Tyr was used, cells highly expressed NP cells markers, i.e Aggrecan and Collagen II, and weakly expressed Collagen I. In contrast, cells encapsulated in hydrogels with a low HA-Tyr content weakly expressed these NP cell markers.
Taken together, these results show that Collagen/Hyaluronic Acid Composite Hydrogels with a high HA content (2%) mimic the physical properties of the Nucleus Pulposus and promote the differentiation of MSCs into NP cells. Hence, these hydrogels could be useful for IVD regeneration"
52354503848
Loss of large, vacuolated notochordal cells (NCs) from the human intervertebral disc (IVD) is thought to initiate degeneration and associated back pain. It is therefore hypothesised that implantation of NCs may halt or reverse degeneration and thus relieve back pain. However, NCs are lost in early childhood, therefore iPSCs differentiation to NCs offers a clinically-viable cell source. Here we aimed to characterise the proteomic profile of the human foetal NCs, versus surrounding annulus fibrosus (AF) cells. FACS sorting for CD24, a known NC marker, was used to isolate NC (CD24+) and AF (CD24-) cells from microdissected human IVDs (14-15 weeks post-conception, n=3). Following iTRAQ isobaric tagging, TripleTOF mass spectrometry and subsequent bioinformatics, differential protein expression was validated by immunofluorescence. Our study revealed 100 up-regulated and 8 down-regulated proteins in the CD24+ population (-1.5≥FC≥1.5, P≤0.1), including known (e.g. keratins 8 and 19) and novel phenotypic markers. Ingenuity Pathway Analysis (IPA) revealed pathways known to play an important role in NCs and IVD homeostasis, such as NRF2-mediated oxidative stress response and caveolar-mediated endocytosis, and putative upstream regulators known to be active in NCs e.g. TGFβ1 and SMAD3. The same analysis predicted many previously unknown proteins, pathways and regulators to be active or repressed in NCs. Together, these results validate our method as a powerful tool for isolation and proteomic analysis of foetal NC cells and reveal novel proteins and pathways of potential use in the development of future strategies for the study and treatment of IVD degeneration, including in the development of protocols to direct differentiation of pluripotent stem cells towards NCs.
83767255989
Recent efforts are beginning to explore the application of extracorporeal shock wave therapy (ESWT) to near lung tissue and transcranial indications. This is driven by a desire to expand the well establish therapeutic and regenerative benefits to a new group of patients. Through mechanotransduction of the incoming pressure pulse, a cascade of biochemical responses is triggered within the cells. Simultaneously, there is a non-linear response in the physical bulk properties of the targeted structures. Either one of those processes may be both beneficial or detrimental for the treatment outcome. We present findings on the efficacy of expanding ESWT to these areas currently consider counterindications. Treating near lung or brain tissue using low energy shockwaves holds an unlocked regenerative potential but has to be seen in the light of potentially destructive tensile forces imparted on the tissue. Based on reference measurements, computational simulations, and in-vivo experiments we explore competing considerations in moving towards a clinical use of ESWT in the potential treatment of a wide range of new indications.
52419503306
Impaired wound healing and infections present a major concern for our health systems. Combined with the steady increase of antibiotic resistances and lack of new antibiotic drugs,
there is a pressing need for alternative treatment options, such as antimicrobial light-based therapies. While the germicidal properties of UV light have already been used for a long time, its negative side effects, for example DNA damage, photoaging and carcinogenicity, limit the possibilities and potential fields of application. Alternatively, blue light with wavelengths between 400 – 500 nm has shown promising results regarding the inactivation of a variety of microorganisms such as bacteria, yeasts and fungi viruses with the additive potential to improve wound healing (1). While the underlying mechanisms of blue light therapy have not been completely resolved yet, it has been proposed, that intrinsic chromophores e.g. porphyrins or flavins, generate ROS upon irradiation which destroy lipids, proteins and nucleic acids.
The aim of this study was to investigate the effects of LED devices emitting “soft” blue light with wavelengths > 430nm (provided by Repuls, Vienna, Austria) on the inactivation of gram-negative as well as gram-positive bacterial strains. It was revealed that the susceptibility towards the treatment differed greatly, presumably associated with the gene recA. In most bacterial strains, the protein RecA is responsible for homologous recombination, DNA repair and induction of the SOS response. Therefore, we hypothesized that the inactivation of the protein would increase the susceptibility of resistant strains and enhance the antimicrobial effects of the treatment. Although the potentiation of the therapy via RecA inhibitors was only partially successful, the influence of recA was confirmed by rescuing susceptible bacterial strains with the insertion of a plasmid carrying the gene. However, further experiments are needed to uncover the mechanisms behind the tolerability of blue light therapy and the importance of the SOS response. Nonetheless, progress was made towards a better understanding of the antibacterial actions of blue light. The modulation of the bacterial SOS response would not only benefit aBL therapy, but also antibacterial drug treatments, which are still the standard protocol for the care of infected wounds. Creating synergistic effects by combining these therapies into one singular treatment would likely increase therapeutic efficacy and is therefore a promising strategy to overcome the dilemma of antibiotic resistance.
83767240505
"INTRODUCTION: Iron oxide based magnetic nanomaterial, magnetic nanoparticles (MNPs) and magnetic nanowires (NW) are versatile tools in biology and medicine. MNPs-mediated drug delivery is tested for regenerative medicine (RM) purposes as well as for tumour treatment and diagnostics. Stem cell mediated delivery represents a modality to target remote, metastatic tumors or regenerative sites for controlled drug delivery. We recently reported that emote controlled actuation of MNPs-loaded cells can deliver micro-mechanical stimulation for modulating mesenchymal stem cell differentiation potential. NW platforms can be used to deliver topographical cues as well as magnetomechanical stimulation to differentiating MSCs.
METHODS: Human adipose derived stem cells (ADSCs) and Wharton jelly derived MSCs (WJMSC) loaded with proprietary were tested for viability, proliferative capabilities, culture induced senescence (beta galactosidase assay) and magnetic properties. In vitro osteogenic adipogenic potential of ADSCs-MNP as well as chondrogenic potential of ADSC and WJMSC –MNP exposed to magnetic field (MF) was tested. ADSC cultured on NW substrates with or without MF exposure were tested for viability and differentiation potential to mesenchymal lineages.
RESULTS: ADSCs-MNP and WJMSC-MNP complexes retain cell viability and proliferative capabilities compared to non-loaded and become controllable within MF due to high iron content. MNP presence decrease stem cells culture induced senescence. ADSCs–MNP display increased osteogenic and decreased adipogenesis when exposed to alternating magnetic field in a time, modality of exposure and MF intensity manner. ADSC-MNP displayed increased chondrogenesis compared to WJMSCs further increased by MF exposure. NW substrate supports attachment and viability of ADSC. Osteogenic conversion of ADSC cultured on NW substrates was found to be increased compared to plastic culture dish.
DISCUSSION & CONCLUSIONS: ADSCs-MNP display increased osteogenic and decreased adipogenic potential under alternating MF dependently on exposure protocol. ADSC-MNP but not WJMSC display increased chondrogenesis in vitro, further increased by MF exposure. NW substrate can be used to enhance osteogenic potential of ADSC in vitro. While this findings need to be confirmed in vivo experiments, MNP loading and NW substrated can be used to design innovative modalities for engineering of implantable bone and cartilage.
ACKNOWLEDGEMENTS:
This work was supported by a grant of the Ministry of Research, Innovation and Digitization, CNCS/CCCDI - UEFISCDI, project number ERANET-EURONANOMED-3-OASIs, within PNCDI III"" and:"" NUCLEU Program (PN 19 28 01 01)"".
"
41883603997
"INTRODUCTION: When assessing the interaction between nanomaterials and cells, an important step is to image the effects induced by the material to the cell membrane. In order to evaluate the results of the interaction process, scanning electron microscopy (SEM) is typically used. Commonly, an important step during the preparation of biological cell samples for SEM is represented by the critical point drying, which involves the replacement of the alcohol used for dehydration with an inert gas. This conventional drying method is not only hard to accomplish, but it can lead to sample destruction if specific parameters are not met. On the other hand, when assessing nanomaterials adhered to the cell membrane, the integrity of the cell is not necessarily important, so a little cell deflation doesn’t affect the intended purpose of the evaluation. Here, we describe a simple and more cost efficient method to prepare biological samples for SEM imaging that preserves cell integrity and can be used to describe nanomaterials interaction with cell surface.
METHODS: Cells were grown on sterilized silica chips, after which the evaluated nanomaterial was added to the cell culture media at least 24h for incubation. Afterwards, the samples were washed to eliminate non-adhered nanomaterials, fixed with glutaraldehyde and osmium tetroxide, and dehydrated with increasing concentrations of alcohol. The silica chips were then air dried in the biological safety hood and in vacuum, followed by a sputter coat film of 5 nm of gold. The samples were imaged with a scanning electron microscope.
RESULTS: We were able to obtain well preserved biological cell samples, both with and without nanomaterials adhered to the cell membrane surface. Nanomaterials such as magnetic nanoparticles and magnetic nanowires were easily traceable on cell surface. Furthermore, the nanomaterials were clearly observed in the images obtained, while the cell surface was not affected by the drying process applied. Although the samples obtained using our method were characterized by a slight deflation of the cells, the morphology of the cells is well preserved and the method is suitable for the evaluation of the interaction between nanomaterials and cell surfaces.
CONCLUSIONS: We have described a novel cost efficient and easy to perform method for processing biological samples for SEM imaging that preserves cell morphology and can be used for analyzing nanoparticle and nanowires interaction with cell surface.
Financial support by UEFISCDI Contract no. 502PED/2020 (Project PN-III-P2-2.1-PED-2019-3442 / OPTIMAG) and a grant project (ERANET-EURONANOMED-3-OASIs, within PNCDI III) is gratefully acknowledged."
41883663368
"Introduction
Mechanotransduction is a key process in many developmental, physiological and pathological processes in bone. Mechanosensitive ion channels such as Piezo1 and TREK1 are the bonafide mechanotransducers that are critical for various biological processes, plays a critical role in bone regeneration1, 2. We already reported that the Magnetic ion channel activation (MICA) technology could apply mechanical force directly to mechanosensitive ion channels on the cell surface targeted with antibody functionalised magnetic nanoparticles for stimulating mechanotransduction and downstream processes3. Recently there is an augmented interest has been aroused to exploit Graphene Oxide (GO) and its derivatives for various biomedical applications. The abundant oxygen-containing groups in GO provides an excellent platform for further modifications using functionalised antibodies to facilitate targeted binding4. Therefore, we have developed a potential nanoplatform using magnetic nanoparticles and GO (GOMNPs) for enhanced osteogenic differentiation through the MICA based non-invasive remote activation. GOMNPs targeting the mechanosensitive Piezo1 and TREK1 ion channels were developed and their osteogenic potential under MICA application is studied with osteoblast-like MG-63 cells.
Experimental details
Characterisation of synthesised GOMNPs was done using TEM, XRD, Raman spectroscopy and VSM. All the cell culture experiments were done on MG-63 human osteosarcoma cells using GOMNPs. Effect of MICA stimulation on osteogenic differentiation was studied with ALP activity, Alizarin red and PCR studies of GOMNPs functionalised with Piezo1 and TREK1 under MICA application for 1h daily for 7,14 and 21 days.
Results & Discussion
The biocompatible GOMNPs were prepared using a simple, versatile strategy. The size and morphology of the as-prepared magnetic nanoparticles and the GO were confirmed using TEM and the structural features of the GOMNPs were evaluated by X-ray diffraction XRD and Raman patterns. The XRD and Raman patterns indicating the peak positions and the relative intensities of MNPs, GO and the GOMNP nanocomposites are successfully obtained. The obtained results also suggested that the synthesised GOMNPs showed excellent biocompatibility with superparamagnetic behaviour. The enhanced ALP activity and Alizarin red staining data indicated the positive effect of MICA stimulation on MG63 cells towards osteogenic differentiation. MICA stimulation mediated osteogenic gene expression by quantitative real-time PCR also confirmed the differentiation of MG-63 cells at mRNA level and improved expression of a panel of osteogenic markers, Runx2, ALP and OCN were obtained upon 7th and 14th days under MICA application
Conclusion
The preliminary in vitro results demonstrated the ability of functionalised GOMNPs for osteogenic differentiation under MICA treatment. The studies also proved that the functionalised GOMNPs were able to successfully bind with the mechanosensitive TREK1 and Piezo1 ion channels and was able to enhance the osteogenesis through mechanotransduction via MICA.
References
1. Henstock, J. R.; Rotherham, M.; El Haj, A. J. J Tissue Eng 2018, 9.
2. Sun, W. J.; Chi, S. P.; Li, Y. H.; et al. Elife 2019, 8.
3. Hughes, S.; McBain, S.; Dobson, J.; El Haj, A. J. J R Soc Interface 2008, 5, (25), 855-863.
4. Sasikala, A. R. K.; Unnithan, A. R.; Thomas, R. G. et al. Adv Funct Mater 2018, 28, (8)."
31412768808
"Introduction: Inflammation is a physiological process in healing however, persistent inflammation signals hold deleterious consequences to the tissue and contributes to the inhibition of regeneration. Resolving inflammation remains an unmet challenge with great impact in the management of chronic inflammatory disorders and for the treatment of tissue injuries. Interleukin 4 (IL4) is a well-known key regulator of macrophage function by stimulating M2 phenotype (1), which is associated with the resolution of inflammation and structural tissue healing, and to dampen macrophage responsiveness to inflammation via IL4-pSTAT6 pathway. Despite IL4 promise towards tissue regeneration, hurdles in IL4 associated to in vivo instability and diminished bioactivity, demand for alternative vehicles to efficiently deliver IL4 and modulate macrophage functions, fostering resolution of persistent inflammatory cues with coordinated action over inflammatory cascades. Magnetic technologies offer promising tools that would precisely deliver biomolecules and control their action via IL4-pSTAT6 pathway (2), while providing contactless control, local retention and real time traceability using conventional imaging techniques. Thus, we propose to use superparamagnetic iron oxide nanoparticles (SPION) as magnetic field-responsive carriers for IL4 presentation, to remotely control immunoregulation of macrophages to favor the M2 phenotype via IL4-pSTAT6 pathway.
Methodology: Commercially available superparamagnetic iron oxide nanoparticles (SPION) were conjugated to a M2 macrophage promoter (IL4) via carbodiimide chemistry (SPION-IL4). The system was characterized according to dimension, shape, and charge as well as for IL4 binding efficiency. THP1-derived macrophages were used to investigate viability and the expression of immune-modulatory molecules in the presence of SPION-IL4. Two time-points (1h or 24h) were investigated, and two SPION concentrations (30 or 100 µg/mL) studied to insight on the impact of IL4 to drive M2 macrophages. These outcomes were compared against exogenous IL4 (Exo IL4)-stimulated THP1 (control group). A magnetic field was provided by a magnefect device (nanoTherics Ltd, UK) (350 mT/ well) for magnetic guidance and IL4 presentation to macrophages.
Results: Magnetically guided SPION-IL4 were shown to contribute for immune strategies participating in M2 polarization via IL4-pSTAT6 pathway. After 24h, our results have shown the levels of pSTAT6 trended higher in THP1 cells treated with SPION-IL4 comparing to Exo IL4. Furthermore, SPION-IL4-treated macrophages showed increased expression of M2 genes: IL10 and ARG1, and of M2 related proteins: CCL2 and IL1Ra, in comparison to Exo IL4, highlighting the effectiveness and impact of SPION-IL4 driving M2 signals.
Conclusions: This work reports the contribution of SPION-IL4 in IL4 mediated actions, taking advantage of SPION-IL4 magnetic responsiveness to deliver IL4 to macrophages and to promote M2 switch with the participation of STAT6. These findings show that SPION-IL4 influence pro-regenerative features in macrophages, and that SPIONs hold potential to be explored as a magnetically controlled system for targeted delivery of immunomodulatory triggers or combined with more sophisticated systems aiming at strategies for improved tissue healing.
Acknowledgements: NORTE-01-0145-FEDER-000021; ERC CoG MagTendon No.772817; EC Twinning project Achilles No.810850; FCT Doctoral Grant SFRD/BD/144816/2019.
References: 1. Stein, M et al. J Exp Med, 1992, 176(1), 287-92; 2. Gonçalves, A.I., et al, Biomed Mater, 2018."
83767226884
"Introduction: The currently available treatments for inflammation often target the symptoms but not the causes, leading to an ineffective management of persistent inflammatory conditions.
Due to the central role of macrophages (Mφ) in the inflammatory response, and overall in healing, innovative strategies to fine-tune the states and functionalities of Mφ may unveil the pathophysiology of chronic inflammation with great promise for a wide range of human afflictions.
MicroRNAs (miRNAs) are powerful materials to program cell responses, offering immune-regulatory possibilities for resolution and prevention of uncontrolled inflammation (1). Nevertheless, efficient and precise delivery systems are still a challenge for translatable RNA-based technologies. A major obstacle is to find carriers that overcome the RNA instability and enhance intracellular release.
Magnetically-assisted strategies hold potential to modulate cell and tissue responses combining contactless control and tissue penetration for tracking, local retention, and real time monitoring (2). However, superparamagnetic iron oxide nanoparticles (SPIONs) have been scarcely explored for targeted delivery and cell programming. Therefore, we theorized that loading miRNAs onto previously functionalized SPIONs to transport them into cells may overcome the instability of miRNAs. Specifically, our aim is to magnetically deliver and study miRNA molecules in the modulation of Mφ responses by suppressing a miRNA sequence (miR-155-5p), known to be overexpressed in inflammatory states, and consequently increasing anti-inflammatory mediators.
Methodology: Commercially available SPIONs were conjugated with polyethylenimine and miRNA (miR-155-5p) to form magnetically-responsive complexes via electrostatic complexation (hereafter referred as magnetoplexes). The system was characterized according to dimension, shape, and charge as well as for miRNA binding efficiency. Stationary (SMF) and pulsed-electromagnetic field (PEMF) using MagnefectNano and MagnetoTherapy devices, respectively, were investigated for internalization and delivery of the magnetoplexes via magnetofection. THP1 cells were primed to an inflammatory state (Mφ1) with lipopolysaccharide and interferon-γ (100 and 20 ng/ml, respectively). Mφ1 viability and the expression of immune-modulatory molecules were assessed in the presence of the magnetoplexes. Two time-points (1 and 4 days) were studied together with different miRNA cargos (0.05 or 0.15 µg) to determine the impact of miRNA in inflammatory mediators. The outcomes were compared against non-treated Mφ1-primed THP1 (Ctrl).
Results: The magnetoplexes were successfully produced with 76±2nm, and 26.8±0.5mV. Iron as low as 40ng in magnetoplexes was effective for miRNA loading. An improved cell uptake was observed in SMF-stimulated cells comparing to PEMF. For that reason, a 20-minute SMF stimulus was used throughout the experiment. Additionally, intracellular magnetoplexes were detected by confocal microscopy. Four days after magnetoplexes treatment, anti-inflammatory molecules as ARG1 trended higher in Mφ1, independently of miRNA mass, comparing to Ctrl. These outcomes suggest that controlled delivery of the miRNA to Mφ1 via magnetoplexes enables precision functional pro-healing changes.
Conclusions: The work combines contactless with high precision control to reprogram Mφ1 profiles, whose outcomes will contribute to advanced targeted and guided macrophage communication favoring a pro-regenerative environment and contributing to improved healing outcomes.
Acknowledgements: NORTE-01-0145-FEDER-000021; ERC CoG MagTendon No.772817; EC Twinning project Achilles No.810850; FCT Doctoral Grant SFRD/BD/144816/2019.
References: 1.Peng, B, et al. Adv. Drug Deliv. Rev.,88, 108-122(2015);2.Gonçalves,A.I., et al. Biomed Mater(2018)."
52354540386
The COVID-19 pandemic has shown how revolutionary treatments based on gene therapeutics has helped overcome a once-in-a-century pandemic and has given new momentum to gene therapy research for a myriad of applications. The field of regenerative medicine is well placed to be a beneficiary whereby, for example, gene therapy might be a valuable tool to avoid the limitations of local delivery of growth factors. While non-viral vectors are typically inefficient at transfecting cells, our group have had significant success in this area using a scaffold-mediated gene therapy approach for regenerative applications. These gene activated scaffold platforms not only act as a template for cell infiltration and tissue formation, but also can be engineered to direct autologous host cells to take up specific genes and then produce therapeutic proteins in a sustained but eventually transient fashion. Similarly, we have demonstrated how scaffold-mediated delivery of siRNAs and miRNAs can be used to silence specific genes associated with reduced repair or pathological states. This presentation will provide an overview of ongoing research in our lab in this area with a particular focus on gene-activated biomaterials for promoting bone, cartilage, nerve and wound repair. Focus will also be placed on advances we are making in using 3D printing of gene activated bioinks to produce next generation medical devices for tissue repair.
52419503804
"The development of hydrogels for regenerative medicine has progressed to the point where they are now considered one of the best options for successfully regenerating injured tissues. Their structural similarities to the extracellular matrix (ECM) and their versatility make them excellent candidates to mimic a native environment. Indeed, they are easily chemically modified and can be tuned to exhibit adequate degradation profile and mechanical integrity, as well as to incorporate growth factors or cytokines, making them suitable microenvironments to guide cell infiltration, proliferation, migration and differentiation, as well as innovative delivery systems of cells, extracellular vesicles or nucleic acids. Injectable hydrogels are the most extensively studied as they offer unmatched advantages compared to other biomaterials. As fluid materials, they have the ability to set in situ by physical or chemical crosslinking to form 3D microenvironments, thus simplifying their injection during minimally invasive surgery. Moreover, the development of dynamic chemistries now allows the use of hydrogels as nearly-physiological matrices to recapitulate the dynamic interactions of native environments.
In this talk, we will first provide an overview of the polymers, chemistries and fabrication techniques that are used to develop injectable hydrogels. We will highlight promising strategies that are used for tissue regeneration, notably in the field of joint diseases. We will then look at cell microencapsulation approaches with natural polymers (hyaluronic acid, alginate). Recent advances in droplet-based microfluidics and micromolding technologies will be discussed. We will also highlight the requirements in terms of diffusion and size properties, outline the 3D microenvironments we have recently developed in our lab using soft lithography technique, and discuss their relevance in the context of osteoarthritis and intervertebral disc treatment. "
94355104417
"Introduction: A major challenge in cartilage tissue engineering (TE) is to develop scaffolds capable of providing an instructive biomimetic environment to effectively drive mesenchymal stromal cells (MSCs) differentiation [1]. Hydrogels have emerged as promising biomaterials for this purpose, due to their biocompatibility and ability to mimic the tissue extracellular matrix [2].
Recently, graphene oxide (GO) emerged as a promising nanomaterial for cartilage TE due to chondroinductive properties when embedded into polymeric formulations [3]. It has been also shown that piezoelectric nanomaterials, like barium titanate (BaTiO3) nanoparticles, can be exploited as nanoscale transducers capable of inducing cell growth/differentiation [4].
Ultrasound waves are an interesting tool to facilitate chondrogenesis. In particular, it has been demonstrated that low-intensity pulsed ultrasound (LIPUS) regulates the differentiation of adipose mesenchymal stromal cells (ASCs) [5].
The aim of this study was to investigate whether dose-controlled LIPUS is able to direct chondrogenic differentiation of ASCs embedded in a 3D piezoelectric hydrogel.
Methodology: Human adipose mesenchymal stromal cells at 2*106 cells/mL were embedded in 3D VitroGel RGD® hydrogel with or without nanoparticles (GO, 25µg/ml, BaTiO, 50µg/ml) and exposed to LIPUS stimulation (frequency: 1 MHz, intensity: 250 mW/cm2, duty cycle: 20% , pulse repetition frequency: 1 kHz, stimulation time: 5 min) every 2 days, until day 10 of culture.
Hydrogels were cultured and chondrogenic differentiated for 2,10 and 28 days. At each time point cell viability (Live&Dead), cytotoxicity (LDH), gene expression of collagen type 2 (COL2), aggrecan (ACAN), SOX9, and collagen type 1 (COL1), electron microscopy, histology and immunohistochemistry (COL2, aggrecan, SOX9, and COL1) were evaluated.
Results: In both 3D hydrogels we evidenced that LIPUS treatment did not affect negatively the viability of the embedded cells. LIPUS boosted the chondrogenic differentiation of ASCs laden in 3D piezoelectric hydrogel: the chondrogenic genes and proteins markers (COL2, aggrecan and SOX9) were increased while the fibrotic marker COL1was decreased compared to control samples (non piezoelectric hydrogels and piezoelectric hydrogels not stimulated with LIPUS).
Conclusions: These results suggest that the combination of LIPUS and piezoelectric hydrogels push the differentiation of ASCs encapsulated in a 3D hydrogel and represent a promising tool in the field of cartilage TE."
62825420404
"Introduction:
The high number of vascular diseases demand for vascular grafts in various clinical application. Nonetheless, a big hurdle for the use of autologous tissue-engineered vascular grafts (TEVG) is represented by the usually long manufacturing time.Therefore, we here present a biohybrid vascular graft and a bioreactor that can provide distinct conditions such as flow, pressure, and temperature allowing the in vitro bioreactor conditioning of TEVG within four days. For in situ monitoring, we established reliable non-invasive imaging methods to monitor the degradation of the synthetic structural elements, ECM production, and signs of inflammation by molecular magnetic resonance imaging and ultrasound.
Methods:
A polyvinylidene fluoride (PVDF) tubular textile mesh was used as a permanent scaffold and coated with biodegradable superparamagnetic iron oxide nanoparticles (SPIONs) labeled PLGA fibers. TEVGs were prepared by a molding process which consists of the scaffold, fibrin gel, and arterial smooth muscle cells (SMCs). After molding an endothelialization process and bioreactor conditioning mimicking physiological blood flow and pressure values followed. Burst strength and suture retention strength of TEVG were measured and compared before and after bioreactor conditioning. The ECM production was studied in TEVG after 14 days of maturation using elastin- and collagen type I-targeted MR molecular gadolinium-based probes and immunohistology. The αvβ3 integrin expression as a marker of inflamed endothelium was assessed by molecular targeted US using RGD-poly(butyl cyanoacrylate) microbubbles and compared to RAD-control microbubbles1.
Results:
After four days of conditioning in a close loop bioreactor, 617±85 mm Hg of burst pressure and 2.24±0.3 N of suture retention strength were achieved. The bioreactor provided a suitable environment to the TEVG in which the cells could proliferate and produce extracellular matrix. The immunohistological findings proved the development of smooth muscle actin, Collagen I, Collagen IV, and continuous endothelial linings within the TEVG’s lumen. The presence of collagen was further identified by MRIusing a targeted contrast agent. The expression of integrin in TEVG was identified by selective binding of RGD microbubbles only after mimicking an inflammatory state..
Conclusion:
We introduce a biohybrid TEVG with a coated scaffold for longitudinal monitoring by non-invasive molecular imaging methods. After 4 days of bioreactor cultivation, this graft provides sufficient stability for implantation and the possibility of longitudinal monitoring in situ.
"
52354542408
"Introduction
The use of non-covalent self-assembly to construct materials has become a prominent strategy in biomaterial science offering practical routes for the construction of increasingly functional materials for a variety of applications ranging from cell culture and tissue engineering to in-vivo cell and drug delivery.[1] A variety of molecular building blocks can be used for this purpose, one such block that has attracted considerable attention in the last 20 years is de-novo designed peptides. The beta-sheet motif is of particular interest as short peptides can be designed to form beta-sheet rich fibres that entangle and consequently form very stable shear-thinning (injectable) hydrogels. The intrinsic biocompatibility and low immunogenicity of these materials makes them ideal for TERM applications. [2-8]
Methodology
We explored the unique shear thinning properties (injectability) of a family of short beta-sheet forming peptides (8-10 amino acids long). Through in-depth structural characterisation (AFM, TEM, SAXS, FTIR) and detailed rheological studies (shear rheometry and SIPLI) and modelling of dynamic behaviour (Standard mechanical models) we were able to develop a fundamental understanding of how design affects injectability. This understanding was then used to develop injectable system for the delivery of cell for a range of TERM applications.
Results & Discussion
Due to the self- assembled nature and dynamic properties of this family of peptides we were able to design readily injectable systems. We showed how the beta-sheet fibre edges properties (hydrophilic vs hydrophobic) could be modified by adding lysine end-residues. This allowed to design highly dynamic systems that were able to “liquify” (shear-thin) upon application of a large strain (e.g.: pressure) and then recover instantaneously their gel-like properties upon removal of the strain. [3] We showed that these hydrogels allowed the successful delivery of cells through injections using very small needle gauges.
In recent work performed in the context of nucleus pulposus repair and heart regeneration using a rat model we showed the potential of these systems as injectable functional materials for TERM applications. [4-6] In addition, we also used 3D-briorinting approaches to show that these shear-thinning materials are ideal bioinks for the 3D printing of cells. [7-8]
Conclusion
The intrinsic biocompatibility and non-immunogenic nature of these system combined with their unique shear-thinning properties allowed us to develop a family or injectable system for TERM applications. We demonstrate how design rule can be manipulated to tailor the properties of these materials to the application intended.
References:
1. Zhang S. G., Nature Biotechnology 2003, 21, 1171.
2. Gao J. et al., Biomacromolecules 2017, 18, 826.
3. Wychowaniec J. et al., Biomacromolecules 2020, 21, 2285.
4. Corimo L. et al., Acta Biomaterialia 2019, 92, 92.
5. Cosimo L. et al., Acta Biomaterialia 2021, 127, 116.
6. Burgess K. et al., Materials Science & Engineering C 2020, 119, 111539.
7. Raphael B. et al., Materials Letters 2017, 190, 103.
8. Chiesa I. et al., Frontiers in Medical Technology 2020, 2, 571626."
62825435106
"INTRODUCTION
Islet transplantation is a recognised treatment for type 1 diabetes: islets are transplanted into the liver and the procedure can stabilise blood glucose levels. A major limitation for successful clinical islet transplantation is the significant loss of islets post-transplantation due to an immune mediated inflammatory reaction. Etanercept, a TNF blocker that binds specifically to TNF-α, is currently provided as a systemic injection to patients on the first, third and fifth day post-transplantation. Whilst administration of etanercept has improved efficacy of clinical islet transplant, it is important to achieve targeted and controlled delivery to support islet engraftment locally. Encapsulation of etanercept within microparticles (MPs) may provide a suitable route for delivery. However, MPs are typically fabricated with commercially available polymers that are non-specific to the liver resulting in inefficient delivery.
Asialoglycoprotein receptor (ASGPR) exhibits high affinity as a galactose receptor and is the only liver-specific receptor identified on hepatocytes thus far. Herein, we demonstrate targeted delivery to the liver by synthesis of a novel poly(lactic-co-glycolic acid) (PLGA) polymer by covalent conjugation with galactose moieties. MPs fabricated from this polymer demonstrated sustained and controlled release of etanercept that reduced the inflammatory response suitable to promote islet survival.
EXPERIMENTAL METHODS
PLGA 85:15 (55 kDa) was functionalised with amino-functionalised lactobionic acid through amide bond formation in dimethyl sulfoxide for 16 h. One gram of galactosylated PLGA (Gal-PLGA) MPs encapsulated with 10 mg etanercept was fabricated using standard water-in-oil-in-water double emulsion method. In vitro functional drug release was measured using ELISA assay from aliquots of MPs suspended in PBS and gently rocked at 37 ºC for up to 7 days. Immunomodulatory response of MPs was performed using THP-1 differentiated macrophages. MPs were assessed in vivo following delivery via the portal vein.
RESULTS AND DISCUSSION
Gal-PLGA was synthesised to provide specific binding site to hepatocytes; MPs fabricated from Gal-PLGA provided an enhanced MPs retention (>85%, compared to 40% with conventional PLGA) in vivo. MPs fabricated with a mean size of 13 µm resulted in less hepatic necrosis when compared to larger particles. MPs exhibited a mean controlled daily release of 0.3 µg of protein per 1 mg of MPs between 2 and 7 days of in vitro release. Immunomodulatory response of MPs performed using macrophages suggest functional release of etanercept in the in vitro setting binds with TNF-α and potentially reduces inflammation observed by down-regulated pro-inflammatory genes (CXCL2, IL-1β) as well as reduced release of soluble inflammatory cytokines (TNF-α, IL-6). Preliminary investigation in vivo has shown no adverse safety concerns in mice.
CONCLUSIONS
We demonstrated a novel synthesis route for galactosylation of commercial PLGA and the subsequent fabrication of Gal-PLGA MPs as a novel protein carrier for targeted liver delivery with controlled functional release kinetics of etanercept. Macrophages have shown reduced inflammation co-culture with etanercept MPs in vitro. Preliminary in vivo study has displayed no adverse safety concerns post-injection.
ACKNOWLEDGMENTS
This project is funded by the UK Regenerative Medicine Platform 2 (UKRMP2) [MR/R015651/1].
"
73296368139
"Statement of Purpose: The development of a hydrogel that could be injected and cured in vivo has gained increasing attention. Collagen has been widely investigated as a thermogel in which there are a lot of ionic interaction, hydrophobic interaction, and hydrogen bonding; however, it must be chemically crosslinked also. Diels-Alder cycloaddition occur under mild conditions without the need for any catalyst, toxic solvent or external activation like UV-irradiation which makes it a pragmatic choice for biomedical application and in particular for developing in situ crosslinked injectable gels. Hence, taking into account the kinetic characteristics of the Diels–Alder reaction, hydrophobic diene-terminated collagen was synthesized and then injectability experiments were performed on fabricated gels to investigate its potential applications in minimally invasive surgery.
Methods: Diene-terminated collagen was prepared employing the nucleophilicity of the ε-amino group of the lysine and arginine side chain. Hydrogels were then fabricated by mixing modified collagen stock solution with PEG maleimide in which the final concentration of modified collagen in the gel was considered to be 2% (w/v) To evaluate the injectability/shear-thinning properties of the hydrogels, viscosity under continuous flow was measured with increasing the shear rate (from 0.01 to 100 s−1), using 20 mm stainless steel parallel plate geometry on hydrogels extruded directly on the rheometer plate from a syringe. Shear-thinning experiments were performed immediately, 4h and 48h after mixing all the hydrogel components. Furthermore, cardiac fetal stromal cells (CFSCs) were encapsulated in the gel network and cytocompatibility of the gels investigated with live/dead viability kit.
Results: Based on our experiments, all modified collagen gels were found to be extrudable (extrusion from a syringe) and injectable (extrusion through a 27G needle), whereas with increasing the shear rate, viscosity decreased. It should also be noted that linkage between diene and dienophile in Diels-alder reaction have dynamic nature. By increasing the temperature, the reaction rate of retro Diels-alder reaction increase, shifting the equilibrium towards the breaking of the reversible bonds. Additionally, instead of thermal energy, mechanical energy also makes the reversible polymer network becomes more dynamic, leading to retro Diels-alder reaction of cycloadducts into polymer chains. Our hypothesis is that injecting with needle act as a force-actuator, resulting in a mechanochemical coupling. From this point onward, the Diels-alder adduct should be coupled to the mechanical force which eventually trigger the retro Diels-alder reaction to release diene-terminated collagen and PEG-maleimide.
Conclusion: Given details mentioned above, the slow crosslinking of Diels-alder reaction and force-induced retro Diels-alder allows modified collagen gels to be injectable at least up to 48 hours post mixing of the gel components when stored at room temperature. Hence, this property is highly beneficial for potential clinical applications in terms of handling and administration given than the hydrogel could then be prepared prior to the surgery and then brought into the surgical room. Additionally, cell viability was higher than 80% for fabricated hydrogels for up to at least 7 days after cell encapsulation, suggesting that the engineered modified collagen gels had no cytotoxicity with CFSCs."
20941881909
Human liver tissue models are of great interest for toxicology analysis in drug development and as liver disease models. All solid tissues depend on sufficient oxygen supply for survival and on controllable oxygen tension for their proper function. Liver tissue models pose a particular challenge in oxygenation given the high oxygen consumption rate of hepatocytes and the well documented gradient in oxygen tension along the liver sinusoid. The oxygen gradient is thought to be key in establishing liver zonation that is required to mimic the in vivo compound metabolism. We have addressed this challenge of reproducing the vasculature function of liver tissue by microengineering massively parallel microfluidic 3D networks in materials open to oxygen- and nutrient-diffusion. The liver-like 3D tissue is cultured between the perfusion channels, thereby shielding the sensitive hepatocytes from shear stresses of the medium flow. In collaborations with cell biologists, the developed technology platform has been employed for the culture of primary human hepatocytes at in vivo-like cell densities for weeks with retained hepatic function as well as culture of human induced pluripotent stem cell-based liver-like cell tissues for months resulting in improved tissue maturation. Gradients in oxygen tensions naturally develop within the cultured tissue due to cellular oxygen consumption, and the cellular oxygen consumption rate depends on the changing local oxygen tension, which makes numerical modeling of the oxygen distribution within tissues highly uncertain without access to a ground truth for validation. We overcome this limitation by development of an optical non-contact method for mapping the actual oxygen concentration in 3D within tissues during culture. The method is based on initial co-seeding of tissue cells with oxygen sensing microbeads in the culture platforms and readout of the oxygen distribution using confocal phosphorescence lifetime microscopy (PLIM).
73387304968
Age-related macular degeneration (AMD) is a chronic eye disease and the leading cause of irreversible vision loss in millions of elderly people world-wide. The ocular system implicated in this disease is the outer blood-retinal barrier (oBRB) comprised of the retinal pigment epithelium (RPE), the Bruch’s membrane (BrM), and the choriocapillaris (CC). The oBRB plays a pivotal role in maintaining the eye homeostasis by regulating the transport of nutrients and metabolic wastes from choroid to the sub-retinal space. In AMD patients, several morphological and structural changes occur in the oBRB resulting in its disfunction and a failure of such homeostasis. At present, due to the multifactorial nature of the AMD, the exact disease pathogenesis remains poorly understood. As a result, although palliative cares exist for only some forms of AMD, no effective treatments exist. In order to investigate the pathophysiological process underlying AMD and to validate novel drug candidates, several in vivo and in vitro models have been proposed. However, none of these have proven to be reliable to mimic the complex cellular interactions in the oBRB with physiological realism and great predictive value. Therefore, herein we present a novel oBRB-on-a-chip model as a biomimetic platform for AMD understanding and for new therapeutic agents development. The device is a 3D microfluidic platform consisting of a biomimetic blood vessel network mimicking the CC and of a novel BrM-mimetic bio-membrane both housed within a single-chamber which resembles the intraocular space and enables the co-culture of human RPE and endothelial cells above the BrM and inside the CC respectively. The microfluidic network, designed starting from Optical Coherence Tomography (OCT) scans , was fabricated from polydimethylsiloxane (PDMS) through a novel manufacturing method established to provide a time-saving and cost-effective alternative to the common lithographic-based techniques. The interior surfaces of the microfluidic channels were subsequently coated with chemically crosslinked gelatin to promote cell adhesion and long-term culture. The engineered BrM was fabricated from chemically crosslinked gelatin by electrospinning process to get porous, ultrathin and nanofibrous membranes mimicking the mechanical, chemical and physical properties of the native substrate. The co-culture chamber with a common internal footprint with the wells in standard 24-well plates was fabricated from PDMS via demoulding process. Perfusion tests were successfully performed for validating the overall microfluidic platform. Human embryonic stem cell-derived RPE cells and HUVECs cells were cultured on the engineered BrM and on PDMS-gelatin substrates respectively to evaluate cells adhesion and proliferation under static conditions. Immunofluorescence microscopy demonstrated that engineered BrMs supported functional RPE monolayer formation, while HUVECs cells shown good adhesion and proliferation on the PDMS-gelatin substrates. Tests of dynamic seeding and static/dynamic co-culture of the cellular species involved are in progress to set out standard protocols. Taken together our findings are encouraging, showing that we successfully designed a physiologically relevant oBRB model to study AMD in vitro, in which patient-derived cells could be used for the identification new drugs paving the way towards personalized medicine.
62903407479
"Introduction
Gut microbiota is able to communicate with the brain through complex bidirectional routes constituting the so-called microbiota-gut-brain axis (MGBA). Gut microbial dysbiosis increases local inflammation also thanks to the secretion of bacterial lipopolysaccharides (LPS), which disturbs the gastrointestinal and also the blood-brain barrier (BBB) permeability thus propagating inflammation to the brain 1. A fluid-clearance pathway in the brain, the glymphatic system (GS), was recently discovered with increasing evidence of its role in neurodegeneration by controlling the clearance of neurotoxic proteins through convective exchange of flows 2. An in vitro model of GS in the scenario of neuroinflammation is missing. Here, we developed a GS model based on tunable levels of brain fluids clearance in a 3D brainlike environment, to be integrated in a MGBA engineered multiorgan-on-a-chip platform developed within the ERC project “MINERVA”, which aims at studying the intestinal microflora impact on brain functionality 3. The presence of a GS model in the brain unit of the multiorgan platform, combined with gut epithelial and BBB on-a-chip systems will allow to recapitulate LPS-mediated neuroinflammation in the presence of pathophysiological brain fluids drainage representing an innovative tool to study MGBA and GS role in dementia.
Methodology
The hydrogel based 3D brain model was engineered starting from a previously validated formulation 4.
Interstitial flow levels and molecules transport within the 3D brain model were predicted by multiphysics computational analysis and subsequently validated by measuring dextran molecules transport at different molecular weights (4, 40 and 70 kDa). The biological validation was performed by measuring cell viability for the cell models used to model the gut and BBB barriers as well as brain inflammation: intestinal epithelial cells (CaCo2); endothelial cells (bEND.3); astrocytes (C8D1A); neuroglioma cells (H4). Transepithelial Electrical Reistance (TEER) was measured to evaluate gut epithelial and BBB barriers integrity. Fluorescent LPS was tracked along the platform by fluorescence intensity detection. Interleukin 6 (IL-6) production by H4 cells at different fluid clearance levels was measured by ELISA.
Results
Computational analysis predicted flow velocities profile inside the 3D matrix and a physiological range of 1-10 uL/min was selected with clearance velocities corresponding to 0.14-1 um/s. Solutes clearance was validated with higher molecular wheight molecules being entrapped inside the hydrogel at low level of flows. The platform sustained the long-term culture of all cells models.
After LPS stimulation of the barrier models, TEER values decreased and IL-6 detectable levels inside the 3D brain model were measured. Preliminary results showed IL-6 production being modulated at different clearance flows levels suggesting an active role of fluid drainage in controlling neuroinflammatory response.
Conclusions
The integration of GS in a MGBA engineered multiorgan platform represents a suitable tool for modelling in vitro both physiological and pathological drainage of fluids inside the brain by recreating tuned GS interstitial flows within MGBA inflammation models.
References
"
20941827909
"Introduction
Organ-on-a-chip (OoC) technology shows great potential to accelerate drug discovery and advance personalized medicine. Induced pluripotent stem cells (iPSCs) may enhance the predictivity of the OoC in the assessment of patient’s response to a pharmacological treatment and related toxicities. Nowadays, there are no body-on-a-chip fully based on iPSCs used for drug screening. In this context, our PEGASO project aims to develop the first iPSC-based multiorgan-on-chip for Alzheimer’s disease drug development. The full platform will be composed of six OoC devices connected and loaded with iPSCs-derived models representing the main body systems involved in dementia drug pharmacokinetics (microbiota/gut, immune system, liver, blood-brain-barrier and brain tissue). Here, we present preliminary characterization of an in vitro 3D iPSCs-based liver model to be hosted into our innovative PEGASO OoC device.
Methodology
An in vitro 3D model with commercially available human iPSC-derived hepatocytes and endothelial cells has been chosen as a liver tissue model for the dynamic culture inside the PEGASO OoC, where hepatocytes encapsulated into a collagen-poly(ethylene)glycol hydrogel and endothelial cells have been cultured interconnected once seeded on each side of a porous membrane hosted into the OoC.
A 2D model with seeded membrane was used as control. Cell viability was assessed with MTS assay while albumin and urea produced by hepatocytes with ELISA and urea colorimetric assay respectively.
The mRNA expression level of CYP3A4 in hepatocytes was evaluated with RT-PCR while the protein expression of albumin and HNF4α with Western Blot and immunofluorescence.
The shear stress and oxygen concentration to which the cells are exposed under dynamic conditions have been assessed by a computational model developed with COMSOL Multiphysics ®.
Results
Hepatocytes grown inside the hydrogel 7 days after plating exhibited comparable metabolic activity with cells in 2D control condition. Differently, the liver-specific functions, referred to albumin and urea synthesis, resulted significantly higher in the 3D model with respect to the control.
The protein expression of albumin and HNF4α, that are key hepatocytes markers, analysed with Western Blot and immunofluorescence was increased in the 3D condition.
The expression of CYP3A4 indicated a higher detoxification ability by hepatocytes in 3D condition with respect to the control.
To select the optimal flow rate for the dynamic culture of the iPSC-derived liver in our organ-on-a-chip, a computational simulation was performed with the software COMSOL Multiphysics ®, tailoring the model for hepatocytes specific requirements, that are shear stresses <0.2 Pa and oxygen consumption of 0.3 nmol/s*m3[1,2].
The numerical simulation indicated 30 µl/min as the proper medium flow rate, leading to adequate shear stresses (range 0.01-0.03 mPa) and oxygen concentrations (range 0.18-0.2 mol/m3).
Conclusions
Taken together, the biological and computational results suggest that our 3D liver model is a suitable iPSC-derived model to be hosted and cultured under perfusion in the PEGASO OoC.
Acknowledgments
PEGASO received funding from the Ministero dell’Istruzione, dell’Università e della Ricerca (MIUR) under the FARE 2019 program (project code R18WWPCXLY).
References
1) MA, L. D, et al. Lab Chip, 18, 2547-2562 (2018).
2) Kang, Y. B. Biotechnol Bioeng, 112, 2571–2582 (2015)."
31412716105
"INTRODUCTION
Rheumatoid arthritis (RA) is an autoimmune disease that affects diarthrodial joints, characterized by a systemic inflammatory response that leads to progressive joint destruction [1]. Various immune cells are involved, but the exact mechanism of RA development is still unknown, and it is well established that none of the currently available animal models fully represent human RA onset and progression [2]. In this scenario, organs-on-chip provide a valuable solution to evaluate the involvement and the interaction of different cell types in RA conditions. To this aim, we developed a novel microfluidic platform allowing to study the cross-talk between immune cells involved in RA.
METHODOLOGY
The platform consists of two separate culture areas, whose communication is controlled through normally closed valves. The first compartment is intended to host cells seeded in a 3D matrix, e.g. macrophages, while the second one is composed of a single channel designed to precisely locate and culture cells in suspension, e.g. T cells. The second compartment encompasses a novel technology, named “sieving valve”, which relies on normally closed valves with underneath microfluidic channels that allow perfusion of fluids but impair cell escaping when closed. CD14+ macrophages and CD4+ T cells isolated from human buffy coat were independently seeded in the two separate compartments and stimulated with TNFa and IL15, respectively. Upon central valve opening, recruitment of CXCR6+ T cells operated by inflamed macrophages expressing CXCL16 was evaluated [3].
RESULTS
A protocol was first optimized to seed and trap CD4+ T cells inside the microfluidic platform, taking advantage of the sieving valve technology. Cells were then successfully stimulated inside the platform exploiting the separation of the compartments. As proven by immunofluorescence staining, CXCL16 expression was enhanced in macrophages after treatment with TNF-α, while CXCR6 expression was up-regulated in T-cells after stimulation with IL-15. After stimulation, migration of T-cells towards macrophages occurred spontaneously upon opening of the communication valves, as quantified through live imaging.
CONCLUSIONS
The proposed device offers an innovative solution to trap immune cells inside microfluidic chips and to study the cross-talk between different cell types, having the possibility to stimulate them separately. The platform was validated replicating a known mechanism in RA, involving resident macrophages and T cells [3]. The use of the platform to elucidate the role of RA-patient specific circulating immune cells on synovial membrane is currently under evaluation, and will eventually increase the understanding about unknown mechanisms in RA progression. Moreover, the technology is highly versatile and can be potentially applied to assess the interaction of immune system in manifold diseases and more complex models.
REFERENCES
1. Lee et al, Lancet, 358, 903-911 (2001)
2. Paggi et al, Nat Rev Rheumatol (2022)
3. Tu et al, Front Immunol, 12, 1-8 (2021)
ACKNOWLEDGEMEBNTS
We thank Dr. Carlotta Catozzi for her technical help in the experiments. This work was supported by Fondazione Cariplo-uKNEEque - Rif. 2018/0551 and has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement No 841975."
52354536639
"Breathing exposes lung cells to continual mechanical stimuli, which is part of the microenvironmental signals directing cellular functions. Therefore, developing in vitro model systems that incorporate physiomimetic mechanical stimuli is urgent to fully understand cell behavior. This study aims to introduce a novel in vitro culture methodology combining mechanical stimuli that simulates in vivo breathing in 3D cell culture platforms in the form of recellularized human lung extracellular matrix (ECM) scaffolds and precision cut lung slices (PCLS) from rats.
To this end, we have constructed a device for controlled cyclic stretch, mimicking amplitudes and frequencies of distensions seen in the breathing human lung. For its validation, we cultured H441 lung epithelial cells in decellularized human lung slices exposed to 16 stretch cycles per minute with a 10% stretch amplitude. Cell viability (resazurin reduction), proliferation (Ki-67) and YAP1 activation were evaluated at 24 and 96 hours by immunohistochemistry, while the expression of SFTPB, COL3A1, COL4A3 and LAMA5 was evaluated by qPCR.
Cyclic stretch induced an increase in SFTPB expression after 24 hours without a concomitant increase in the stretch responsive gene YAP1. The ECM milieu lowered the expression of the basement membrane protein genes COL4A3 and LAMA5 compared to tissue culture plastic control cultures, without any additional effect induced by the mechanical stimuli. Additionally, we show compatibility of the device with PCLS culture showing preserved morphology and metabolism in rat PCLS after 72 hours of mechanical stretch. Thus, we present a novel device and methodology for the study of lung tissue slice cultures subjected to physiomimetic mechanical stimuli, which shows promise for future studies of cell and tissue function in a lung ECM milieu with physiological or pathological mechanical stimuli."
73296325005
Multicellular tissues such as spheroids, organoids can be assembled in vitro from clusters of cells in a spatio-temporal manner, mimicking in vivo physiology and tissue microenvironment, which can leverage pre-clinical evaluation of the efficacy and safety of potential new drugs for diseases such as hereditary, neurodevelopmental, infectious and cancer. In one approach, immortal, primary or differentiated pluripotent cells are seeded in three-dimensional (3D) constructs leading to assembled multicellular structures, whereas the other approach involves the self-organization of cells that are subjected to a variety of external stimuli such as physical, chemical, mechanical and electrical. One of the greatest challenges is the regulation of spatio-temporal organization and/or differentiation of cells within a 3D microenvironment. In this study, the impact of microgravity in self-assembly of cells was evaluated in a bioengineered system that provided controlled dynamic flow conditions, supported growth factor diffusion, metabolite exchange in larger sizes and survival. The formation of multicellular structures was not only investigated on cellular level including morphology, proliferation, adhesion but also on functional level in terms of recapitulating the physiology. Reproducible scaling of these engineered spheroids and organoids in consecutive batches will allow high-throughput screening of emerging therapeutics and combinational therapies as preclinical models at an industrial scale, which is envisaged to accelerate in the near future.
31412745855
TBA
"Introduction: The acquisition of neural signals from the brain cortex has always been of high relevance in the field of neuroscience in order to analyze and interpret brain processes as well as individuate neurological diseases and disorders [1]. Nowadays, the design of a neural interface conformable with the brain tissue is one of the major challenges, since the inadequate conformability might lead to inaccurate signal recording and potential misdiagnosis [2].
Methodology: In this research, we design and produce a soft neural interface composed of polyacrylamide hydrogel loaded with plasmonic silver nanocubes to provide the system with good electrical properties. The hydrogel nanocomposites are surrounded by a template of two layers of silicon-based materials (i.e. polydimethylsiloxane and soft skin adhesive) as supporting elements for guaranteeing a tight and stable neural-hydrogel contact, while allowing a stable recording from specific locations of the brain cortex.
Results: The morphological, chemical, electrical, and mechanical properties of the platform are evaluated. The hydrogel nanocomposites show superior conductivity properties, while mimicking the brain tissue mechanical characteristics. Furthermore, in vitro biological tests performed by seeding neural progenitor cells reveal the biocompatibility of the hydrogel-based system as well as neural differentiation and proliferation. In vivo experiments on a mouse model demonstrate that the hydrogel nanocomposite-based neural interface permits the efficient recording of neural signals with augmented amplitude. Additionally, chronic neuroinflammation tests reveal no adverse response towards the proposed platform.
Conclusions: The biocompatible conductive hydrogel nanocomposite-based device is a promising candidate as neural interface for brain signal acquisition without provoking neuroinflammation. The potential exploitation of the proposed conductive hydrogel platform in electronic devices for Electrophysiological Recording of Electrocorticographic or Electroencephalography Recording will be investigated in the near future.
Acknowledgments
This study was supported by the First TEAM grant number POIR.04.04.00-00-5ED7/18-00, which is conducted within the framework of the First TEAM programme of the Foundation for Polish Science (FNP) and co-financed by the European Union under the European Regional development Fund.
References
[1] Nam, J. et al., ACS Nano 14, 664−675, (2020).
[2] Rinoldi, C. et al., Biomacromolecules 22, 3084-3098, (2021)."
31412714088
Background: intrahepatic islet transplantation in patients with T1D is limited by donor availability and lack of engraftment. To overcome these limitations, based on our experience with decellularized rat lung as scaffold for the generation of Vascularized Islet Organ (VIO, lung scaffold repopulated by murine islets and HUVEC cells), we engineered an upgrade based on human blood-derived endothelial cells (BOECs - Blood Outgrowing Endothelial Cells) and immature neonatal porcine islet clusters (NPIs). Methods: NPIs and BOECs phenotype profile was assessed by flow cytometry, insulin secretion test and tube formation assay. Rat lung was decellularized with SDS and Triton and seeded with NPIs and BOECs, generating a Vascularized Endocrine Pancreas (VEP). VEP was cultured for 7 days in a customized bioreactor specifically designed to allow cell integration. The β cell death in mature VEPs was estimated during ex vivo organ maturation evaluating miR-375 expression ddPCR compared to batch matched NPIs in standard condition. Matured VEPs and control NPIs function were measured by dynamic glucose perifusion and insulin quantification (by ELISA/IF). Thus, VEPs were subcutaneously transplanted in diabetic immunodeficient NSG recipients and compared with matched NPIs transplanted in different implantation sites: kidney capsule (KC-NPIs), deviceless (DL-NPIs) and liver (LV-NPIs). Results: Matured VEPs showed a regenerated vascular network (CD31+) with NPIs (insulin+) integrated. miR-375 was expressed in NPIs but not in BOECs, as expected. VEP was able to significantly reduce β cell death (p<0.05). Matured VEPs were able to sustain NPIs engraftment, survival and significantly improve insulin secretion during the maturation process compared to batch matched NPIs cultured in standard conditions (AUC VEPs first phase: 3.765±0.90; NIPs 1.60±0.25 p<0.01). In long-term transplants in diabetic mice, VEPs demonstrated a significant NPIs engraftment with a prompt function after implantation and the reversal of the glycaemia within 2 days until 60 days after implantation, showing significant superior function compared to all the internal controls (KC-NPIs, DL-and LV-NPIs). Conclusions: VEP technology is able not only to foster the NPIs functional endocrine maturation in vitro but also to immediately perform in vivo upon transplantation for over 2 months, compared to normal performance within 8 weeks after implantation in different state of the art preclinical models. Given recent progress in genetic engineering of NPI donor pigs, this technology may enable assembly of immune-protected functional personalized endocrine organs. VEP is the first organ to our knowledge assembled with relevant source of endocrine and endothelial cells suitable for future clinical translation.
62825415955
"Four-dimensional (4D) bioprinting (i.e., fabrication via additive manufacturing of scaffolds characterized by a programmed change, over time, under a predefined stimulus [1]) can be exploited to produce active scaffolds that can modify their shapes upon desired stimulation, thus potentially recapitulating biological processes such as morphogenesis.
In this study, we exploited the 4D bioprinting approach to design and fabricate an innovative smart scaffold for in vitro modeling the development of the neural tube (NT), the structure from which the central nervous system stems in the embryo, with the final aim to guide stem cells towards neural differentiation. The smart scaffold is able to self-fold in time, mimicking the neural plate folding to create a hollow tube, namely the NT [2].
The requested behavior is achieved exploiting the differential swelling properties of bilayer films [3]. Indeed, the different volumetric swelling of the two layers, when dipped in water, creates a deformation mismatch in the film that leads the folding of the film itself. In this study, the two layers were made of the same bulk material (i.e., gelatin crosslinked using (3-Glycidoxypropyl)methyldiethoxysilane, GPTMS-GEL), thus guaranteeing a chemical bond between the layers and avoiding delamination. The swelling behavior of the layers was tuned through the modification of the GPTMS and GEL concentrations.
GPTMS-GEL-1 monolayer films, with the higher volumetric swelling, were fabricated by solvent casting. Then, lines of GPTMS-GEL-2, with lower volumetric swelling, were deposited on the GPTMS-GEL-1 film by Extrusion-Based Bioprinting. The presence of precisely oriented lines (as second layer of the bilayer film) provides a constrain and, as a consequence, a complete control over the film folding direction.
When dipped in water the film self-folds, maintaining its shape in time and, as expected, the orientation of the folding depends on the printed line direction.
Cellular tests have been performed to verify the properties of the smart scaffold, using human induced pluripotent stem cells (iPSCs) directed toward neural progenitor fate via Dual SMAD inhibition. iPSC-derived neural progenitor population uniquely recapitulates in vitro the onset of the founder population of the developing NT. Indeed, the simultaneous application of these cellular and bio-engineering technologies will provide a platform to assess complex phenomena such as NT folding and cellular polarization in a dynamic 4D environment. This pioneering platform will provide an innovative standpoint to unravel neural tube defects and their clinical impact.
[1] Agarwal T., et al. (2021) J. Mat. Chem B, 9,7608-7632.
[2] Vieira, C., et al. (2009) Int. J. Dev. Biol. 54.1,7-20.
[3] Kim, S. H., et al. (2020) Biomaterials, 260,120281"
94238144919
"Micro- and nanofibrous materials have attracted the interest of the scientific community for the better part of ~30 years, due to their unique properties and potential for applications. Electrospinning is a technique that can produce micro- and nanofibers fast, repeatedly, and with tailored morphology. When fibres are electrospun from biocompatible polymers then they can be used in numerous medical applications such as tissue engineering, regenerative medicine, implant coatings, medical textiles, etc. This academic focus has slowly translated into a number of medical devices containing electrospun biomaterials becoming clinically available. The particular area of interest for this presentation is the use, substitution, and improvement that electrospun textiles offer in the field of medical textiles as metal stent coatings.
A brief overview of the state-of-the-art of woven textiles as well as in use in metal stents will be given, as well as a summary of existing electrospun metal stent coverings available now or in clinical trials.
At The Electrospinning Company Ltd we design, develop and manufacture advanced micro- and nanofibrous materials. These biocompatible materials can be used in a range of different biomedical applications, including wound care, cell therapy, and cardiovascular devices. Currently our materials are being used in at least four FDA-approved medical devices that are in the clinical trial stage.
Together with our partners we are developing different types of electrospun textiles for various metal stent applications. We will present data on direct comparisons between traditional textiles and electrospun textiles and their performance in different metal stent applications, such as EVAR, TEVAR, occluders, and covered vascular stents. Examples of the parameters discussed are diffusion across the coverings, blocking of particles, as well as adhesion to the metal frame.
As a centre of excellence in electrospinning biocompatible materials, we have an in-depth understanding on the relationship between material characteristics, like fibre morphology and architecture, and their suitability for specific applications. Therefore, it is paramount to ensure that these characteristics remain stable while the adhesion of the fibres on the different substrates is enhanced."
83767241505
"Bone tissue regeneration (BTR) has been trying to mimic the bone environment in biofabricated platforms. Given the complex bone metabolism, creating a functional, differentiated and biologically compatible platform that stimulates tissue formation in an autonomous way is currently a challenge [1]. Therefore, the development of multifunctional micro-platforms easy to produce and to translate into the clinic while promoting BTR is a top priority. In this work, we boosted a recently developed platform fabricated in combination with metal coordination and gelling properties of gelatin [2], providing a suitable microenvironment for applications in BTR.
Using gelatin modified with two different catechol analogues (Hydroxypiridinone-HOPO, and Dopamine-DA) as building blocks on the creation of liquefied protein-based microcapsules (mCap), we encapsulated bone-marrow human mesenchymal stem cells (BM-hMSC) using the electrospray technique. The presence of HOPO allowed the formation of the micro-hydrogel shell through metal coordination, while DA was inserted by its affinity for calcium ions allowing the mineralization of the system by deposition of calcium-phosphate crystals (e.g. hydroxyapatite-HA) [3].
With this strategy, we created the desired microenvironment for mineralization and osteogenesis without the demand of osteogenic inducers. With BM-hMSCs organizing themselves inside the inner wall of the gelatin shell, mCap created an encouraging environment for cell communication and differentiation. The inclusion of DA into the system prompted accelerates the differentiation process, with osteoblastic stages being reached in early periods of culture. The prompt differentiation into osteoblasts might be related to the bioactive properties of the mCap, autonomously promoting the deposition of HA crystal. A more detailed analysis revealed the formation of a dense mantle in the interior of this enclosed system, exposing matrix deposition covering osteoblasts. The fact that osteoblast can merge within the new bone matrix suggests that this micro-platform can potentiate the formation of osteocytes, supporting this as a suitable biomimetic platform to closely resemble bone morphology. Therefore, the achieved bone-like microcapsules are a promising bioengineering platform that induce autonomously and in a fast way osteogenesis. By recreating part of the bone cellular microenvironment and architecture, this platform can be explored to bioengineering more closely the bone niche, addressing a variety of bone defects.
"
52354566648
"Introduction
Tissue growth in defects is controlled by the local curvature of the substrate and is traditionally regarded to follow the same process of cell organization from large to small defects [1]. At defect dimensions of 100s of µms to a few mms, tissue growth typically follows a centripetal process driven by an inner ring composed of contractile and proliferative cells. In contrast, the closure of very small defects with a diameter of 10s of µms, cell protrusions seem to contribute to defect closure. Here, we investigated how curvature drives cells from the previously described layer-by-layer tissue growth into defect closure and we reveal a curvature-driven “cell suturing” process that is most pronounced in stromal cells.
Methodology
The response of different cell types (i.e., fibroblasts, mesenchymal stromal cells, osteoblasts, pre-osteoblasts and endothelial cells) to surface curvature was characterized using micro-engineered cell culture substrates featuring half-cylindrical environments with controlled curvature variation. Collective cellular self-organization and gap closure was analyzed inside well-defined 3D cylindrical pores as well as in a biomaterial environment featuring channel-like pores with more stochastic geometrical conditions. Lastly, micro-wounds were introduced in an in vitro tissue growth setup to observe the transition between different modes of cell organization during wound healing.
Results
Cellular response to curvature is dependent on the cell type and degree of curvature. While on large curvatures (i.e., diameter > 300 µm) stromal cells prefer to align following the direction of higher curvature, as the curvature increased (i.e., diameter < 300 µm), cells adapt to curvature via two well-differentiated mechanisms: alignment towards the minimum curvature or lifting from the substrate along the maximum curvature. The occurrence of cell lifting was correlated with the distribution of the focal adhesions around the cell and can be regulated by the cytoskeletal stress state or the stiffness of the substrate. Cells without lifting capability lead to a process of centripetal gap closure. However, cellular processes involving cell lifting induced a significantly faster suture-like wound healing mechanism compared to the previously described centripetal gap closure.
Conclusion
Fundamental differences were found in how distinct cell types respond to curvature within gaps of few 100s of micrometers. In living tissues, such gaps could represent meso-scale defects occurring i.a., after tissue delamination consequence of mechanical overloading or injury, but such micro-defects may also be engineered in synthetic porous materials. Based on our findings, cells may be classified into types capable to close micro-defects through an extremely efficient suture-like process and cell types that favor a layer-by-layer gap filling associated with a circular lumen shape and slower defect closure. Addressing the fast gap closure resulting from the cell suturing-mode in biomaterial strategies is regarded advantageous for material-driven tissue healing and regeneration. This can be achieved by implementing appropriate curvatures into porous materials that provoke cell suturing for the individual cell type of interest [2].
References
83767269288
"Introduction: One of the most challenging and daunting task of tissue engineering is the assembly and integration of functional microvasculature systems within the biofabricated tissue equivalents. Having functional vasculature is of the utmost importance to promote the rapid integration of the host’s microvasculature with the engineered one present in the graft, thus enabling an efficient transport of nutrients/removal of wastes to/from the graft. To date, a plethora of strategies have been explored to build such vasculature networks and, in the last decade great progresses have been achieved. In particular, it has been demonstrated that one can generate a vascular network via vasculogenesis – the de novo assembly of endothelial progenitor cells into capillaries – using microfabrication approaches such as microfluidic technology, 3D co-culture models (spheroids and organoids), and biofabrication via 3D bioprinting strategies. However, these milestones are still unsatisfactory and, to date, efficient protocols for the manufacturing of vascularized artificial tissues are still missing.
Methodology: To integrate a functional microvasculature within skeletal muscle tissue, we have developed a microfluidic-assisted 3D co-axial wet-spinning strategy. This technique allows to biofabricate core-shell fibres that were deposited on a rotating drum. Such fibers were composed of alginate (shell) and fibrinogen (core, bioink). Within the bioink, we loaded skeletal muscle precursors (C2C12) and gelatin methacrylate (GelMA) microbeads coated with endothelial cells (EC-GelMA) that acted as endothelium micro-seed units. Such beads were massively produced using a millipede step-emulsification microfluidic device.
Results: Applying the correct bioinks formulations, viable vascularized constructs were obtained, and vasculature micro-seeds supported the formation of a capillary-like network Interestingly, we have noticed that by increasing the core stiffness in the constructs, the endothelial cells have been able to generate vessels with different calibers switching from larger (150 microns) to smaller (50 microns) capillaries.
Conclusions: Our results have shown that: i) 3D co-axial wet-spinning is a valuable strategy to biofabricate and integrate in vitro micro-vessels within a secondary tissue; ii) the stiffness of the matrix microenvironment plays a key role over the fate of endothelial cells influencing the size/number – i.e. architecture - of the resulting microvascular network; iii) Micro-vascular seeds are an interesting solutions for engineering microvascular network, easy to manufacture and use in combination (e.g. by simply resuspension in a bioink) with other biofabrication strategies."
20941831055
3D culture and organoid technologies have been developing rapidly in the last decade, and already found widespread applications in biology and medicine. While cells, and stem cells in particular, have tremendous self-organization potential, most applications benefit from further engineering of the cellular microenvironment in order to guide the morphogenesis.
We will describe several recently developed technologies that exploit dynamic physical processes to support or guide morphogenesis in defined 3D cultures. In one instance, we formed hydrogels with reversible, dynamic bonds, in order to impart polyethylene glycol (PEG) hydrogels with stress relaxation and self-healing properties. The resulting hydrogels could support intestinal stem cell expansion as well as differentiation to budding organoids in a single defined hydrogel. In another instance, we exploited a dynamic aqueous-aqueous phase separation triggered by PEG cross-linking in order to gain control over the pore structure of the PEG gels. The method is simple, cost-effective, compatible with injection and cell encapsulation, yields clear hydrogels, and easily tuned to obtain pore sizes of any relevant size, from less than 1 to more than 100 micrometers. These hydrogels proved optimal to support the formation of functional 3D neural networks in a defined environment. Finally, we will show how morphogenesis can further be guided with dynamic patterning of morphogens / growth factors, using a recently developed two-photon patterning method, compatible with sensitive biomolecules, live cells, and both natural and synthetic extracellular matrices.
We believe such tailored microenvironments, exploiting dynamic processes to guide morphogenesis, will be key to the development of 3D engineered tissues with a higher degree of cellular organization.
20967804648
Growth factor (GF) based therapies in regenerative medicine are limited by the high cost, fast degradation kinetics, and lack of specificity as consequence of the multiple functions of GF in the cell. One common GF therapeutic scenario is the administration of VEGF to support vascularization during tissue regeneration. The therapeutic window for VEGF treatment is narrow: low doses are safe but not sufficient to yield a therapeutic benefit, and slightly higher doses lead to the growth of angioma-like vascular structures. Moreover, angiogenesis in vivo occurs by sustained angiogenic stimulus over a month to achieve stable vessels, and the outcome of the process is highly dependent on the spatiotemporal distribution of the proangiogenic signal. In this talk different approaches to control the presentation of angiogenic signals in biomaterials for tissue regeneration using phototriggers will be presented.
Introduction:
Recently, tissue engineering still lacks thorough vasculature, which represents a major drawback in developing physiologically relevant tissue constructs. Among others, the design and the biofabrication of blood vessels at the microscale remain challenging, due to their role in nutrient and oxygen exchange, but also waste removal. Parallelly, fiber-based biofabrication techniques such as 3D-(bio)printing are considered time-consuming approaches in the field of microvascular tissue engineering. In this work, vessel-like 3D core-shell bundles have been rapidly fabricated using a novel wet-spinning system, allowing for the collection of cell-laden hydrogel-based fibers onto a rotating drum to reproduce the native architecture of the microvascular network.
Methodology:
Therefore, two different hydrogel formulations were optimized. First, a fibrinogen- and an alginate-based solutions were studied in terms of material characterization. Rheological measurements on the pre-polymer solutions and swelling test on the fibrous scaffolds were carried out to investigate the behavior of these biomaterials. Then, SEM analysis was assessed to evaluate the microstructure of the core-shell yarns. Afterwards, the fibrinogen-based formulation loaded with a co-culture of human umbilical vein endothelial cells (HUVECs) and mesenchymal stem cells (MSCs), and the alginate biomaterial ink were simultaneously extruded from a microfluidic co-axial nozzle immersed in a CaCl2 coagulation bath to produce tissue-specific core and supporting tubular shell structures, respectively. Both motor speed and flow rates were adjusted and tuned to create fibers with a diameter dimension of around 300 μm. Upon instantaneous gelation of alginate, wet-spun fibers were collected to form densely packed fascicles. To further stabilize the bundles, a secondary crosslinking was performed by immersing the yarns in a thrombin solution to induce the enzymatic polymerization of the core. Thus, the engineered constructs were incubated at cell culture conditions for up to 21 days. The metabolic activity of encapsulated cells was evaluated by means of proliferation assay. Subsequently, cell-laden scaffolds were investigated in terms of morphological characterization. Finally, immunocytochemistry will be performed to prove the formation of vessel-like structures.
Results:
The material characterization of the proposed formulations exhibited a Newtonian-like behavior, proving the suitability of non-shear thinning hydrogel-based bioinks for wet-spinning. Tissue-specific wet-spun core-shell fibers supported cell adhesion, migration, and alignment over the culture time, generating packed 3D cell-laden constructs that may recapitulate the microvascular network. The proliferation assay confirmed consistent metabolic activity during the cell-culture period. In addition, scaffolds would likely reveal their endothelialization role, highlighting the potential of the two proposed bioinks in the frame of microvascular tissue engineering.
Conclusions:
In conclusion, wet-spun fibers were produced from a novel co-axial needle and collected by using functionalized hydrogels, thus validating the system for the (bio)fabrication of Newtonian-like hydrogel-based constructs. Herein, encapsulated MSC-HUVEC migrated within the cell-laden hydrogel core towards the wall of the alginate shell, thus aligning along the direction of the microfibers axis to form a cellular layer. This study aims to highlight a new model to promote microvascular networks. The proposed wet-spinning platform can be considered as a potential alternative to 3D-(bio)printed engineered microvascularized constructs.
52354546605
Myotendinous junction disfunctions due to degenerative musculoskeletal diseases or injuries resulting from strenuous physical activities are still considered an ongoing issue in the field of musculoskeletal tissue engineering. Indeed, the main challenge of biofabrication strategies relies on the development of methods enabling the generation of artificial bio-constructs that can replicate the complexity of the muscle-tendon tissue interface. In this work, a core-shell Y-shaped microfluidic chip has been developed to alternatively deliver two different hydrogel-based bioinks that enable the mimicking of the tendon and the muscle tissue, respectively. As a result, core-shell microfibers are extruded and collected on a rotating drum to form heterogenous hydrogel yarns. In order to fabricate a myotendinous-like construct, the time-switching between the two bioinks, the drum rotational speed, and the core and shell flow rate, have been optimized. Once flow rates have been selected, different values of drum rotational speeds (i.e., 20, 30, 40, 50 and 60 rpm) have been tested to evaluate the overall effect on both the microfiber diameter and the core dimension. Moreover, NIH 3T3 fibroblasts and C2C12 myoblasts, used to mimic the tendon and muscle side respectively, were encapsulated in the core bioink and spun at the selected rotational speed values. As a result, the effect of the increasing velocity induced a decreasing of the microfiber and core diameter along with the shell thickness. Furthermore, rotational speeds up to 40 rpm showed high viability and cell alignment along the fiber direction for both NIH 3T3 fibroblasts and C2C12 myoblasts. On the other hand, rotational speeds of 20 rpm and 30 rpm induced low cell alignment and spreading along with a low cell viability, due to the increasing of shell diameter that prevents the oxygen and nutrient exchange. In addition, it was observed that rotational speeds up to 40 rpm were related with higher switching-time that create flow rate turbulences and a reduced ratio of muscle/tendon tissue-only section compared to the tissue interface one. Hence, 40 rpm has been selected as optimal rotational speed velocity for the fabrication of myotendinous-like constructs. Cell-laden heterogenous scaffolds showed high degree of compartmentalization and enabled the recreation of the tissue-specific biological heterogeneity. Moreover, C2C12/NIH 3T3-laden constructs showed high cell proliferation up to 14 days of culture. Finally, immunochemistry analysis will be performed in order to investigate myosin heavy chain as well as collagen I and III expression at the muscle and tendon side, respectively. Thus, such biofabrication method could be validated for the generation of a biomimetic heterogenous scaffold that can recapitulate the biological complexity of the muscle tendon unit.
31412746655
"Introduction: Articular cartilage (AC) defects remain a significant clinical challenge[1]. This is partially due to the challenging nature of recapitulating the complex layered structure observed in the naturally curved AC tissue. While three-dimensional (3D) bioprinting appeared as a promising Tissue Engineering (TE) approach, it has serious limitations in the fabrication of curved constructs[2]. This has motivated the development of four-dimensional (4D) bioprinting as the next generation of biofabrication technologies, combining 3D-bioprinting with time-dependent shape transformation, and introducing time as the fourth dimension[3]. 4D-bioprinting allows for the fabrication of self-bending scaffolds and shape-transforming constructs. In this study, we report an advanced 4D-biofabrication method based on the differential swelling of a multi-material smart bioink.
Methods: Two biomaterial ink formulations with different swelling properties were selected: tyramine-functionalized hyaluronan (HAT, high-swelling) and alginate with HAT (AHAT, low-swelling). Firstly, the inks were characterized with an MCR-501 rheometer (AntonPaar) to measure their storage/elastic modulus, loss/viscous modulus, shear-thinning, and viscosity. BioX-bioprinter (Cellink) was used to fabricate a bilayered scaffold. The bottom zone was made of HAT and the top zone of AHAT. After printing, the bilayered scaffold was crosslinked in 200 mM CaCl2, and then submerged in saline or DMEM medium. Finally, human bone-marrow derived cells (hMSC) were incorporated into AHAT (top zone) before 4D-bioprinting the bilayered scaffolds. The scaffolds, cultured in chondrogenic medium for 28 days, were analyzed by live/dead and histology.
Results: Rheological characterization demonstrated that both HAT and AHAT inks had i) similar elastic, gel-like behaviors, as their elastic modulus was 8x higher than their viscous modulus; ii) shear-thinning behavior, and iii) relatively fast recovery reaching 100% and 65% (respectively) of the storage modulus. After 3D printing, AHAT showed a higher compression modulus than HAT (6.7 vs. 2.1 kPa). Upon 24 h submersion in saline HAT absorbed 2x more liquid than AHAT. The inks were 3D printed into a bilayer. After time (4D), the differential swelling between the two zones led to the scaffold’s self-bending behavior. Different scaffold designs were used to characterize the degree of curvature. The live/dead results demonstrated high cell-viability in the 4D-bioprinted scaffolds. After 28 days, the curvature was still evident, with no delamination observed, and histology suggested an increase in sGAG production.
Discussion and conclusion: A proof of concept of the recently emerged technology of 4D-bioprinting with a specific application for articular cartilage tissue engineering was achieved. We fabricated smart cell-laden scaffolds with self-bending properties for the design of curved structures mimicking the native AC tissue architecture in specific regions. This approach allowed for the fabrication of a curved bilayer made from two biocompatible and commonly used hydrogel-based materials in TE. Further studies should focus on increasing the mechanical properties of the scaffold, as well as improving the tissue formation through incorporation of tissue-specific biological cues.
Acknowledgments: Dutch Medical Delta (RegMed4D) and TUD-EMC Convergence Initiative Health & Technology.
References: [1]DeNiese P.J. et al, Arthrosc Surg Sport Med 1, 16, 2020. [2]Giannopoulos A. et al, Nat rev, 13, 701, 2016. [3]An J. et al, Int J Biop, 2, 3, 2016."
31412704686
"Introduction: Bioprinting is a booming and promising technology to create tissue models, with numerous applications in tissue engineering and regenerative medicine. However, the biomaterials commonly used for bioprinting involve non-physiological stimuli (e.g., sudden changes in temperature, pH, ionic forces) and lack tunability post-printing. These biomaterials are therefore still far from recapitulating the physicochemical and biological characteristics required to create relevant in vitro tissue models. To date, a biologically relevant, ultra-tunable, fast- and easy-to-use bioink platform remains to be invented.
Methodology: We envisioned to create a novel bioink platform with tunable composition and mechanical properties post-printing. To succeed, we hypothesized that a dynamic covalent hydrogel, which can flow, can be modified with a reactive moiety so that mechanical and biochemical properties can be adjusted after printing upon the simple addition of a complementary reactive molecule to the culture medium. Using hyaluronic acid (HA) as a natural polymer of interest, boronate ester crosslinking was investigated for the design of the platform dynamic covalent bioink. Regarding the secondary chemical reaction, we used a « click » reaction, namely the strain-promoted azide-alkyne cycloaddition (SPAAC), able to meet rigorous criteria (i.e., physiological conditions of pH and temperature, no byproducts, no purification, bioorthogonality) and allow post-printing modifications in the presence of cells. By combining these two chemical tools, we successfully created what we called « clickable dynamic bioinks ». We then investigated the feasibility of various post-printing modifications (e.g., stiffening, peptide addition) to drive cell fate and build biologically relevant tissue models.
Results: We demonstrated for the first time that boronate ester crosslinking can be used for the design of printable hydrogels, with non-swelling/non-shrinking, shear-thinning and self-healing properties, tunable viscoelasticity (G’ of 200 to 2500 Pa, at 1 Hz), and in vitro stability over months. We showed that these hydrogels are cytocompatible (>90% viable cells) with various primary human cell types (e.g., MSCs, chondrocytes), and that they can prevent cell sedimentation in a cartridge, circumventing what is a common issue in bioprinting. These new bioinks allowed us to design constructs of various shapes and volumes (tested up to 10 layers). The 3D bioprinted constructs immersed in culture medium can be tuned by simply adding to the medium the SPAAC-modified molecule of interest, which diffuses in the constructs and react with the dynamic network. We showed that the composition of a bioprinted construct can be tuned by adding chondroitin sulfate, low molecular weight HA, gelatin or an adhesive peptide (RGD). This technique also allowed us to increase the rigidity of a construct (G’ increased from 200 to 1200 Pa) or control cell adhesion. Of major value, we demonstrated that these post-printing modifications can be controlled in time and space.
Conclusions: We showed that clickable dynamic bioinks constitute a simple and versatile platform for bioprinting. It carries the hope of easy, fast and cost-effective access to any kind of tissue with adaptable composition and architecture, paving the way to biologically relevant 4D bioprinting, with virtually unlimited tissue engineering applications."
31412715309
"Introduction
Short peptide amphiphiles have been widely reported as building blocks of supramolecular hydrogels for biomedical applications1,2, as they can copycat bioactive protein sequences. However, in the extracellular matrix (ECM), proteins are usually present as glycoproteins with different roles, e.g., storage depots of proteins and co-receptors. In this context, the use of self-assembling glycopeptide amphiphiles is gaining an increasing interest3 due to their ability to form supramolecular structures that mimic the ECM. In addition, the fact that they are maintained by non-covalent interactions (e.g., p-p or CH-p stacking) makes them inherently stimuli-responsive and dynamic systems.2 Here, we synthesised a short glycopeptide amphiphile, i.e., Fmoc-diphenylalanine-glucosamine-6-sulfate (Fmoc-FF-GlcN6S), and evaluated its ability to promote neural regeneration.
Methodology
Fmoc-FF-GlcN6S was synthesised by coupling Fmoc-FF and GlcN6S using DCC-NHS chemistry. Two methodologies were used to prepare the Fmoc-FF-GlcN6S hydrogels: 1) temperature switch (T method) – heating at 90 °C to dissolve the amphiphile followed by cooling to room temperature; and 2) solvent-switch (S method) – dissolving the amphiphile in DMSO followed by its dilution into water. The mechanical properties of the generated hydrogels were assessed by rheology, and their supramolecular structure (i.e., molecular packing/interactions and nanofiber morphology) was evaluated using CD, FTIR and AFM. The effect of the generated hydrogels on human adipose-derived stem cell (hASC) behaviour was evaluated by live/dead analysis, immunostaining and qPCR.
Results
Both methods formed hydrogels composed by entangled nanofibers. The stiffness of the hydrogels was influenced by the preparation method: gels generated by the T method were stiffer than the ones formed by the S method, G’ (T) = 2.4kPa > G’ (S) = 0.5kPa. Under both methods, the morphology of the nanofibers was similar (AFM). Interestingly, CD and FTIR analyses demonstrated that peptide glycosylation altered the secondary structure of the nanofibers from β-sheets (for Fmoc-FF) to α-helixes (for Fmoc-FF-GlcN6S).
hASCs cultured on Fmoc-FF-GlcN6S hydrogels showed distinct behaviour dependant on the preparation method: hASCs spread throughout the surface of the hydrogels prepared by the T method, while cell clusters were observed for hydrogels generated by the S method. qPCR and immunostaining showed that hASCs seeded on both types of hydrogels (i.e., T/S methods) overexpressed GFAP and Nestin on day three and MAP2 and βIII-tubulin on day nine of cell culture.
Conclusions
We demonstrate that Fmoc-FF-GlcN6S is able to self-assemble into biofunctional hydrogels, whose stiffness can be tuned altering the preparation method (i.e., S/T), as well as the concentration of the amphiphile. The Fmoc-FF-GlcN6S hydrogels induce hASC differentiation into neural lineages, being a good indication of their suitability for neural regeneration. In this context, the T method is a more adequate alternative as it does not use organic solvents and the gelation conditions (e.g., gelation timeframe) allows the encapsulation of cells.
Acknowledgements
We acknowledge the financial support from the EC (#668983-FORECAST and #964342-ECaBox) and FCT (PTDC/CTM-REF/0022/2020-OncoNeoTreat, PD/BD/135256/2017, COVID/BD/152018/2021 and CEECINST/00077/2018).
References
1.Ulijn, R. V. & Smith, A. M. Chem Soc Rev 37, 664-675, (2008).
2.Lampel, A. Chem 6, 1–15, (2020).
3.Brito, A. et al. Chem 7, 2943-2964, (2021)."
73296334968
Nanoengineered Mechanically Robust Bioactive Particles Disseminated in Chitosan/Collagen Matrix for Osteoporotic Bone Treatment
Kulwinder Kaur1,, Ciara Murphy1,2,
1Tissue Engineering Research Group, Department of Anatomy and Regenerative Medicine, Royal College of Surgeons in Ireland, Dublin, Ireland
2Advanced Materials and BioEngineering Research (AMBER) Centre, Dublin, Ireland
KulwinderKaur@rcsi.ie
Introduction: Osteoporosis, characterised by depleted bone mass and disrupted bone architecture due to impaired bone remodelling, is the most prevalent metabolic bone disease in the world, causing fractures worldwide at a rate of one every 3 seconds, exceeding health care costs of € 37 billion each year1. Osteoporotic vertebral fractures (OVFs) are the most common complication of osteoporosis and patients determined to have OVFs are 5 times more likely to suffer secondary vertebral fragility fractures2. The clinical gold standard of care for OVFs is vertebroplasty and kyphoplasty, whereby cement is injected into the damaged vertebrae to stabilise the fracture site and reduce pain. These cements are not biodegradable and often leading to complications such as cement leakage and appearance of secondary fractures in adjacent diseased vertebrae1. So, the main aim of this study was to tackle a devastating clinical orthopaedic challenge for which there is currently no reparative treatment specifically OVFs, by developing an advanced mechanically robust biomaterial technology to repair & restore structural integrity and function of disease damaged bone. These materials can be loaded with different agents to promote a targeted delivery, reducing the occurrence of side effects common in conventional treatments.
Methodology: Strontium loaded nano hydroxyl apatite particles (nHAS) functionalized with SWCNTs were prepared by using wet-precipitation method. Thermoresponsive nHAS decorated hydrogels were prepared by using β-GP3 and scaffolds were prepared by freeze dried method4. Physicochemical properties was assessed by using XRD, FTIR, SEM, TGA, DSC,TEM techniques ,degradation profile in PBS and mechanical properties by Zwick Roell testing machine using 5N load. Rat mesenchymal stem cells were seeded on the scaffolds to assess osteogenesis activity via cytotoxicity, proliferation and quantitative RT-PCR to detect key osteogenic markers, ALP activity and calcium deposition. RAW 267.4 cells were used to check the osteoclastogenesis effect of releasing Sr ion via TRAP activity and RT-PCR to detect key osteoclastogenic markers.
Results: Mechanically robust scaffolds for controlled degradation and ion releasing profile were prepared. All the scaffolds was found to be have high water retention ability, porous and bioactive in nature. Scaffolds are found to be non-toxic with enhanced osteogenic differentiation and promote mineralised matrix deposition with increasing content of Sr, and decreased TRAP activity for RAW 264.7. This shows the repair & restore structural integrity of our scaffolds for disease damaged bone without the need of added additional therapeutics.
Conclusion: We developed a therapeutic mechanically robust biomaterial technology that will for the first time, combine mechanically robust carbon nanotubes with antiosteoclastic-ion substituted nano-particles, to target impaired bone remodelling and drive regeneration in a disease compromised load-bearing environment.
References
Svedbom et al., Arch Osteoporos, 2013
2 Sözen et al., Eur J Rheumatol,2017
Kaur et al., Mater Sci & Eng C, 2021
Murphy et al., Biomater, 2010
52354524437
Introduction. Despite considerable developments in the field of orthopedic implants, complications including poor bone ingrowth and implant-associated infections (IAI) persist. Macrophages have recently been acknowledged to be essential for the implant success in the body, partly through intimate crosstalk with mesenchymal stem cells (MSCs) in the process of new bone formation [1]. However, the behavior of these immune cells is known to be affected by environmental cues, including the implant surface properties. Additive manufacturing (AM), surface biofunctionalization, and silver nanoparticles incorporation are promising techniques to achieve orthopedic implants with osteogenic, immunomodulatory, and antibacterial biofunctionalities [2–4]. The osteoimmunomodulatory properties of such implants are, however, not yet well understood. We, therefore, investigated the effects of human macrophages on the human mesenchymal stem cells (hMSCs) when co-cultured in vitro with AM titanium implants biofunctionalized via plasma electrolytic oxidation (PEO) and incorporated with silver nanoparticles (AgNPs).
Methodology. AM generated Ti-6Al-4V implants were biofunctionalized via PEO with/without AgNPs. Surface characterization was performed with a scanning electron microscope (SEM) and silver ion release was measured. The effects of the incorporation of AgNPs at different concentrations on human macrophages and bacterial cells were assessed by evaluating the viability of human macrophages and performing an isothermal microcalorimetry assay where bacterial metabolic activity was measured. The response of human macrophages and hMSCs monocultures to the PEO-treated Ti-6Al-4V implants were subsequently evaluated by measuring mineralization, protein, and gene expression. Finally, an indirect co-culture of macrophage-hMSCs was performed to study the effects of the macrophage response induced by the implants on the hMSCs osteogenic differentiation.
Results. The PEO modification of the AM implants created TiO₂ surfaces with micro- and nano-porosities. AgNPs were successfully incorporated into the TiO₂ layer. A concentration of 0.3 g/L AgNPs was found to be optimal to maintain the viability of human macrophages while imparting sufficient antibacterial properties to prevent bacterial growth on their surfaces. The expression of tissue repair related factors decreased in the specimens containing 0.3 g/L AgNPs as compared to the PEO-treated specimens not incorporating AgNPs. The same trend was observed for the macrophages co-cultured with hMSCs. However, this did not affect the osteogenic differentiation of hMSCs. Both co- and single-cultured hMSCs could osteogenically differentiate without any adverse effects caused by the presence of macrophages that were exposed to the either surface.
Conclusions. Based on the findings of this study, the incorporation of AgNPs into the PEO layers does not compromise the osteogenic differentiation and mineralization of hMSCs when co-cultured with human macrophages, while adding antibacterial functionalities to AM surfaces. Further evaluation of these promising implants in a bony in vivo environment with and without infection is, thus, recommended, and may prove them worthy of further development for potential clinical use.
References:
[1] Chen, Z. et al., Mater. Today 19, 304–321 (2016)
[2] Taniguchi, N. et al., Mater. Sci. Eng. 59, 690–701 (2016)
[3] Van Hengel, I. A. J. et al., Mater. Today Bio. 7, 1–12 (2020)
[4] F. Razzi et al., Biomed Mater. 15, 035017 (2020)
52354545528
"Angiogenesis, the process by which new blood vessels sprout from existing surrounding ones, is essential for the survival of implanted tissue engineered constructs. In fact, the lack of proper re-vascularization and core necrosis is the main pitfall of many developed biomaterials (1). In this study, we describe a highly tunable hydrogel system for the growth of capillaries based on alginate.
Alginate was chemically functionalized with norbornene. Norbornene forms a covalent bond with a thiol group in the presence of a photoinitiator upon exposure to UV light. The degree of functionalization obtained was 4,63%, as measured with NMR. Mechanical properties were analyzed by rheology and hydrogels ranged 10 – 1000 Pa, depending on final polymer concentration and concentration of cross-linker. Photoinitiator LAP was used at 2 mM. To introduce biodegradability to the system, we used matrix metalloproteinases (MMP)-cleavable sequences flanked by two thiol groups as cross-linkers. The speed of degradation can be tuned by modifying the sequence specificity to a range of MMPs. Non-degradable hydrogels were fabricated by using 2000 dalton PEG di-thiol. Degradable hydrogels dissolved when exposed to collagenase, while non-degradable gels remained unaltered.
In a one-pot-synthesis manner, alginate-norbornene, thiolated-RGD, dithiol MMP-cleavable peptide, cells, VEGF165 and photoinitiator were mixed. Solution was casted onto siliconized glass-slides and irradiated with UV for 30 seconds. Human Umbilical Vein Endothelial Cells (HUVECs) and Mesenchymal Stromal Cells (MSCs) were encapsulated in hydrogels in a 1:10 ratio (HUVEC:MSC). Cells were viable after cross-linking and were able to fuse and sprout, recapitulating the process of angiogenesis. Cell elongation was apparent already after 24 hours of culture. Hydrogels were stained against CD31 and imaged with confocal microscopy. Then, networks were analyzed and quantified using Amira and WinFiber3D (2). Vessel parameters were superior in hydrogels with lower degree of cross-linking, and thus softer, and with higher concentration of RGD. Initial studies have shown no major differences depending on the cell-binding peptide used: RGD (derived from fibronectin), YIGSR (a sequence found in laminin) and GFOGER (a collagen-derived peptide).
In conclusion, we have developed a platform that allows the study of the influence of stiffness, matrix degradability and ECM binding motifs on angiogenesis. Further work will focus on the use of these hydrogels as bioinks for 3D printing vascularized tissues and organoids.
References
1. Lesman et al. Mechanical regulation of vascular network formation in engineered matrices. Adv Drug Deliv Rev. 2016.
2. Bonda et al. 3D Quantification of Vascular-Like Structures in z Stack Confocal Images. STAR Protocols. 2020."
41883626205
"Introduction
Fibrin biomaterial is widely used in the clinic as a tissue sealant and in pre-clinical research as a carrier material for growth factor delivery. In these applications, premature fibrin degradation leads to suboptimal tissue adhesion, recurrent bleeding and limited regenerative efficacy. Therefore, fibrinolytic inhibitors are commonly added into fibrin formulations, such as the potent inhibitor aprotinin. Nevertheless, the use of aprotinin in the clinic has been associated to some important side-effects, including some immunogenic reactions related to the bovine origin of the drug. In this project, we characterized the use of an endogenous human fibrinolytic inhibitor, α2-antiplasmin (α2PI), as a potential substitute for aprotinin. We first tested α2PI as an antifibrinolytic agent for applications in tissue sealants and for growth factor delivery in diabetic wound healing. Then, we assessed α2PI as a stand-alone hemostatic agent to reduce blood loss during surgery.
Methodology
We produced α2PI as a recombinant his-tagged protein in Human Embryonic Kidney (HEK) 293 mammalian cells and purified it by affinity-based chromatography. The longevity of α2PI-supplemented fibrin hydrogels was assessed in vitro in presence of plasmin, and in vivo upon subcutaneous implantation in mice using an in vivo imaging system (IVIS). Next, the delivery of angiogenic growth factors via α2PI-supplemented fibrin hydrogels was tested in a wound healing model in the db/db diabetic mouse and was quantified by histomorphometric analyses. More specifically, wound regeneration was assessed in terms of wound re-epithelialization, granulation tissue formation and wound angiogenesis. Finally, we used a tail vein bleeding model in mice to evaluate α2PI hemostatic properties upon intravenous injection.
Results
Incorporation of recombinant human α2PI into fibrin biomaterials significantly prolonged their duration in vitro and in vivo. Upon subcutaneous implantation, α2PI-supplemented fibrin implants remained present for over 30 days, thus vastly outperforming the implants supplemented by aprotinin. In the diabetic wound healing model, the delivery of angiogenic growth factors by α2PI-supplemented fibrin significantly enhanced granulation tissue formation and wound angiogenesis as compared to the delivery of growth factors in absence of α2PI. In addition, we observed positive trend toward enhanced wound re-epithelialization (p-value = 0.09). Finally, we demonstrated that α2PI had similar hemostatic properties than aprotinin in the mouse tail vein bleeding model in vivo, significantly reducing blood coagulation time and blood loss as compared to non-treated animals.
Conclusion
In conclusion, α2PI showed strong efficacy in vivo, both as an anti-fibrinolytic and as a hemostatic agent, therefore appearing as a highly competitive human-derived substitute to the bovine aprotinin. Indeed, α2PI successfully increased the longevity of fibrin implants, enhanced growth factor delivery in diabetic wound healing and reduced bleeding time and loss upon intravenous delivery, respectively mimicking the 3 main applications of aprotinin in fibrin sealants, fibrin-mediated drug delivery and in surgery."
20941842648
"Immunohistochemistry and immunofluorescence for vascular network analysis play a fundamental role in basic science, translational research, and clinical practice. Due to their versatility and specificity, these techniques are also widely used in the tissue engineering context, where the study of neovasculature is crucial to assess the function of artificial tissue surrogates. However, identifying vascularization in histological tissue images is time-consuming and markedly depends on the operator's experience. This study introduces ""Blood Vessel Detection – BVD"", an automatic and ready-to-use morphometrical tool for quantitative analysis of vasculature in fluorescent histological images. BVD is based on the extraction and analysis of low-level image features and spatial filtering techniques, which do not require a training phase. The performance of the BVD algorithm was tested on four different datasets: a set of phantom images and on three sets of histological sections from three separate in vivo studies that specifically focused on the characterization of angiogenesis. The first study analyzed was an example of a rat abdominal wall defect treated by a polymeric patch loaded with microparticles able to release an angiogenic factor1. The second case was a rat infarction model treated with a bilayer biohybrid patch composed of polymer and extracellular matrix2. Finally, the algorithm was utilized to quantify angiogenesis in the case of ectopic organ regeneration3. Collectively, 173 independent images were analyzed, and the algorithm's results were compared to those obtained by human operators. The developed BVD algorithm proved to be a robust and versatile tool, quantifying the number, the diameters, the perimeters, the areas, and the spatial distribution of blood vessels within all the considered datasets. BVD is provided as an open-source application working on different operating systems. BVD is supported by a user-friendly graphical interface designed to facilitate large-scale analysis.
1 D'Amore, A. et al. Tissue Engineering Part A 24, 889-904 (2018).
2 Silveira-Filho, L. et al. JACC: Basic to Translational Science 6.5, 447-463 (2021)
3 Francipane, M. G. et al. The American journal of pathology 190, 252-269 (2020)"
41883639768
"Introduction: Inducing axial vascularisation of tissue engineering constructs is a well-established method to allow an adequate support of large 3-dimensional tissues. Progenitor cell chemotaxis towards axially vascularized tissues and its role in inducing neo-vascularisation and tissue regeneration has not been well characterized.
Methodology: In a prospective randomized controlled study including 32 male syngeneic Lewis rats we investigated the native capability of the axially vascularized constructs to specifically attract systemically injected bone marrow mononuclear cells (BMMNCs). The underlying mechanism for progenitor cell homing was investigated focusing on the role of hypoxia and the SDF1-CXCR4/7 axis. Sixteen animals were used as donors for BMMNCs. The other 16 animals were subjected to implantation of a tissue engineering construct in the subcutaneous groin region. These constructs were axially vascularized either via an arteriovenous loop (AVL, n=6) or via uninterrupted flow-through vessels (non-AVL, n=10). BMMNCs were isolated, labelled with quantum dots (Qdot® 655) and injected 12 days after surgery either via intra-arterial or intravenous routes. 2 days after cell injection, the animals were sacrificed and examined using fluorescence microscopy.
Results:The Qdot® 655 signals were detected exclusively in the liver, spleen, AVL constructs and to a minimal extent in the non-AVL constructs. A significant difference could be detected between the number of labelled cells in the AVL and non-AVL constructs with much more cells detected in the AVL constructs specially in central zones (p <0.0001). The immunohistological analysis showed a significant increase in the absolute expression of HIF-1 in the AVL group in comparison to the non-AVL group. The PCR analysis also confirmed a 1.4-fold increase in HIF-1 expression in AVL constructs. Although PCR analysis showed an enhanced expression of CXCR4 and CXCR7 in AVL constructs, no significant differences in SDF1 expression were detected via immunohistological or PCR analysis.
Conclusions: At the examined time point, the AVL constructs were capable of attracting systemically injected BMMNCs in a mechanism probably related to a state of controlled hypoxia associated with a robust tissue formation."
83767214526
"Introduction: Neo-angiogenesis describes the development of new blood vessels and takes place during bone healing in the fracture gap. Angiogenesis and the establishment of a functional vascular network is closely associated with bone formation and critical for scar-free healing. Type H vascular endothelial cells (ECs), characterized by high expression of CD31 and endomucin (Emcn) have been identified as key regulators for angiogenesis-osteogenesis coupling. However, the underlying mechanisms that drive revascularization, especially in the early phases from hematoma formation to cartilage development largely remain unknown. Here, we especially want to draw attention to the role of mechanosensors YAP/TAZ in ECs and how they affect angiogenesis and osteogenesis. By modulating YAP and TAZ in ECs we further want to unravel the different phases of angiogenesis during bone healing.
Methodology: We analyzed vessel ingrowth and organization under different fixation stabilities during bone regeneration. Female, 12 weeks aged mice with endothelial YAP/TAZ dKO were sacrificed 7 days or 14 days post-osteotomy, respectively. Soft callus formation, cell organization and ECM deposition within the fracture gap was analyzed by immunohistological protocols and second harmonic imaging (SHI). To characterize H type vessel formation, CD31 and endomucin antibodies were used. Confocal microscopy was employed to analyze the target proteins during the onset of neo-vascularization in bone regeneration. Endochondral ossification was analyzed in areas of bone healing and compared against areas in the growth plate.
Results: We could show that H type vessels (CD31hiEmcnhi) are present in the osteotomy gap, suggesting that they play a key role in de novo angiogenesis during pathological and regenerative processes. Further, we could show that H type vessels are surrounded by osterix expressing pre-osteoblasts, supporting their important role in angiogenesis-osteogenesis coupling during bone healing. Conditionally knocking out YAP/TAZ in ECs leads to an increase in bone vasculature in the hypoxic microenvironment of the osteotomy gap. Further, EC YAP/TAZ dKO induces more vessel crosses, suggesting that vessels fail to stabilize and build a functional basement membrane. Whereas in the pre-cartilage phase complete vascular invasion can be observed, vessels regress in areas of cartilage development in later stages. This suggests that two different mechanisms of angiogenesis exist during bone healing. Angiogenesis via endochondral ossification in the osteotomy gap shows similarities to the bone formation process at the growth plate, however, the latter being more organized.
Conclusion: Next to a downregulation of inflammation, successful bone regeneration requires angiogenesis. We explored here the role of EC mechanosensors YAP/TAZ and their ability to regulate the build-up of a capillary network. We could further show, that revascularization during bone healing occurs via two distinct pathways. Neo-angiogenesis at the pre-cartilage phase follows different mechanisms than angiogenesis via a cartilage template in later stages of bone healing. Understanding the mechanisms of angiogenesis and the effect of EC YAP/TAZ is essential to develop new therapies how to best accelerate bone healing, which would particularly important for the treatment of bone fractures in the elderly.
References:
Neto, F. et al., eLife; 7:e31037 (2018)
Kusumbe, A. et al., Nature 507, 323–328 (2014)"
73296317367
"Introduction
Tissue engineering of human blood vessels is pursued as a clinical revascularization therapy as well as to develop in vitro human blood vessel models with native-vessel like organization. Clinical revascularization strategies that use autologous vessels are hampered by poor quality and limited availability. Synthetic vascular scaffolds may be used as alternative, but for replacement of smaller vessels (<4 mm range) this is associated with high thrombogenicity and patency loss. Pre-cellularization of the polymer constructs with autologous vascular cells could protect smaller diameter grafts against patency loss and improve long term graft survival upon implantation. In vitro seeded vascular grafts using patient-derived induced pluripotent stem cells (iPSCs) could be used for disease modeling or therapeutic screening. Here, we use iPSCs vascular Organoid Derived Endothelial Cells (ODECs) and Mural Cells (ODMCs) as a (autologous) vascular cell source for in vitro tissue engineering of small diameter biodegradable vascular grafts.
Methodology
The 3D vascular organoid culture method described by Wimmer et al. [1] was further developed to isolate pure populations of ODECs and ODMCs, which can be cryopreserved and expanded in traditional 2D culture without loss in cell pool purity, viability, and proliferative capacity. 2D ODEC culture was used in dynamic flow and TEER experiments to determine the shear stress responsiveness of the cells. Both ODECs and ODMCs were subsequently seeded on 3 mm diameter degradable solution electrospun polycaprolactone-bisurea (PCL-BU) scaffolds and exposed to flow for 48 hours.
Results
The 2D dynamic flow experiments and TEER experiments showed that ODECs are shear stress responsive and were able to establish and restore the endothelial barrier after thrombin stimulation. Additional experiments demonstrated that ODECs and ODMCs could be successfully seeded in bilayer configuration on the PCL-BU scaffolds, forming the luminal endothelium and underlying medial layer that partially mimicked the layered structure of a human native vessel. Exposure of the resulting human organoid derived tissue engineered vascular graft (TEVGs) to lumen perfusion in a flow-bioreactor setup showed integrity preservation of the bilayer configuration and endothelial attachment to scaffold substrate after subjection to flow.
Conclusions
In conclusion, iPSC derived vascular organoid cells can be successfully used as a source of functional, flow-adaptive vascular cells for tissue engineering of perfused macrovascular grafts. Therefore, our protocol offers a TEVG based solution for replacement of small caliber native human macrovessels using patient derived cells.
[1] Wimmer, R.A., Leopoldi, A., Aichinger, M. et al. Human blood vessel organoids as a model of diabetic vasculopathy. Nature 565, 505–510 (2019)."
62825402527
"Introduction: Endochondral ossification (EO) is the process of bone development via a cartilage template. It involves multiple stages, including chondrogenesis, mineralization and angiogenesis. Importantly, how angiogenesis contributes to EO is not fully understood. To characterise the interaction between human mesenchymal stromal cells(hMSCs)-derived cartilage and blood vessels, we designed an in vitro co-culture model comprised of tissue engineered hMSCs derived cartilage pellets that undergo mineralisation, and adipose-derived stromal cells (ASCs) and human umbilical vein endothelial cells (HUVECs) that form blood vessel structures. After characterisation of the pellets, we assessed the effect of their conditioned medium on angiogenesis using HUVECs migration and proliferation assays. Finally, we co-cultured the pellets, HUVECs and ASCs in a fibrin hydrogel to develop a comprehensive system to study cartilage vascularisation.
Methodology: Chondrogenic hMSC pellets (N = 3 donors) were generated by culture with medium containing transforming growth factor(TGF)-β3 (10ng/ml) for 28 days. For mineralized pellets, β-Glycerophosphate (BGP) (10mM) was added from day 7 and TGF-β3 was withdrawn on day 14. On days 7, 14, 21 and 28, conditioned media were produced by culturing the pellets for 24h in basal medium. Then, the pellets were harvested for gene expression and histological analysis. Thionine and Von Kossa stainings were performed to detect glycosaminoglycans (GAGs) and calcium deposits, respectively. Transwell migration and EdU-proliferation assays were employed to evaluate the effect of conditioned medium on HUVECs. To generate a 3D-vascular network, HUVECs and ASCs were simultaneously co-cultured with pellets in a fibrin hydrogel for 14 days. The vessel structures were visualised by immunofluorescent staining for laminin.
Results: Thionine and von Kossa staining evidenced successful in vitro cartilage formation and mineralisation, respectively. BGP exposure induced the formation of mineralised deposits which increased in time, while GAG staining progressively decreased. The mRNA expression of osteogenic (ALPL, IBSP) and angiogenic/remodelling markers (VEGFA, MMP13) in mineralised pellets showed the highest levels on d14 and decreased during late mineralisation. Transwell migration assays showed that conditioned medium from chondrogenic and mineralised pellets stimulates HUVEC migration (24.2-folds and 16.8-folds vs. negative control, respectively). HUVEC proliferation was also increased after exposure to conditioned medium from chondrogenic or mineralised pellets (1.9-fold and 2-fold vs. negative control, respectively). Finally, by co-culturing pellets/ASCs/HUVECs in a fibrin hydrogel, we achieved the successful formation of a 3D vascular network. Confocal imaging analyses revealed contact between microvessels and chondrogenic/mineralised pellets.
Conclusions: In this study, we established an in vitro model of cartilage vascularisation during endochondral ossification. By characterising mineralising pellet cultures, we found that the expression of pro-angiogenic markers and the pro-migratory and pro-proliferative effects towards HUVECs are maximum during early mineralisation and then decrease. Furthermore, 3D in vitro vascular network formation was achieved in the presence of chondrogenic or mineralised pellets. Our in vitro 3D co-culture model can be applied for mechanistic studies on the role of angiogenesis in bone formation and repair, as well as disease modelling."
73296313124
"Introduction
Prevascularization of tissue-engineered constructs before implantation is used to accelerate anastomosis with the host vasculature and thus to enhance successful implantation. To induce prevascularization, the co-culture of tissue-specific cells with endothelial cells is explored. To date, most co-culture studies are still conducted with human umbilical vein endothelial cells (HUVECs). However, this ultimately restricts clinical applications and thus ideally autologous cells are used. Furthermore, the mounting evidence of the endothelium as a regulator of regenerative processes in an organ/tissue-specific manner warrants the use of tissue-specific cells for tissue engineering. Skeletal muscle microvascular endothelial cells (SkMVECs) are an interesting candidate for prevascularizing tissue-engineered skeletal muscle as they are both autologous and tissue-specific. Here, we compare SkMVECs to HUVECs, both in 2D as well as in 3D bio-artificial muscles.
Methodology
Primary human SkMVECs were obtained from 3 different donors. These cells were thoroughly characterized in comparison to the current standard, HUVECs, through immunostaining and bulk RNA sequencing. Next, in vitro sprouting capacity of the SkMVECs was compared to the HUVECs using a conventional spheroid assay and tube formation assay. In addition, endothelial network formation in a fibrin hydrogel was evaluated over 14 days. And finally, direct and indirect co-cultures with human primary myoblasts were set up to evaluate the interaction between the SkMVECs and myoblasts.
Results
SkMVECs were extensively compared to HUVECs through bulk RNA sequencing to evaluate differentially expressed genes and these were annotated to the related pathways. Next, the angiogenic potential was evaluated and SkMVECs were found to sprout less compared to HUVECs in terms of sprout number but sprout length was found to be similar. Also, from the tube formation assay, a similar extent of tube formation was found but HUVECs were found to form tubes more rapidly. However, when both endothelial cell types were embedded in a fibrin hydrogel over a longer period, which is similar to our tissue engineering system, SkMVEC constructs were found to result in more branched endothelial networks compared to HUVECs. In addition, the more rapid proliferation of HUVECs compared to SkMVECs seemed to interfere with stable endothelial network formation after 10 days. Conditioned medium of both cell types was used to dissect the cross-talk between myoblasts and endothelial cells and effect. Finally, to explore the potential of SkMVECs for skeletal muscle tissue engineering, 3D co-culture experiments with autologous myoblasts were performed to evaluate the interaction between the SkMVECs and myoblasts.
Conclusion
Taken together, SkMVECs are capable of forming stable, extensive endothelial networks in a relevant model for tissue-engineering applications. In contrast, the currently used standard endothelial cell type, HUVECs, was found to be highly angiogenic in short-term assays but less suited for long-term endothelial network formation. Furthermore, SkMVECs interact with autologous myoblasts and vice-versa, which further underscores their potential as a suitable endothelial cell source for the prevascularization of engineered skeletal muscle tissue."
31412735886
"INTRODUCTION: Mesenchymal stromal cells (MSCs) are potential candidates in tissue engineering applications. Nevertheless, MSC-based therapies have fallen short of the initial promise and hype due to the low MSC retention rate caused by the disruption of nutrient and oxygen supplies. Our previous studies established that the lack of glucose (but not oxygen) is fatal to human MSCs (hMSCs) because it acts as a pro-survival molecule for hMSCs upon transplantation. As a first step towards the engineering of a glucose releasing hydrogel, this study aims to provide insights into the effects of glucose on hMSCs paracrine function pertinent to angiogenesis in vitro and in vivo.
METHODOLOGY: In vitro experiment: Release of bioactive factors, angiogenic potential, and chemo-attractive potential of conditioned media (CM) from hMSCs towards human umbilical vein endothelial cells (HUVECs) were performed using Multiplex Luminex® Assays, Matrigel-Based Tube Formation Assay, and Incucyte® Live Cell Analysis System, respectively. The CM was obtained by exposure of hMSC to near-anoxia in the presence of glucose at 0, 1, or 5 g/L for 3 days. In vivo experiment: hMSC pro-angiogenic potential was investigated using hMSC-containing hydrogels loaded with either 0, 1, 5, 10, or 20 g/L glucose and hMSC-free hydrogels loaded with 20 g/L glucose, which were implanted ectopically in nude mice. The formation of new blood vessels was quantified using a micro-CT scanner after Microfil® injection at 21 days.
RESULTS: Glucose improves angioinduction of MSCs in near anoxia: Supernatant CM collected from hMSCs cultured with either 1 or 5 g/L glucose in near-anoxia for 3 days increased HUVECs migration and formation of vascular-like structure compared to that in the CM collected from hMSCs cultured without glucose. These data were corroborated by the increased amounts of pro-angiogenic factors (Angiogenin, VEGF-A, VEGF-C, Angiopoietin-1, Endostatin, and CCL2) in the CM collected from hMSCs cultured with glucose. Glucose improves the survival of MSCs and new blood vessels formation post-implantation: Upon ectopic implantation into nude mice, the volume of newly formed blood vessels within hMSCs-containing hydrogels loaded either 5, 10, and 20 g/L glucose exhibited a 2.4, 2.8, and 2.4 fold increase compared to hMSCs-containing hydrogels without glucose at days 21 post-implantation, respectively.
CONCLUSION: These data demonstrate that critical impact of glucose on MSC-mediated angiogenesis both in vitro under near-anoxia and in vivo using an ectopic mouse model. Further investigations are ongoing to determine whether endoplasmic reticulum stress is involved in the positive effects of glucose in MSC-mediated angiogenesis. All in all, the in vivo delivery of hMSCs in the presence of glucose strategy may be broadly helpful to improve not only the survival but also the angioinductive potential of hMSC-based therapies."
41883602164
Despite the success of a handful of companies and the overabundance of research the tissue engineering & regenerative medicine (TE&RM) community has still not delivered the more then 3 decades ago promised health care and commercial break through’s. Yet, rendition of research outcomes into clinical application is still the rallying cry of the modern TE&RM establishment. Deciphering observations from basic investigation protocols (e.g., in silico, in vitro, ex vivo, or in vivo, ,etc.) to address into not even clinical routines but first in human studies has diverse challenges. Exploitation of the vast literature uncritically to defend further research, regardless of their authenticity, clinical significance, vigour or quality, is often the most hurried path to write and fund grant proposals and to obtaining publishable data, even though these data inherit no translational and often little scientific relevance. The research data manufacturing machinery is authorised but real world applicability and impact are often compromised. Muted translational achievements continue to plague most aspects of TE&RM research as the development of bedside to bench and back again approach possess challenges that most academics but also TE&RM companies are not typically trained to overcome. Based on the above arguments this talk will critically review two key research areas in TE&RM, namely scaffold guided tissue engineering and bioprinting.
Gene therapy is the most promising treatment for recessive dystrophic epidermolysis bullosa (RDEB), however genetic cargo delivery efficiency is still a technical limitation. Viruses are the traditional vector of preference for gene therapy, as virus trophism increase tissue specificity. However, drawbacks related with safety and high manufacturing costs have facilitated the expansion of non-viral vectors, such as liposomes and cationic polymers. Our group is focused on the development of highly branched cationic polymers for gene therapy to treat RDEB. Our polymers have demonstrated encapsulation and delivery of a full COL7A1 cDNA with no toxicity, performing better transfection efficiencies than commercial counterparts in RDEB keratinocytes.
In this work, we show the research progress expanding the polymer technology for mRNA, pDNA and ribonucleoprotein complex delivery for developing CRISPR/Cas9 based gene editing therapies for RDEB. Gene edition in vivo has been achieved by a single topical application, obtaining efficiencies comparable with viral vectors (Ad5). Endosomal escape, by peptide polymer decoration is being investigated to improve efficiency in vivo by avoiding endosomal retention. Storage of the nanoparticles, formed by polymers and the genetic cargo, at -20C ensures no reduction in efficiency for more than 6 months. However, in order to avoid cold chain challenges, nanoparticles have been lyophilised, increasing dose concentration and facilitating formulation with skin absorption enhancers for topical application.
Proven ability to transfect stem cells combined with high efficiencies transfecting with multiple plasmids at the same time, should contribute to the success of prime editing strategies to pursue permanent correction of potential >89% of all described EB disease-associated mutations. The developed polymer platform shows high potential to be adapted to a wide range of genetic approaches for RDEB, including the most novel ones, that can be expanded to other EB subtypes, other genodermatoses and other rare genetic disorders such as cystic fibrosis.
73387306587
"INTRODUCTION: There remains a substantial unmet clinical need for tissue engineered strategies to heal large volume bone defects. The delivery of microRNAs from biomaterial-based scaffolds presents a promising approach: whereby the scaffold provides a structural support to bone tissue while the microRNAs (miRs) induce the endogenous cells to produce relevant therapeutic proteins and genes at physiological levels while shutting off aberrant effects 1,2. However, the effective delivery of miRs is frequently jeopardized by their poor stability, requiring a suitable vector which would protect them from degradation guaranteeing their effective intracellular delivery and transient secretion of osteogenic proteins by host cells. In this study, collagen-nanohydroxyapatite (coll-nHA) scaffolds1,2,3, previously optimized for bone repair within our lab, were coupled with self-assembling, amphiphilic, cell-penetrating RALA peptide4 as a delivery non-viral vector yielding a scaffold-based system for simultaneous delivery of miR-26a mimic1,2 and miR-133a inhibitor5 for bone repair.
METHODS: miRs were complexed with cationic RALA peptide5, incorporated (1μg or 3 μg) into coll-nHA scaffolds1 which were assessed in terms of calcium release, loading efficacy, distribution and release of nanoparticles (NPs). 3×105 human mesenchymal stem cells (hMSC) were seeded onto the miR-activated scaffolds and the expression of miRs, metabolic activity, DNA content, ALP activity and calcium deposition were quantified. The scaffolds were implanted into calvarial defect in male rats, the total bone volume and tissue mineral density were assessed at week 4, 8 and 12 of the study.
RESULTS: The NPs were successfully incorporated into scaffolds and worked effectively delivering miRs to the hMSCs in controlled manner. The miR-activated scaffolds cultured in cell-free media showed sustained release of miRs, uptake of calcium, and an increase in compressive modulus. The scaffolds delivered the miR-26a mimic or miR-133a inhibitor, either alone or combined, to the hMSCs resulting in a silencing effect and an enhanced ALP activity. The miR-activated scaffolds enhanced the healing in rat calvaria generating greater amount of bone compared to the scaffold alone.
CONCLUSION: This study describes the development of scaffold system using self-assembling, amphiphilic, cell-penetrating peptide for sustained delivery of therapeutic microRNAs for treatment of bone defects. The miR-activated scaffolds transfected the hMSCs with miRs enhancing the osteogenesis of the cells3,5. The miR-scaffold system has potential to be used as a next generation therapeutic for repair of large bone defects offering precise and transient gene editing with minimal immunogenicity. The novel miR co-delivery scaffold-based system is versatile and has the potential for a myriad of applications beyond bone repair by tailoring the individual miRs delivered – as well as the scaffold composition.
ACKNOWLEDGEMENTS: National Science Foundation- Science Foundation Ireland (NSF-SFI) US-Ireland R&D Partnership Programme (NSF_ 17_US_3437). JMS benefits from a Marie Skłodowska-Curie Individual Fellowship from the European Commission through the H2020 project GAMBBa (Project ID: 892389).
REFERENCES: 1)Mencía Castaño I. et al., J. Control. Release., 2015 200: 42-51, 2)Raftery R. et al., Adv. Mater., 2016 28: 5447-5469, 3)Mencía Castaño I. et al., Sci. Rep., 2016 6: 2794, 4)McCarthy H et al., J Control Release, 2014 189: 141-9, 5)Li L. et al., Biomaterials, 2013 34: 5048-58."
52354518008
"Introduction:
Cystic fibrosis (CF) is a lethal autosomal recessive inherited disease caused by mutations in the CFTR gene encoding the CF transmembrane conductance regulator (CFTR) protein, and has no cure to date. CFTR gene mutations lead to abnormal chloride ion transport in epithelial tissues, which changes the hydration and pH of fluids and mucus, affecting respiratory (main cause of morbidity and mortality) and digestive systems among others [1].
Gene therapy is the most suitable approach for treating this disease. Gene replacement strategies for CF have been previously developed but failed due to low gene delivery efficiency combined with brief expression in epithelial cells. Although gene therapy based on viral vectors has been proven to be efficient, safety risks and immune response are important limitations [2]. The rise of non-viral vectors has taken place to overcome these drawbacks, however, clinical trials with liposomes have been performed and finally abandoned.
In this work, we propose a different approach that consists in the development of a non-viral gene therapy based on highly branched poly(β-amino ester)s (HPAE), which family has been previously shown to perform high transfection efficiency in other genetic conditions [2], and offers advantages compared to liposomes such as easier functionalization and higher stability and retention of the cargos [3].
Methodology:
A library of cationic HPAE polymers was developed, characterized, and optimized by selection of the best molecular weights, enhancement of the branched structure and the biocompatibility, and terminal group performance evaluation. Then, a screening of the candidate polymers was carried out in vitro in CF disease model cell lines in terms of cell viability and plasmid DNA transfection efficiency. Moreover, optimized CFTR gene-containing DNA plasmids were constructed to be combined with the HPAEs developed.
Results:
A family of HPAEs with different monomeric combinations and terminal groups was created and their physicochemical features were characterized. Among all these polymers developed for plasmid DNA delivery, some of them showed higher transfection efficiency than others commercially available in CF lung epithelial cell lines, with similar or higher levels of cell viability. In addition, different CFTR gene-containing DNA plasmids with different combinations of promoters and enhancers were successfully obtained as an alternative system to the gene replacement strategies currently available.
Conclusions:
The basis for the development of a promising non-viral therapy for CF has been laid. This has been possible thanks to the synthesis of efficient gene delivery tools as are HPAE polymers combined to optimized genetic systems to restore the CFTR protein levels, regenerating thus the lung epithelial cells function. This approach offers a new perspective from the clinical trials performed to date with other non-viral vectors and is expected to be further tested in vivo as an inhalation therapy. In addition, these systems have got a high potential for future commercialization and as bench-to-bedside research.
References:
41883619327
"Introduction: Osteoporosis (OP) is characterized by a loss in bone mass and mineral density1. The stimulation of the canonical Wnt/β-catenin pathway has been reported to promote bone formation by increasing the osteogenic potential of mesenchymal stem cells (MSC)2. This pathway is controlled by several regulators as secreted frizzled-related protein-1 (Sfrp-1) that acts as an antagonist3. Thus, Sfrp-1 silencing therapies could be suitable for enhancing bone formation. However, the stimulation of this pathway at a systemic level has been correlated with adverse cardiovascular events. The use of nanoparticles (NPs) for oligonucleotide targeted delivery could be effective avoiding these undesirable effects. Aptamers (Apt) can recognize and bind specific structures based on their three-dimensional conformation. This work hypothesizes the systemic administration of lipid-polymer NPs (LPNPs) functionalized with a MSC specific Apt and carrying an SFRP1 silencing GapmeR, could favor bone formation in OP.
Methodology: Different pegylated-LPNPs formulations were prepared and the effect of pH, GapmeR encapsulation and aptamer functionalization on their physicochemical properties was evaluated by DLS and TEM. The oligonucleotide encapsulation efficiency and its in vitro release at variable temperatures were evaluated using fluorescently-labeled GapmeR. Adequate SFRP1 GapmeR-loaded Apt-LPNPs (Apt-LPNPs-SFRP1) were then evaluated in terms of cytocompatibility and gene silencing efficiency. Finally, the developed systems were administered in vivo in osteoporotic mice and their biodistribution and bone induction capacity was evaluated using radiolabeled LPNPs or by the femurs’ histological, histomorphometric and immunohistochemistry assays after three months, respectively.
Results: The developed LPNPs show an adequate average diameter of approximately 160 nm independently of the GapmeR encapsulation. Moreover, these formulations presented low polydispersity indexes (< 0.3). The incorporation of the aptamer in the nanoparticles surface, as expected, led to a decrease in the ζ-potential. Both LPNPs-SFRP1 and Apt-LPNPs-SFRP1 exhibited a spherical core-shell structure, characteristic of LPNPs. Their GapmeR encapsulation efficiency was 64 ± 4.32 % for both LPNPs showing a biphasic release pattern. The treatment of MSCs with LPNPs at variable concentrations did not show any toxicity. On the other hand, the treatment of MSCs with LPNPs-SFRP1 decreased the sfrp1 expression but less than the positive control (Damafect). Nevertheless, for cells treated with Apt-LPNPs-SFRP1, sfrp1 expression levels were similar to positive control. Moreover, the aptamer functionalization modified the LPNPs biodistribution profile showing a four-fold increase in the bone accumulation and a ten-fold decrease in the hepatic accumulation compared to naked LPNPs. The femurs histological evaluation revealed evident changes in bone structure and microarchitecture observing a more compact trabecular bone and a cortical bone thickness increase, in the Apt-LPNPs-SFRP1 treated mice compared to control (saline solution). Moreover, the immunohistochemical analysis of Col-I and OCN, revealed increased immunoreactivity for both markers in the Apt-LPNPs-SFRP1 treated mice.
Conclusions: Aptamer functionalized LPNPs loaded with SFRP1 silencing GapmeR showed adequate properties and biodistribution profiles leading to an enhancement on the bone density of osteoporotic mice.
1. Yadav V.K. et al., Nat. Med. 16(3), 308-12 (2010).
2. Houschyar K.S et al., Front. Cell Dev. Biol. 6, 170(2018).
3. Bodine P.V. et al., J. Mol. Endocrinol. 18(5), 1222-37 (2004)."
20941856088
"Trauma is the leading global cause of mortality and disability. Patient management after trauma can be challenging, particularly in a multiple trauma setting, a condition characterized by several severe injuries. Multiple trauma can elicit major immunological responses, such as systemic inflammatory response syndrome, which lead to a deterioration in the patient’s condition. The complement system, being a central component of the immunological response after trauma, plays an important role therein. The activation of the complement system in multiple trauma, and subsequent regulation of inflammatory cascades, are therefore of clinical interest. MicroRNAs, important post-transcriptional gene regulators, may play a regulatory role in the activation and progression of immunological and regenerative responses in multiple trauma. The aim of this study was therefore to examine microRNA expression in the systemic circulation, and the sites of injury, in a porcine multiple trauma model. In this model, two different trauma treatment methods were compared, as well as one separate treatment group which was administered a combination of a C5-convertase inhibitor to inhibit complement system activation, and an anti-LPS receptor tore inhibit PAMP activation pathways.
The porcine multiple trauma model consisted of blunt chest trauma, liver laceration, bilateral femur fracture, and controlled haemorrhagic shock. Animals were operatively and medically stabilized, and monitored under ICU-standards for 72 hours, after which they were sacrificed. The control group consisted of six animals. Two trauma treatment methods were applied, early total care (ETC, n=7), and damage control orthopaedics (DCO, n=8). Furthermore, a separate group (n=4) was subjected to ETC and treated with a C5-convertase inhibitor and anti-CD14. For this study, fracture hematoma, bone from the fracture site, bone from an unfractured long bone (humerus), and blood plasma were sampled. MicroRNAs were isolated, transcribed and pooled for qPCR array analysis.
The array data revealed distinct microRNA expression levels, specific to the trauma treatment method and the application of anti-complement/anti-CD14 medication. Overall, anti-inflammatory microRNAs were upregulated in the ETC group as compared to the DCO group. The ETC group that received anti-complement/anti-CD14 medication showed a reduced expression of several of these anti-inflammatory microRNAs. In all treatment groups, the expression levels of pro-fibrotic microRNAs were lower than the expression levels of anti-fibrotic microRNAs. A major difference was observed between the fracture site samples from the ETC and DCO groups. MicroRNAs related to fibrosis were mostly downregulated in the DCO group, as compared to the upregulation of fibrotic microRNAs in the ETC group. Plasma microRNA expression revealed uniformly expressed circulating microRNAs, as well as multiple trauma specific microRNAs, and treatment specific microRNAs.
This study revealed microRNA expression profiles in fracture hematoma, bone, and plasma samples from a porcine multiple trauma model, linked to key processes in inflammation and fracture healing. Furthermore, the immunological response after multiple trauma seems to be represented in the systemic circulation through the expression of specific circulating microRNAs. Further research will focus on target analysis of the microRNA data, and in vitro fracture healing models in which mimics and antagomirs will be applied as possible regenerative and immunologic modulative therapy."
62825446719
Mesenchymal stem cells (MSCs) have been studied for the treatment of Osteoarthritis (OA), the most common chronic disease of joint cartilage. A potential mechanism of MSC-based therapies has been attributed to the paracrine secretion of trophic factors, where extracellular vesicles (EVs) may play a major role. It is suggested that MSCs from younger donor sources compete are optimal with respect their EV production capabilities. Therefore, MSCs generated from induced pluripotent mesenchymal stem cells (iMSCs) may represent a promising cellular source for the manufacture of EV therapeutics. In this study, we isolated and tested the efficacy of EVs secreted by MSCs and by iMSC for treatment of OA using an in vitro model.
To obtain high-quality EVs, we optimized the culture conditions for MSCs and iMSCs, the supernatant collection time, and EV extraction methods. MSCs and iMSCs were cultured in vitro in serum-free clinical grade condition. The cells were characterized for surface expression pattern, proliferation ability, senescence rate and differentiation capacity during long term-expansion. The culture media were collected continuously during the cell expansion, and EVs were isolated using an FPLC-anion exchange chromatography (AEX) approach. Nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), and western blots as well as non-conventional flow cytometry were used to identify EVs. We evaluated the biological effects of MSC and iMSC-derived EVs on IL-1αtreated human chondrocytes, to mimic the OA environment.
We observed that the use of a serum-free, chemically defined medium for isolation and culture of hMSCs allowed us to expand a population with a stable phenotype from early to late passages. It is already well known that MSC proliferation, differentiation and function decline with passaging, in fact, after 3 passages we observed a drastic impact on cell growth and differentiation. Paracrine activity of hMSCs during long-term expansion was also evaluated. The number and size of vesicles released by hMSCs increased proportionally with their age in culture. EVs collected during hMSC long-term expansion retained tetraspanin (CD9, CD63 and CD81) expression and did not vary with parental cells age. Anti-inflammatory activity of MSC-EVs were evaluated in an in vitro model using osteoarthritic chondrocytes; administration of hMSC-EVs showed positive effects for early passages-derived vesicles only. The expression of IL-6 and IL-8 was significantly reduced after treatment with hMSC-derived EV at passage3. Over time in culture, the dimension of the vesicles increased while their anti-inflammatory effect was reduced. Concurrently, the expansion of iMSCs in serum-free conditions in vitro was optimized to define the best culture conditions to maintain the cells and to define the best time window in which to isolate EVs with maximum biological activity.
Despite the promising potential of EVs for therapeutic applications, robust manufacturing processes that would increase the consistency and scalability of EV production are still lacking. The focus of our study was directed on determining the optimal range of time in which MSCs and iMSC are biological functionally with respect to production of EVs in a serum-free culture system. This paracrine application may represent a novel therapeutic approach for the treatment of OA.
94238126164
"Human long-lived plasma cells (LLPCs) are terminally differentiated effector cells of the B-lymphocyte lineage that reside in specialized niches in the human bone marrow (BM) harboring many different microenvironmental niches. LLPCs play an essential role in the humoral immune protection by maintaining constant high-affinity antibody levels against pathogens and their toxic products, independently of antigen presence. So far, the in vitro long-term cultivation of BM LLPCs is challenging since they reveal a brief survival time ex vivo. Thus, this project aims to develop an in vitro model that mimics the physiological microenvironment of their niche in the BM and enables long-term cultivation of LLPCs.
Our previously developed 3D model based on a hydroxyapatite-coated zirconium oxide-based ceramic can be used to maintain hematopoietic stem and progenitor cells for up to 8 weeks in their undifferentiated state (CD34+CD38-) when co-cultivated with BM mesenchymal stromal cells (MSCs) (Sieber et al. 2018). Based on this data, we aim to adapt the model to establish a microenvironment to support the survival of functional LLPCs in vitro. Human plasma cells (PCs) (CD38+CD138+) are isolated via magnetic activated cell sorting (MACS) from femoral head after mechanical preparation and introduced into the ceramic pre-seeded with MSCs building up a BM microenvironment. The survival capacity of functional PCs is assessed by flow cytometric analysis and detection of secreted antibodies by Bioplex.
So far, we are able to maintain PCs for up to 21 days in our in vitro system built up by the 3D scaffold pre-seeded with MSCs. Due to limitations of efficient extraction of PCs embedded in the 3D microenvironment after cultivation for flow cytometric analyses, their survival is shown indirectly by making use of their ability to secrete immunoglobulins. The cultured PCs remain functional and maintain their ability to secrete immunoglobulins (IgG1, IgA, IgM) over the culture time.
The established survival niche model could serve as a system to study niche interactions and will pave the way to establish disease models for diseases like multiple myeloma or autoimmunity to analyze changes in the microenvironment that promote the maintenance of pathogenic PCs. The better understanding of survival mechanisms of pathogenic PCs could disclose new targets for specific therapies."
62825476779
INTRODUCTION: Within the field of tissue engineering, biomaterial scaffolds augmented with gene therapeutics have emerged as a promising treatment strategy for tissue regeneration. To date, the majority of ‘gene-activated’ scaffolds in tissue engineering have utilised plasmid DNA as the gene therapeutic of choice. Recently, messenger RNA (mRNA) has emerged as an attractive alternative to DNA-based therapeutics due to its increased safety profile and faster protein expression. The aim of this study is to optimise the delivery of mRNA to difficult-to-transfect mesenchymal stem cells (MSCs) and incorporate optimised particles into collagen scaffolds to create a platform that can be used for multiple tissue engineering (TE) applications.
METHODS: A wide range of non-viral gene delivery vectors were screened for their ability to encapsulate and condense mRNA. The complexes were characterised in terms of physicochemical properties before being brought forward to transfection studies using rat MSCs. All mRNA complexes were compared in terms of transgene (luciferase) expression and cytotoxicity in MSCs grown both in 2D monolayer and in 3D on collagen-based scaffolds. In addition, three different types of mRNA – unmodified mRNA (uRNA), modified mRNA (modRNA) and self-amplifying mRNA (saRNA) (BioNTech) were screened to determine the effect of mRNA type on expression for TE applications.
RESULTS: Various polymeric and lipid-based vectors were found capable of successfully delivering mRNA to MSCs. It was found that both the vector and mRNA type used had a significant impact on transgene expression in our cell type. Overall, the base-modified mRNA achieved the highest levels of protein expression in MSCs demonstrating a 1.2-fold and 5.6-fold increase versus uRNA and saRNA respectively in 2D monolayer studies. When delivered from a collagen-based scaffold, lipid-based vectors (e.g. MessegerMax/RNAiMAX) outperformed polymeric vectors (e.g. jetPEI) and achieved high levels of protein expression in the MSCs ( 2.3x106 relative light units).
CONCLUSION: Messenger RNA represents a promising tool for tissue engineering applications. This study highlights the optimised transfection conditions for mRNA delivery to mesenchymal stem cells in 2D and 3D. It is hoped that this work will serve as a template for future translational research within the field.
20941822305
/The abstract will be added later due to patenting/
20967801404
Embryos and regenerating systems produce very complex, robust anatomical structures and stop growth and remodeling when those structures are complete. How do cell collectives know what to build and when to stop? Control of this process would provide a radical solution to injury, cancer, and perhaps aging. In this talk, I will discuss new tools and data that are revealing how groups of cells use bioelectric networks to coordinate the building and repair of organs. I will show examples of molecular modulation of bioelectric signaling in vivo that enables regeneration of limbs and correction of complex birth defects. I will also discuss computational tools that are now enabling us to design combinations of ion channel drugs (electroceuticals) as a roadmap to regenerative medicine.
Covalent carbohydrate conjugates, referred to as glycans, decorate the surfaces of all mammalian cells and are found on nearly half of all mammalian proteins. Proteins that recognize specific glycan structures, known as lectins, play a central role in decoding the information stored within glycans and converting this information into signals that direct changes in cell behavior. Galectins are a specific subset of secreted lectins that can act as signaling molecules in healthy and pathological processes throughout all stages of life. Our research program employs supramolecular biomaterials as scaffolds to interrogate and manipulate galectin-glycan interactions. Supramolecular biomaterials are an ideal scaffold for these efforts because they allow us to mimic the multivalent architectures of galectins and glycans that are critical for their binding and biological activity. For example, we are developing beta-sheet peptide nanofibers modified with glycan appendages as synthetic glycoprotein mimetics. By tailoring glycan density or glycan chemistry on the nanofibers, we can tune their galectin binding affinity or specificity. We can use these glycopeptide nanofibers to disrupt or augment galectin signaling by changing the manner in which they are presented to cells (e.g. solution versus solid-phase). We also employ alpha-helical coiled-coils as scaffolds for multivalent galectin display. Using these scaffolds, we can create tools to study valency-function relationships of galectin-3. We can also use these scaffolds to create delivery vehicles that anchor a therapeutic enzyme at an injection site by endowing the enzyme with extracellular glycan-binding affinity. Finally, we can use coiled-coil scaffolds to create new anti-inflammatory therapeutics by recombining different galectins into non-natural multivalent architectures. Collectively, the examples presented in this talk will highlight the broad potential of supramolecular biomaterials to interrogate and manipulate galectin-glycan interactions, and identify new opportunities to exploit galectin-glycan interactions for tissue engineering.
52419502605
"Regulatory T cells (Tregs) are immuno-suppressive cells which have been recently rediscovered as pro-regenerative cells. When accumulating in injured tissue, Tregs express numerous pro-healing factors such as anti-inflammatory molecules and cytokines. Interestingly, we found that a growth factor (GF) is expressed by Tregs across multiple injured tissues in the mouse. Therefore, we hypothesized that delivering a recombinant form of the GF into damaged tissues would promote healing.
In order to optimize the activity of the GF following delivery in tissues, we first aimed at improving its signalling, utilizing rational protein engineering. As a strategy, we modified the receptor-binding site of the GF taking inspiration from the receptor-binding site of a high-affinity ligand of the same receptor, in order to generate a new high affinity variant (GFhi). The improved affinity of GFhi for the receptor was confirmed with various binding assays. A cell line was used to investigate the nature of the signalling through experiments of receptor internalization/degradation and phosphorylation. The regenerative capacity of GFhi compared to the wild-type GF was tested in mouse models of bone regeneration (calvarial defect), muscle regeneration (volumetric muscle loss of quadriceps) and skin repair (full-thickness wound). The GFs were delivered via a fibrin hydrogel in bone and muscle and via intradermal injections in the skin. To understand the mechanisms behind GFhi regenerative capacity, we tested its ability to affect proliferation of stem/progenitor cells which are important for the healing process such as keratinocytes, myoblasts, mesenchymal stem cells, and endothelial cells. We also investigated if GFhi could modulate macrophage activity, since they are key cells involved in tissue repair and regeneration. On this regard, we assessed GFhi regenerative potential in mice lacking its receptor on myeloid cells.
We show that GFhi has a higher binding affinity for its receptor compared to the wild-type GF. GFhi also shows a higher ability to induce receptor internalization/degradation and phosphorylation compared to the wild-type GF. In vivo experiments show that local delivery of GFhi promotes a more complete healing compared to the delivery of the wild-type GF in bone, muscle and skin models. Mechanistically, GFhi showed to have some proliferation effect on stem/progenitor cell. More interestingly, preliminary data where GFhi was delivered in mice lacking the GF receptor on myeloid cells indicates that the engineered GF likely promotes regeneration via modulating macrophage activity. Overall, this study shows that delivering a factor highly expressed by Tregs is able to induce tissue regeneration. Moreover, engineering the activity of key Treg-derived factors is a promising strategy for regenerative medicine applications."
52354502655
"INTRODUCTION:
Multiple organs consists of macroporous structures such as the lung, bone, and kidney. However, when creating porous materials, added factors are quickly washed away, thus there is a need to bind them to the material.
Elastin-like Recombinamer (ELR) hydrogel is a type of biomaterial that has proved to have excellent biocompatible properties [2], where we here create a macroporous version using the cryogelation technique and functionalize it with recombinant glycosaminoglycans to have a controlled release of growth factors.
METHODS:
ELR is a two-part solution, which are modified so that one part has an alkyne modification while the second part has an azide modification. When these two parts are mixed a covalent bond is formed resulting in a hydrogel. ELR cryogel was created by forming the hydrogel in subzero temperatures. Azide containing glycosaminoglycans were covalently added during the formation to slow the release of growth factors.
RESULTS:
ELR cryogels were subcutaneously implanted in mice, where cryogels with added growth factor only showed an increased blood vessel formation but the addition of recombinant glycosaminoglycans in combination with growth factor showed a change in the immune response going from a more inflammatory state to a more regenerative state shown by the shift of present macrophage phenotypes.
CONCLUSION:
The data showed that an ELR-based cryogel is a promising synthetic scaffold for tissue engineering, mimicking the 3D environment of the extracellular matrix and that recombinant glycosaminoglycans can be added for a controlled release of growth factors.
ACKNOWLEDGMENTS:
Financial support was received from the Swedish Heart-Lung Foundation, Swedish Research Council, Crafoord Foundation, Royal Physiographical Society of Lund, Österlund Foundation.
REFERENCES
[1] A. Ibanez-Fonseca et al. J Tissue Eng Regen Med. 2018;12:e1450–e1460."
41883627606
Background:
The repair and treatment of large bone defects in patients with compromised bone metabolism due to ageing and medical conditions such as osteoporosis present often a clinical challenge. Therefore adjunctive methods to enhance bone healing are needed. Bone tissue engineering with application of nanotechnology allows to construct biomaterials with desired properties being osteoconductive, osteoinductive and osteogenic.
Aim/Hypothesis:
The aim of our study was to promote bone regeneration using functionalised scaffold with Rhamnogalacturonan-I pectins (RG-I) in vitro and in vivo using aging and osteoporotic rodent models.
Material and Methods:
The biomaterials were poly(l-lactide-co-ε-caprolactone) scaffolds and the RG-I was from potato. The chemical and physical properties of functionalised biomaterials with RG-I nanoparticles were characterised using confocal and atomic force microscopy. Functionalised scaffolds with RG-I (tested sample) were evaluated in vitro with human osteoblasts from osteoporotic patients and their response was tested using real-time PCR. In vivo evaluation was performed using criticalsize calvaria bone defect model in ageing and osteoporotic rat models. Scaffolds were implanted randomly in the calvaria defects of aged female Wistar rats (11-12 months old) and osteoporotic female Wistar rats induced by ovariectomy. The control was scaffold without RG-I. After 2 and 8 weeks animals were euthanised. Harvested samples were analysed for osteogenic and inflammatory markers using real-time PCR. Bone formation was evaluated radiographically and histologically. The data was analysed using one-way ANOVA.
Results:
The chemical and physical properties results indicated success of the functionalisation of scaffolds with RG-I. Osteoblasts response suggested osteogenic (upregulation osteopontin, osteocalcin, collagen1, bone sialoprotein) and anti-inflammatory properties (downregulation IL-1, IL-8, TNFalpha) on the scaffold functionalised with RG-I. The in vivo results in aged and osteoporotic rat calvaria model of early (2 weeks) bone regeneration showed increase of osteogenic markers and decrease of proinflammatory markers and RANKL, compared to control. In osteoporotic rat model at week 2 and 8 and in aged rat model at week 8, the mean percentage of BV/TV (bone volume/tissue volume) in the defect with RG-I scaffold was significantly greater than the defect with control. The histological evaluation in both rat models revealed larger areas of new bone formation in RG-I scaffolds than in control.
Conclusion and Clinical implications:
In conclusion, the plant-derived nanoparticles significantly increased osteogenic and decreased pro-inflammatory response in vitro and in vivo. These finding may have a crucial impact on bone repair process especially in elderly and osteoporotic patients.
20941838684
Three-dimensional (3D) bioprinting has emerged as a class of promising techniques in biomedical research for a wide range of related applications. Specifically, vat-polymerization techniques such as digital light processing (DLP), are highly effective methods of bioprinting, which can be used to produce high-resolution and architecturally sophisticated structures. Nevertheless, conventional DLP bioprinting systems are hampered by several key limitations such as their bulky footprints, their insufficient multi-material bioprinting capacities, and their usual requirements on mechanically strong materials for volumetric bioprinting due to the layer-by-layer fabrication mechanism. In this talk, I will discuss our recent efforts on developing various DLP-based platforms that successfully tackle these challenges. These platforms will likely provide new opportunities in constructing functional regenerative and tissue modeling products in the future.
20967806797
Leveraging advances in biomaterials and tissue engineering, it is becoming possible to develop successful reparative, regenerative and tissue modelling solutions. Over more than two decades, we have made progress with studying biodegradable materials for reparative medicine. For example, biodegradable screws and plates were translated to the clinic as osteosynthesis implants. Scaffolds for in situ tissue engineering were investigated and applied for guided tissue regeneration, some of which were also translated to the clinic. We have also developed osteoconductive scaffolds for cell-based tissue engineering. To make scaffolds biomimetic, we developed nanofiber-based scaffolds made of different polymers and optimized their processing techniques using electrospinning. Because different biodegradable polymers elicit inflammatory reaction during their degradation process, controlled tissue response properties were added by the inclusion of anti-inflammatory drugs in these scaffolds. To improve the two-dimensional structure of the scaffolds, three-dimensional (3D) composite constructs were developed and their effect on preserving cell phenotype was demonstrated. To better control cell distribution in scaffolds, 3D bioprinting was pursued for developing cell-laden constructs. To improve cell survival in engineered scaffolds novel bioink based on improved nanostructure was developed and effect on improved cell survival was sown. In addition, the development of oxygenated bioink was pursued and its effect on enhanced cell survival was achieved. A new osteopromotive cell-survival enhancing bioink was developed. In addition to advances, dynamic flow is needed to better mimic native tissue environment. Integration of microfluidic systems and other construct processing techniques is needed to be able to produce advanced biomimetic tissue constructs useful for clinical applications as regenerative tools or as tissue models for disease studies and drug development. To achieve this, multidisciplinary approach and sustained funding are required.
"Introduction
Laryngeal cancer is often diagnosed at an advanced stage when treatment options are limited and most often restricted to laryngectomy. As this procedure requires a permanent tracheostoma, it is associated with additional complications such as a high risk of infection, fistulae formation and loss of the natural voice. In addition, restoration of the most important laryngeal function, protection of the airways, usually leads to deterioration of other functions like speaking or swallowing. To date, no method exists to simultaneously restore all laryngeal functions, resulting in a poor quality of life in the long term.
In future, a tissue engineered autologous laryngeal replacement could provide a way to ensure airway protection and voice production in parallel. A tissue engineered vocal fold would represent a milestone in developing a complete laryngeal replacement. As the native vocal folds undergo a broad variety of mechanical stresses in vivo such as tension, shear and impact, they exhibit specific biomechanical characteristics that also have to be met by tissue engineered constructs. In this study, we therefore developed a bioreactor that combines vibrational stimulation and stretching for the in vitro culture of vocal fold replacement tissues.
Methodology
To fulfil the tissue’s in vivo properties, we targeted a bioreactor with a frequency range between 100 and 300 Hz in combination with 20 % tensile strain, allowing alternating stimulation patterns with resting periods. Compatible scaffolds for cell seeding include elastic membranes and hydrogels. In addition, we aimed for a reusable, sterilizable and straight-forward design that is easy to implement. Electrical components were designed to avoid contact with the humidified incubator atmosphere, thus enabling long-term cultivation over several days to weeks.
Results
We developed a vocal fold bioreactor consisting of a transparent polymethyl methacrylate (PMMA) cylinder, polyoxymethylene (POM) scaffold holders and POM based housing parts. A linear module connected to a stepper motor implements the stretching of the scaffold while piezoelectric patches transmit vibration. Appropriate oxygenation of the medium is achieved via a silicon tubing loop. Cell compatibility of the bioreactor was evaluated for culture periods of up to 7 days.
Conclusion
Our bioreactor offers new perspectives for in vitro studies on mechanobiological processes in regenerating tissues. In addition, it represents a first step towards developing a vocal fold replacement tissue that may in the future provide new treatment options for laryngeal cancer patients after total laryngectomy."
94238112159
"Background: Hypospadias is a common congenital abnormality with varying severity. There may be lack of local tissue during hypospadias surgery. Oral mucosa (OM) is widely used for urethroplasties and repair after failed hypospadias in adults. Little is known about the applicability of OM in prepubertal boys because of eventual hormonal effect of puberty on OM. We investigated the androgen receptor (AR) localization and testosterone sensitivity in OM.
Materials & Methods: Small waste OM fragments of adult patients undergoing urethral surgery were collected under local biobank protocol. AR staining was performed on tissue sections and tissue was used for isolation of oral keratinocytes. Cells were serum starved, exposed to testosterone for different time points followed by immunostaining for AR.
Results: OM of patients with normal testosterone levels showed nuclear AR localization in the basal layer, and cytoplasmic AR localization in the apical layer, whereas OM of an adult hypospadias patient showed low cytoplasmic AR expression. The basal layer was Ki-67 positive, associated with cell proliferation. Oral keratinocytes of adult hypospadias patient exposed to testosterone showed switch from cytoplasmic to nuclear localized AR after 60 minutes, indicating that testosterone signaling was activated.
Conclusions: Oral keratinocytes are sensitive to testosterone. These findings suggest that OM is responsive to testosterone. Therefore an autograft transplant of OM in prepubertal boys might be suitable and able to keep up with the testosterone induced penile growth. In addition, this is an indication that OM cells can be used in tissue engineering for urethral reconstruction in pediatric patients."
52354511697
"Skin ageing is a multifactorial process attributed to both intrinsic and extrinsic factors. Intrinsic ageing is associated with changes that occur with age such as cumulative molecular and cellular damage, while extrinsic ageing exacerbates these changes through the UV exposure. Nowadays, ageing has become an important issue in the world: life expectancy and population have increased, while social and psychological factors have motivated a stronger desire for healthy ageing and a youthful appearance. Alongside other environmental factors, solar UV radiation is associated with extrinsic skin ageing, as it drives up to 80% of premature skin aging1 and it is one of the most potent carcinogens known. UV radiation induces structural and functional changes in both the epidermal and dermal compartments, such as erythema sunburn, tanning, DNA damage, inflammation, remodelling of the extracellular matrix, changes in epidermal barrier function, cellular proliferation and differentiation. The extent of these effects are mainly due to the degree of constitutive pigmentation of the skin.
Human neonatal skin cells were used to generate in vitro human skin equivalents as previously described in Roger et al.2. A UV irradiator system was used to simulate UV exposure on skin equivalents and they were characterised using a colorimeter, histology, immunofluorescence, melanin quantification and advanced microscopy.
UV-irradiated non-pigmented skin equivalents, which lack melanin protection, reveal structural epidermal changes, sunburn cells, DNA damage in form of cyclobutane pyrimidine dimers, apoptotic cells, and decreased expression of the differentiation marker filaggrin compared to the sham-irradiated non-pigmented skin equivalents. This effect appears to be dose-dependent, with a greater response identifiable following chronic irradiation. Conversely, UV-irradiated pigmented skin tans following chronic exposure, which is confirmed by an increased melanin content and demonstrates the protective effects of melanin by demonstrating a well-differentiated and organised epidermis and an absence of UV-induced damage. This melanin photoprotection is related to the tone of the pigmented skin equivalent, where the lightest skin tone has a greater UV impact than darker skin types.
We describe the characterisation of UV-induced skin equivalents, which recapitulate UV exposure of human skin and the role of melanin, providing a platform to test new and current formulations for cosmetic products designed to protect and treat the skin from harmful UV-exposure and ageing.
20941810604
Introduction: Three-dimensional bioprinting (3DBP) relies on extrusion-based methods for the printing process, however such methods are known to reduce cell viability due to the introduction of shear forces during extrusion. Additionally, the incorporation of solid elements into bioinks such as ceramic particles that may support bone growth, leads to increased shear forces during 3DBP leading to additional decreases in cell viability [1]. An innovative approach to overcoming these limitations is to develop hybrid bioinks where the inorganic osteopromotive components will be chemically linked with the organic matrix. To achieve the greatest degree of homogenization between the osteopromotive component(s) and the polymer matrix, it is required an in situ synthesis method. This work aims to utilize an innovative approach that combines a methacryloyl functionalized collagen-derived gelatin (GelMA) with an Ag-doped bioactive glass (GAB) to deliver a novel osteopromotive and antibacterial hybrid hydrogel material. Chemical, structural, and antibacterial characteristics of the new hybrid material GAB were studied. Methodology: The synthesis of GelMA is performed as described in the literature [2,3] and then the solution was frozen and lyophilized before storage. For the synthesis of GAB hybrid material, the lyophilized GelMA is dissolved in DMSO, a coupling agent 3-Glycidyloxypropyl trimethoxysilane (GPTMS) is added to the solution. The sol-gel process was used to synthesize the Ag-doped bioactive glass (Ag-BaG) following previously described methods [4]. The combination of the solutions leads to precipitation, then washes and lyophilization to prevent the materials characteristics from changing during storage. Prior to the further use, lyophilized GAB was dissolved in phosphate buffered saline (PBS) along with the photoinitiator and photopolymerized producing GAB hybrid hydrogels. Structural characterization, performance, printability, and antibacterial properties were studied. Results: Rheological evaluation found the GAB exhibited shear thinning behavior, which is a preferential characteristic for printability. The incorporation of the Ag-BaG was found to be homogenous at the molecular level that led the GAB to exhibit less amount of swelling and the slow degradation behavior compared to GelMA alone. Significant antibacterial inhibition was achieved by GAB against MRSA. Conclusions: GAB is expected to be suitable for extrusion-based 3DBP technologies and expected to improve cell viability of the 3D printed constructs due to the absence of particulate components. The antibacterial characteristics can advance the performance and success of the printed constructs.
20941856968
INTRODUCTION: Vascular endothelial growth factor (VEGF) is the principal regulator of angiogenesis. Tightly bound gradients to extracellular matrix determine the microvascular endothelial cells fate during organogenesis and tissue regeneration and uncontrolled expression can lead to abnormal vascular growth and vascular tumours 1. Despite advances in tissue engineering, tight spatio-temporal control of VEGF remains a challenge hindering its therapeutic application. Nanoclay-gels have established potential in tissue engineering due to their capacity to sequester proteins for sustained and localised bioactivity 2, 3. The current study reports a biomimetic method to applying self-assembling nanoclay-gels comprising 3D gradients of VEGF to support localised spatio-temporal formation of microvasculature.
METHODOLOGY: Hydrous suspension of Laponite®, a synthetic smectite clay, were added to a solution containing biomolecules to facilitate the structured gels assembly via reaction-diffusion. The assembled structures were loaded with punctured VEGF gradients. Its spatial distribution and concentration were confirmed with fluorescent imaging and ELISA, respectively. The biocompatibility and bioactivity were assessed with a human umbilical vein endothelial (HUVEC) tubule formation assay and a 28-day murine subcutaneous implantation. A contrast agent was injected to visualise the new blood vessel formation via µCT and corroborated with immuno-staining.
RESULTS: Structured gels were able to controllably pattern the distribution of VEGF in 3D with a resolution of 40 - 120µm depending on assembly conditions. Patterned gels supported tubule formation of HUVECS grown on gel surfaces and µCT analysis of the in vivo study indicated vascularization of gels within regions of VEGF patterning. This was confirmed by histological analysis showing progressive cell invasion and degradation of the gel, and microvessel formation within the projected area of the VEGF pattern.
CONCLUSIONS: Clay-based structured gels are a promising delivery system of growth factors for tissue engineering applications with clinical relevance. Here we demonstrated for the first time its capacity to hold VEGF gradients for localized bioactivity.
REFERENCES:
1. Gianni-Barrera, R. et al. Stem Cells Transl Med. 9, 433-444 (2020).
2. Dawson J, I. et al. Adv Mater. 23, 3304 - 3308 (2011).
3. Page D, J. et al. Acta biomater. 100, 378-387 (2019).
94238144105
"Introduction
We have derived horse embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). Pluripotent stem cells undergo unlimited self-renewal and can differentiate into every tissue of the body; properties which can be utilised for therapeutic applications and disease modelling.
We have investigated their application in tendon injuries and catastrophic bone fractures, conditions that have major welfare and economic impacts on equine industries. Tendon injuries are a leading cause of retirement in horses taking part in a range of disciplines and catastrophic fracture is the number one reason for euthanasia during Thoroughbred racing.
Adult tendon injuries repair through scar tissue formation which predisposes horses to high rates of re-injury and novel therapies to aid tendon regeneration are required. Equine ESCs turn into tendon cells following their injection into the injured tendon1 and we have established 3D in vitro culture methods2 to define their properties and compare them to adult cells.
The risk of catastrophic fracture is influenced by environmental and genetic factors. A better understanding of the genetic risk factors would enable improved identification and management of high risk horses. Equine iPSCs can be derived from horses with different genetic backgrounds. This has allowed us to establish an in vitro model3 to understand the genetic basis of catastrophic fracture risk.
Methodology
We have established protocols to differentiate ESCs and iPSCs into tendon and bone (osteoblast) cells using both 2D and 3D culture methods.
Global gene expression profiling using RNA sequencing has been used to make comparisons between ESC-derived, adult- and fetal-tenocytes, and between iPSC-osteoblasts derived from horses at high and low genetic risk of fracture.
Other properties of ESC-tenocytes such as cell migration and response to inflammation have also been measured. Molecular biology techniques to understand the impacts of DNA variants associated with fracture are also being used.
Results
We have demonstrated that ESC-tendon cells more closely resemble fetal than adult tenocytes4 but that they have properties that are unique to them, such as resistance to inflammation5 and unpublished data.
iPSC-osteoblasts taken from horses at high and low risk of fracture were found to have 112 differentially expressed genes. Some of which have known roles in bone formation or fracture. Pairing gene expression information with whole genome sequencing data allows us to identify putative causal single nucleotide polymorphism (SNPs) that may be responsible for these differences and we highlight results for COL3A1 that demonstrate the power of this approach.
Conclusions
Pluripotent stem cells from horses not only provide a source of cells for potential therapeutic use, but they can also be used for disease modelling. They therefore hold great promise to allow the future development of novel interventions and therapies.
References
1. Guest, D.J.et al. Equine vet. J., 2010. 42(7): p. 636-642.
2. Barsby, T., et al. Tissue Eng Part A, 2014. 20: p. 2604-2613.
3. Baird, A., et al., Biology Open, 2018. 7(5): p. bio033514.
4. Paterson, Y., et al., Mechanisms of Development, 2020. 163: p. 103635.
5. McClellan, A., et al., Sci Reports, 2019. 9(1): p. 2755."
83871201986
"Background Synovial membrane-derived mesenchymal progenitor cells (SM-MPCs) are a promising candidate for the cell-based treatment of osteoarthritis (OA) considering their in vitro and in vivo capacity for cartilage repair. However, the OA environment may adversely impact their regenerative capacity. There are no studies for canine (c)SM-MPCs that compare normal to OA SM-MPCs, even though dogs are considered a relevant animal model for OA. Therefore, this study compared cSM-MPCs from normal and OA synovial membrane tissue to elucidate the effect of the OA environment on the MPC numbers indicated by CD marker profile and colony-forming unit (CFU) capacity, and the impact of the OA niche on tri-lineage differentiation.
Methods Normal and OA synovial membrane were collected from the knee joints of healthy dogs and dogs with rupture of the cruciate ligaments. The synovium was assessed by histopathological OARSI scoring and by RT-qPCR for inflammation/synovitis-related markers. Presence of cSM-MPCs in the native tissue was further characterized with flow cytometry, RT-qPCR and immunohistochemistry, using the MPC markers; CD90, CD73, CD44, CD271, and CD34. Furthermore, cells isolated upon enzymatic digestion were characterized by CFU capacity, and a population doublings assay. cSM-MPCs were selected based on plastic adherence, expanded to passage 2 and evaluated for the expression of MPC-related surface markers and tri-lineage differentiation capacity.
Results Synovial tissue collected from the OA joints had a significantly higher OARSI score compared to normal joints, and significantly upregulated inflammation/synovitis markers S100A8/9, IL6, IL8 and CCL2. Both normal and OA synovial membrane contained cells displaying MPC properties, including a fibroblast-like morphology, CFU capacity, and maintained MPC marker expression over time during expansion. However, OA cSM-MPCs were unable to differentiate towards the chondrogenic lineage and had low adipogenic capacity in contrast to normal cSM-MPCs, whereas they possessed a higher osteogenic capacity. Furthermore, the OA synovial membrane contained significantly lower percentages of CD90+, CD44+, CD34+ and CD271+ cells.
Conclusions The OA environment has adverse effects on the regenerative potential of cSM-MPCs, corroborated by decreased CFU, population doubling and chondrogenic capacity compared to normal cSM-MPCs. OA cSM-MPCs may be a less optimal candidate for the cell-based treatment of OA than normal cSM-MPCs."
20941815455
"Background Osteoarthritis (OA) is a common degenerative joint disease, that affects the whole joint. Knee joint distraction (KJD) has been proposed as an alternative joint-preserving treatment strategy for relative young patients with end stage OA. Although there is evidence for the clinical and structural benefits of KJD, the regenerative mechanisms behind KJD remain unclear. This study explores the role of the synovial membrane (SM), an important, but to date limited researched part of the distracted joint, by studying the SM secretome and its effect on chondrocytes.
Methods Osteoarthritis was bilaterally induced using the groove OA model and allowed to develop for 10 weeks in 12 dogs. Subsequently, KJD was applied unilateral to the right hindlimb for 8 weeks. To determine the role of the SM during KJD, the SM and conditioned medium of the synovial membrane of OA (OA-CM) and OA+KJD joints (KJD-CM) were investigated directly after KJD treatment (n=4), and after 10 weeks of follow-up after KJD treatment (n=8) using RT-qPCR analysis and a canine-specific multiplex ELISA. Furthermore, clinically normal articular chondrocytes (n=3), synovial membrane (n=6) and CM (normal-CM) from healthy dogs were collected for comparison. The effect of OA-CM and KJD-CM on isolated normal articular chondrocytes was investigated using RT-qPCR and Nano luciferase response element reporter assays for targeted signalling analysis of pathways involved in anabolic, catabolic and inflammatory processes during OA.
Results In the synovial membrane and conditioned media the expression of pro-inflammatory cytokines (CCL2, IL-6, IL-7, IL-15, and IL-18) was increased directly after KJD compared to the normal SM. After 10 weeks of follow-up, these cytokines were still elevated, except for IL-6, compared to directly after KJD. Directly after KJD, the OA and the KJD-CM had a clear catabolic effect on articular chondrocytes, as established by decreased expression of the cartilage matrix genes ACAN and COL2A1 and increased expression of the catabolic genes MMP13 and ADAMTS5. After 10 weeks of follow-up, this effect shifted towards a less catabolic response. The reporter assays showed an upregulation of CRE and SIE signalling after stimulation with OA-CM and KJD-CM compared to normal-CM. SRE and SRF signalling was downregulated in the presence of the KJD-CM at follow-up compared to the KJD-CM from directly after KJD.
Conclusion OA and KJD result in inflammation of the SM, characterized by increased levels of pro-inflammatory cytokines and chemokines. The SM secretome directly influences cartilage matrix metabolism and cellular response, corresponding with the catabolic environment in the cartilage that is found directly after KJD. After follow-up the catabolic influence of the SM decreases. Therefore, the SM may contribute to the effect that joint distraction exerts on the OA joint. However, as the effect is small, in order to gain further insights, the sample size should be increased. Furthermore, more cartilage protective pathways should be investigated to investigate the role of the SM in the regenerative mechanisms behind joint distraction."
41883615355
"Introduction:
Intervertebral disc (IVD) degeneration is among the leading cause of low back pain, disability and morbidity worldwide. As the world population ages and only symptomatic treatment exist, IVD regeneration is a major public health challenge for the upcoming years. Human IVD cells are difficult to obtain, especially healthy ones, and murine IVDs present numerous differences with human ones (size, mechanical loading, presence of notochordal cells). The sheep spine exhibits biological and biomechanical similarities with the human one and is thus recognized as an appropriate model for translational applications1. With the goal of reducing our reliance on animal models, and for economic, regulatory, and ethical reasons, we have set up an in vitro platform based on sheep annulus fibrosus (AF) and nucleus pulposus (NP) cells to evaluate cell and extracellular vesicles (EVs) therapies.
Methodology:
Cells from both the AF and NP were isolated from the IVDs of five young sheep (≈ 3 months old). Their metabolic activity and gene expression were evaluated by CCK-8 assay and RT-qPCR. To simulate a degenerative IVD microenvironment, disc cells were treated with either recombinant sheep IL-1β (10 ng/mL) or H2O2 (500 µM) or maintained in culture for over 10 passages. EVs from human adipose-derived mesenchymal stromal cells (hASCs) were produced in a turbulent flow as previously described2 and their effect on degenerative-like NP and AF cells was evaluated. In a separate experiment, hASCs cells were co-cultured with NP or AF cells, in direct contact or transwells. The use of two species allowed us to analyze RNA expression from disc cells in direct co-culture with hASCs by using sheep-specific primers.
Results:
Prolonged culture and treatments with IL-1β or H2O2 led to a significant overexpression of inflammatory cytokines (IL6, CXCL8), matrix metalloproteinases (MMP1, MMP2, MMP3, or MMP13 depending on the treatment), and downregulation of key components of the extracellular matrix (COL1A1 & COL2A1) at the transcriptional level. While EVs consistently increased basal metabolic activity of both AF and NP cells at early and late passages, they had little effect on gene expression. On the other hand, direct cocultures with human ASCs profoundly affected disc cells’ transcriptional profile. Notably, both types of cocultures led to a drastic downregulation of CXCL8 in disc cells, reduced by over 60% in indirect coculture and even undetectable in direct coculture, while we observed an upregulation of COL1A1 but also MMP1. Surprisingly, IL6 expression showed a slight increase with hASCs on transwells but a sharp decrease by over 80% when hASCs were in direct contact.
Conclusion:
We demonstrated that healthy sheep cells expressed markers of degeneration after IL-1β and H2O2 treatment, or after numerous passages in culture. They showed biological responses to hASCs and, to a lesser extent, to hASC-derived extracellular vesicles. These results confirm the suitability of sheep disc cells to model IVD degeneration in vitro and assess biotherapies.
"The phenotypic characterization of multipotent mesenchymal stromal cells (MSC) still suffers from deficits and the resulting heterogeneity of MSC used in different preclinical and clinical studies hamper the translational success. In search for novel MSC characterization approaches to complement the traditional trilineage differentiation and immunophenotyping assays reliably across species and culture conditions, this study explored the applicability of lipid phenotyping for MSC characterization and discrimination. Human peripheral blood mononuclear cells (PBMC), human fibroblasts, and human and equine adipose-derived MSC were used to compare different mesodermal cell types and MSC from different species. For MSC, cells cultured in different conditions, including medium supplementation with either fetal bovine serum or platelet lysate as well as culture on collagen-coated dishes, were additionally investigated. After cell harvest, lipids were extracted by chloroform/methanol according to Bligh and Dyer. The lipid profiles were analysed by an untargeted approach using liquid chromatography coupled to mass spectrometry (LC-MS) with a reversed phase column and an ion trap mass spectrometer. In all samples, phospholipids and sphingomyelins were found, while other lipids were not detected with the current approach. The phospholipids included different species of phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI) and phosphatidylserine (PS) in all cell types, whereas phosphatidylglycerol (PG) species were only present in MSC. MSC from both species showed a higher phospholipid species diversity than PBMC and fibroblasts. Few differences were found between MSC from different culture conditions, except that human MSC cultured with platelet lysate exhibited a unique phenotype in that they exclusively featured PE O-40:4, PG 38:6 and PG 40:6. In search for specific and inclusive candidate MSC lipid markers, we identified PE O-36:3 and PG 40:7 as potentially suitable markers across culture conditions, at which PE O-36:3 might even be used across species. On that basis, phospholipid phenotyping is a highly promising approach for MSC characterization, which might condone some heterogeneity within the MSC while still achieving a clear discrimination even from fibroblasts. Particularly the presence or absence of PG might emerge as a dinstinctive criterion for future MSC characterization.
This study has recently been published under the creative commons license in Front Cell Dev Biol. (2021 Dec 1;9:784405; doi: 10.3389/fcell.2021.784405)."
83767231959
Introduction:
Immunomodulation and immunogenicity of mesenchymal stem cells (MSCs) may influence their efficacy and safety, thus being key for their therapeutic use. The immune regulatory mechanisms of MSCs depend mostly on the secretion of different mediators which are not only important for the MSC therapeutic mechanisms but also to facilitate their escape from immune recognition when administered allogeneically. Actually, a highly relevant paradigm change is that MSCs are not truly immune-privileged but immune-evasive, and thus, their recognition and elimination by the immune system in the allogeneic setting should be considered. Since the horse is highly valuable as both patient and translational model, further knowledge on equine MSC immune properties is required. This study analyzed how inflammation, chondrogenic differentiation and compatibility for the major histocompatibility complex (MHC) influence the MSC immunomodulatory-immunogenicity balance by studying the changes elicited in vitro by equine MSCs on relevant lymphocyte subpopulations.
Methodology:
Equine MSCs in basal conditions, pro-inflammatory primed (MSC-primed) or chondrogenically differentiated (MSC-chondro) were co-cultured with either autologous or allogeneic MHC-matched/mismatched lymphocytes. Two types of co-cultures were used: immunosuppressive assays to study MSC immunomodulatory potential, and one-way modified mixed leukocyte reactions (MLRs) to assess MSC immunogenicity. Lymphocytes were stained with carboxyfluorescein succinimidyl ester (CFSE) and with a panel of antibodies to study changes in the frequency and proliferation of T cell subsets (cytotoxic, helper and regulatory) and B cells by flow cytometry.
Results:
Overall, MSC-primed were superior suppressing the proliferation of lymphocytes, followed by MSC-chondro and MSC-naïve. The proliferation of CD3+ T cells was reduced in the presence of all types of MSCs and for all the combinations (autologous and allogeneic MHC-matched and mismatched). When looking at specific lymphocyte subsets, MSC-primed showed higher regulatory potential of the proliferation of cytotoxic and helper T cells and B cells, while inducing T reg cells in the one-way MLRs. However, MHC-mismatched MSC-primed can also elicit a proliferative response in lymphocytes likely due to increased MHC expression. Interestingly, equine MSC-chondro maintained their regulatory ability and did not increase their immunogenicity, but showed less capacity than MSC-primed to induce regulatory T cells and further stimulated B cell proliferation.
Conclusions:
Priming MSCs with proinflammatory cytokines activates their regulatory potential. However, inflammation can also increase the immune recognition of these cells through induction of MHC expression, thus making the allogeneic MHC-mismatched MSC-primed more likely to be targeted by the immune system. Importantly, equine MSCs do not lose their regulatory ability neither increase their immunogenicity after chondrogenic differentiation, but have reduced capacity to stimulate Treg cells and can stimulate the proliferation of B cells.
Even though lymphocyte proliferation assays are important tools to assess both the immunomodulatory and immune evasive properties of MSCs, subsequent in vivo studies are needed to elucidate the complex interactions between MSCs and the recipient immune system, which is critical to develop safe and effective therapies.
31412730807
"EVs in equine regenerative medicine – challenges and potential therapeutic implications.
Mesenchymal stem/stromal cell (MSC) derived EVs have shown an equivalent therapeutic potential to their donor cells, making them a promising tool for regenerative medicine. However, non-standardised EV isolation methods and the limited availability of cross-reacting markers for most animal species restrict comparability and standardisation of animal experiments.
We therefore performed a study to establish an EV isolation and characterisation protocol for equine MSC-derived EVs and define minimal criteria based on the ISEV criteria, which will facilitate reproducibility and comparison of equine derived EVs between studies in the future. Furthermore, we studied the therapeutic effect of equine bone marrow MSC (bmMSC) derived EVs on inflamed horse tenocytes in vitro and evaluated the influence of inflammatory preconditioning (pc) of the EV donor MSC on the content and biological activities of their EVs.
Equine bmMSC dericed EVs from 3 donors were isolated using two different techniques, size exclusion chromatography (SEC) and ultracentrifugation technique (UC). To establish minimal criteria for equine EVs, we validated markers (CD63 and CD9) for Western blot analysis and tested a combination of methods including techniques that do not require antibodies, such as Nanoparticle Tracking Analysis (NTA) and trans electron microscopy (TEM), with Fluorescence-triggered flow cytometry (FT-FC).
To evaluate their therapeutic effect, EVs were obtained from supernatants of bmMSCs preconditioned in serum free media with (pcMSC) or without (npcMSC) addition of 10ng/ml IL1β and 10ng/ml TNFα. Chemically inflamed (10ng/ml IL1β and 10ng/ml TNFα ) tenocytes (400.000 cells) were treated with 1ml of medium containing 1x10^9 autologous pcMSC or npcMSC derived EVs. Untreated chemically inflamed tenocytes and healthy tenocytes served as control groups. RNA-Sequencing (RNA-Seq) of EVs and tenocytes was performed. Adjusted p-value was set at 0.25.
The isolated particles stained positive for CD9 and CD63 as demonstrated by Western blot and for CD81 using FT-FC. FT-FC, NTA and TEM confirmed the EV-appropriate size range (between 30 and 150 nm) and TEM the presence of the characteristic lipid bilayers surrounding EVs.
Application of pcMSCs as well as npcMSC derived EVs significantly reduced expression of inflammation markers like CXCL6 (-1.4 and - 1.6 logFC), CSF3 (-3.3 and - 4.1 logFC), CXCL8 (-2.2 and - 2.2 logFC), CXCL6 (-1.4 and -1.6 logFC), TNFAIP6 (-1.4 and - 1.4 logFC) in treated tenocytes as compared to untreated controls. miRNA-Seq of the EVs showed significant down-regulation of miR-146a (-0.6 and -0.7 log2FC) which has been shown to play an important role in the negative regulation of inflammation in rheumatoid arthritis.
The obtained data allows to suggest minimal criteria for the standardized isolation and characterization of equine MSC derived EVs. The results further indicate that the cargo of pcMSC-EVs and npcMSC-EVs may reduce tenocyte inflammation and may have an immunomodulatory effect on the recipient cells which is independent of pre-conditioning of the EV donor cells."
52354523526
"Introduction
Tendon injuries in humans are a major healthcare concern but they also occur spontaneously in other species including horses. The superficial digital flexor tendon (SDFT) of the horse is a highly relevant model for studying tendinopathies because it is a functional homologue of the Achilles tendon with similarities in aetiopathogenesis and associated risk factors (1,2). Previous studies with cyclical loading of tendon explants have revealed that injured SDFTs produced high levels of pro-inflammatory cytokines and 10-1000-fold overexpression of matrix metalloproteinases with impaired resolving mechanisms, especially in older horses (3). In combination, these markers are hallmarks of senescent cells. Senescent cells are generated by many pathways (both cell division dependent and independent) all of which produce viable cells that (a) overproduce pro-inflammatory cytokines collectively known as the Senescence Associated Secretory Phenotype (SASP) and (b) actively remodel matrix, all of which are implicated in age-related degenerative changes that predispose to tendinopathies. Corticosteroids frequently used clinically to treat tendinopathies initially block the inflammatory component of tenocyte senescence but also may cause therapy-induced senescence and interfere with tendon repair, contributing to the high re-injury rate (4). The aim of this study was therefore to use an in vitro model to investigate the effects of dexamethasone on senescence.
Methodology
Tendon derived cells (TDCs), isolated from the superficial digital flexor tendon (SDFT) of adult horses (11 years old), were cultured in DMEM (supplemented with 10% fetal bovine serum and 1% P/S) in a humidified incubator with 5% CO2 at 37°C. TDCs were treated with 1 and 10µM dexamethasone for 48 hours and then cultured in medium without dexamethasone for a further 24 and 72h. The effect of dexamethasone on cell viability was measured by the MTT assay at 24 h. Senescence was confirmed in treated cells by cytochemical analysis with EdU (Click-iT Plus EdU Cell Proliferation Kit, Fisher) and Ki-67 (Rabbit monoclonal to Ki67, Abcam).
Results
The viability of TDCs was not affected by 1 and 10 µM doses of dexamethasone treatment. However, dexamethasone at both concentrations inhibited cell proliferation and induced cell cycle arrest. Senescence in TDCs was confirmed by reduced expression of proliferative-associated protein (Ki-67) and reduced DNA synthesis (EdU incorporation). Exposure to dexamethasone at both doses for 48 hours rendered more than 50% of the tenocytes senescent.
Conclusion
Dexamethasone at clinically relevant doses induced growth arrest in equine tenocyte cells. These data provide a mechanistic explanation for potential adverse effects of using corticosteroids for the treatment of tendinopathies. The model will enable investigations of novel candidate molecules that can slow or stop the degenerative process by inhibiting senescence in vitro, which could be used to improve the clinical benefits of corticosteroids via its anti-inflammatory effects.
References
1.Lui PP, Maffulli N, Rolf C, et al. Scand J Med Sci Sports 2011;21:3-17.
2.Patterson-Kane JC, Rich T. ILAR J 2014;55:86-99.
3.Hosaka Y, Kirisawa R, Yamamoto E, Ueda H, Iwai H, Takehana K. L. J Vet Med Sci. 2002;64(10):945-7.
4.Poulson RC, Watts AC, Murphy RJ, Snelling SJ, Carr AJ, Hulley PA. Ann Rheum Dis. 2014;73(7):1405-13
31412717204
"MicroRNAs, short non-coding RNAs, are important regulators of skeletal development. Several individual microRNAs have been shown to alter long bone growth with most interest focused on the cartilage ‘specific’ microRNA, miR-140. We made a new miR-140-null mouse and confirmed the skeletal phenotype, identifying a range of target genes that the microRNA controls. The null mice also have increased joint damage during surgically-induced osteoarthritis and cartilage transcriptome analysis has revealed novel pathways in which the microRNA functions during joint destruction.
Less is known about microRNAs and bone development, though serum microRNAs are biomarkers for osteoporosis. We identified a little studies microRNA that appears important in bone ageing. Mice null for this microRNA develop bone thickening with age, with increased cortical and trabecular bone thickening. In the talk I will discuss how this one microRNA fine tunes osteoblast, osteoclast and osteocyte transcriptomes and thus function to regulate bone formation and turnover."
62903402364
"Increasing attention is being paid to the use of RNA molecules as agents to facilitate tissue healing. Messenger RNAs (mRNAs) introduced into the cell induce synthesis of their encoded proteins. This approachholds several advantages over traditional DNA-based gene therapy including improved cell internalization and higher transfection efficiencies, without disruption of the host genome. In the context of tissue engineering and regenerative medicine, the eventual turnover of the exogenous RNA is a further advantage. Because conventional RNAs have strong immunogenicity and low stability, chemical modifications are needed to facilitate their therapeutic application. Moreover, efficient delivery systems are of crucial importance for efficient cell internalization.
This talk will cover progress on mRNA therapeutics with a focus on tissue engineering and regenerative medicine. Particular interest will be placed on applications of mRNA for musculoskeletal regeneration. The presentation will transit from mRNA biology and biochemistry to the internalization of RNA using different biomaterials and non-viral delivery systems, ending with novel applications reported for the regeneration of diverse tissues. In terms of chemical modifications, the use of modified nucleotides, UTRs, and optimized poly(A) tails among other strategies will be reviewed. Regarding RNA delivery, examples of novel lipid and polymer delivery systems will be shown. Also, the advantages of mRNA internalization techniques such as magnetofection will be highlighted. Finally, examples of preclinical studies on mRNA applications to enhance the regeneration of bone tissue will be discussed. Overall, this talk will give an overview of the cmRNA technology from basic aspects to preclinical proof of concepts."
31451703755
Background:
Cell adhesion can be guided through the mechanical signals provided by the biomaterials1,2. These signals are transmitted to the cytoskeleton, to the nuclei and finally to the chromatin3. The chromatin remodeling is highly dynamic and sensitive to these cues and imposes extensive effects on DNA-related metabolism, including transcription. Recently, chromatin has been identified as a mechanosensitive compartment that is shaped as much by external forces pressing down upon it, as internal forces pushing outwards from the chromatin: this dynamic process activate or silence genes. Chromatin therefore has a major role in the fate of the cell, in particular for the stem cell. Inversely, how chromatin communicates cues back to the nuclear membrane is poorly understand. Particularly, whether chromatin structure impacts by itself cell adhesion, more precisely the cell adhesion strength, is an underexplored topic.
Methods:
We have investigated if hyper-condensation of chromatin by ATP depletion (with sodium azide and 2-deoxyglucose, SA2D) or by inhibition of histone acetyltransferases (with anacardic acid, ANA) alters epithelial cell adhesion strength. This adhesion force is measured by fluidic force microscope (FluidFM), an atomic force microscope-driven micropipette4. Through this induced remodeling of chromatin, we investigated the organization of the cytoskeleton and focal adhesion.
Results:
Chromatin compaction within nuclei is induced by SA2D or by ANA. Our results demonstrate that this phenomenon is accompanied by a decrease of chromatin acetylation, an increase of histone H3K27 methylation and a decrease of the nuclear area. This leads to reorganization of the actin cytoskeleton showing small stress fibers localized around the nucleus while focal adhesion contacts decrease in size compare to untreated cells. By videomicroscopy, we show that after drugs remove from to the culture medium, cells morphologic are restored with nucleus decondensed. We observed by FluidFM that chromatin compaction by SA2D or ANA significantly decreases cell adhesion strength compared to untreated cells.
Conclusions:
These results show for the first time that structure of the chromatin physically impact cell adhesion strength. Moreover, this chromatin remodeling is correlated with a global deacetylation of the nucleus and impact the dynamic of the cytoskeleton and the focal adhesion contacts.
A new open question is to determine how cell adhesion strength is mechanically impacted by chromatin structure, especially during stem cell differentiation and thus could provide essential data in epigenetic cell reprogramming for stem cell tissue engineering. Another attractive question will be to determine how a cell rapidly coordinates external forces with internal genomic forces to accomplish mechanosensitive biological processes.
References
1. Rabineau, M. et al. Cell guidance into quiescent state through chromatin remodeling induced by elastic modulus of substrate. Biomaterials 37, 144–155 (2015).
2. Rabineau, M. et al. Chromatin de-condensation by switching substrate elasticity. Sci. Rep. 8, 12655 (2018).
3. Argentati, C. et al. Insight into Mechanobiology: How Stem Cells Feel Mechanical Forces and Orchestrate Biological Functions. Int. J. Mol. Sci. 20, 5337 (2019).
4. Guillaume-Gentil, O. et al. Force-controlled manipulation of single cells: from AFM to FluidFM. Trends Biotechnol. 32, 381–388 (2014).
52354524255
"It is well known that cartilage possesses a low intrinsic regenerative potential, causing tissue damage that remains unhealed and contributes to a high socioeconomical burden for affected patients. New strategies to restore the properties of load bearing, friction-reducing hyaline cartilage are thus timely. Tissue engineering and regenerative medicine approaches must deal with the high donor-to-donor variability typical of primary human cells, including bone marrow derived-mesenchymal stromal cells (BMSCs). Recently, we have demonstrated that the chondrogenic potential of BMSCs can be predicted on a donor-specific basis by the ratio between the gene expression levels of two main TGF-β receptors, namely TGFBR1 and TGFBR2 [1]. Also, a transient downregulation of the TGF-β receptors TGFBR2 and ACVRL1 was sufficient to reverse the phenotype of cells that poorly responded to TGF-β1 stimulation and increased their chondrogenic commitment in pellet culture. In the field of regenerative medicine, major efforts focus on engineering hyaline cartilage of clinically relevant sizes which, in addition to the cell type, requires an adequate 3-dimensional cell culture substrate or scaffold. Therefore, the aim of this study is to validate the translational potential of those findings and explore the feasibility of siRNA delivery in hydrogels to improve chondrogenic differentiation of human BMSCs.
For this purpose, human BMSCs were isolated and expanded from the bone marrow of patients undergoing spinal surgery, with full ethical approval (Bern Req-2016-00141) and written informed consent. Gelatin methacryloyl (GelMA) was synthesized using Gelatin type A from porcine skin and methacrylic anhydride to reach a 50% degree of substitution. Gels were prepared as 8% w/v GelMA in PBS containing 0.3% Irgacure and cured at 365 nm. Fibrin was prepared using Fibrinogen and Thrombin from human plasma (Sigma-Aldrich) to a final concentration of 25 mg/ml fibrinogen and 1 U/ml thrombin, respectively. Cells were encapsulated in either GelMA or fibrin at a density of ~20x106 cells/ml. For siRNA delivery, gels were supplemented with either a negative control or a TGFBR2-targeting siRNA (10 pmol, Thermo Fisher) complexed with Fuse-It-siRNA reagent (Ibidi). Constructs were cultured for up to 3 days for RNA isolation and RT-qPCR analysis. Samples were stained with calcein green and ethidium homodimer for analysis of cell viability at 7 days.
Confocal microscopy was used to evaluate the Live/Dead staining of constructs after 7 days in culture, showing good viability and even distribution of embedded cells. Up to 3 days, gene expression analysis from fibrin constructs showed a consistent downregulation of TGFBR2, resulting in an increase of the TGFBR1/TGFBR2 ratio. This phenomenon was much less pronounced in GelMA, potentially because of the lower migration ability of cells within this hydrogel. Future evaluations will explore the chondrogenic differentiation potential of distinct BMSC donors within different hydrogels and in response to siRNA delivery. We suggest that this method will increase the therapeutic efficacy of patient-specific cell-hydrogel based constructs for cartilage regeneration.
The work is supported by AO Foundation and AO Research Institute Davos. GC was funded by the Chinese-Swiss Young Researchers' Exchange Programme (EG-CN_04-032019).
[1] Rothweiler et al. (2020) Front Bioeng Biotechnol 8:618."
52354515324
"Introduction
Silica based materials have been commonly studied in the field of bone regeneration, due to their bioactivity and osteogenic properties1. However, silica materials have low degradation rates and are brittle, which can be overcome by developing hybrid materials, which include an organic part bound to the silica network2. Despite these can be processed in several shapes, there is a need to fabricate these materials in a custom made morphology to adapt better to the site of defect, producing scaffolds with a desired porosity, shape and size3. However, 3D printing of silica materials normally uses very high temperatures to obtain the final structure which limits the ability to encapsulate bioactive molecules. An alternative to produce silica based materials at low temperatures is the sol-gel method, in which a precursor forms a silica network using pH or temperature as a catalyst4. Therefore, in this study, a printable sol-gel silica-based hybrid has been developed by combining tetraethylorthosilicate (TEOS), and gelatin that were cross-linked with (3-Glycidyloxypropyl)trimethoxysilane (GPTMS).
Methodology
The inks were prepared by adjusting the amount of TEOS and GPTMS in the sol and the proportion of gelatin added and mixing them at 37ºC to obtain printable inks. The developed inks were 3D printed using a Cellink BioX printer, and the different physicochemical properties were analyzed. As control, pristine printed silica was used. The analyzed properties were shape fidelity, water uptake, degradation, mechanical properties and bioactivity. Cytotoxicity and proliferation were assessed using rat mesenchymal stem cells, counting the cells with a PicoGreen assay and observing the morphology with an immunofluorescence assay. In vitro osteogenic differentiation was also assessed using rMSCs and osteogenic media, using pristine silica and PLA as controls. The differentiation was measured by ALP assay. Statistical significance was accepted at p<0.05.
Results
Three different inks were obtained, one of them with a high amount of cross-linker (HC) and two with a lower amount (LC). All inks maintained the shape fidelity of the scaffold design. Regarding the water uptake, degradation, bioactivity and mechanical properties, all of the hybrid conditions showed an improvement when comparing with pristine silica. HC showed cytotoxic effects, but not the LC conditions, which indicates the potential toxicity of the cross-linker. Conditions with gelatin showed an improved adhesion and proliferation of the cells, comparing with the pristine silica. The differentiation showed that the hybrids have lower osteogenic properties than pristine silica, however, when comparing with PLA scaffolds, it showed that, even though the gelatin decreases the ALP activity, it still promotes osteogenic differentiation.
Conclusion
Three different silica-gelatin 3D printable hybrids were obtained. The hybrid inks improved all physicochemical properties when comparing to pristine silica scaffolds. Moreover, the cell adhesion and proliferation was improved by hybrid scaffolds, maintaining the osteogenic activity of these silica-gelatin hybrid scaffolds.
References
(1) Al-Harbi, N., et al., Pharmaceuticals 2021, 14 (2), 1–20.
(2) Jones, J. R., Acta Biomater. 2013, 9 (1), 4457–4486.
(3) Bose, S., et al., Mater. Today 2013, 16 (12), 496–504.
(4) Owens, G. J., et al., Prog. Mater. Sci. 2016, 77, 1–79."
20941831157
Interest in manned spaceflights has increased in recent years and this brings about a surge in the need for more in-depth research into the adverse effects of spaceflights on the human body. In the absence of gravitational forces there are countless negative effects on the immune system. This dysfunction in the immune system can lead to increased susceptibility of infections by astronauts and poor wound healing. In particular, there is a lack of research on the effects of microgravity on immune cells in physiologically relevant cellular microenvironments. Amongst the various immune cell types, dendritic cells (DCs) are the primary mediators between the innate and adaptive immune systems and any dysregulation of these cells can lead to inadequate immune responses, especially in a long-term specific immune response. To gain insight into how DCs’ function is affected under microgravity conditions on Earth, we utilized loose and dense 3D fibrillar collagen matrices to explore the effects of simulated microgravity using Random Positioning Machine (RPM) on cells of the immune system. Immune potency of DCs was assessed in terms of their transcriptome profile, differentiation state, secreted cytokine profile, antigen uptake, and their ability to trigger a T-lymphocyte cell response. The transcriptome profile, using RNA-sequencing, showed that DC differentiation and maturation were altered under simulated microgravity conditions in a matrix density dependent manner. In addition, surface markers, cytokine secretion profile, and functional assays of DCs were reduced upon exposure to simulated microgravity conditions. Overall, our work pinpoints the importance of mechanotransduction in DC differentiation and function under simulated microgravity conditions, which could contribute to the design of immune modulating materials for use in spaceflight. In addition, as microgravity-associated physiological alterations closely resemble those found in the elderly, this data is not only important for space biology but could also be beneficial for a simulated model of aging on Earth.
41883620517
"3D microenvironment maintains the transcriptome profile of T cells but not B cells in simulated microgravity
The adverse effects of space travel on the human body are abundant and with the rise in interest in manned spaceflight over recent years, there is an increased need for more research into these effects. In the absence of gravitational forces, one of the primary systems to be negatively affected is the immune system and its dysregulation leads to increased susceptibility of astronauts to infections. Lymphocytes (T and B cells), the key players of the adaptive immune system, are involved in fighting infections and producing antibodies. Currently, less is known to which extent microgravity affects lymphocyte functions. To address the above mentioned question, we utilized 2D tissue culture plastic and 3D collagen matrices, the latter better mimics the in vivo cellular microenvironment, as cell culture models. T and B cells were cultured on ground and under simulated microgravity conditions. Both cell types were also analyzed under resting and activated states using RNA-Sequencing. Our data indicates that the 3D culture microenvironment appears to maintain the transcriptome profile of T cells but not B cells during early activation under simulated microgravity conditions when compared to ground controls. In T cells, DNA damage and protein degradation were upregulated in 2D cell culture under simulated microgravity conditions whereas the 3D microenvironment prevents these adverse effects. Interestingly, B cells showed a higher number of differentially expressed genes when activated in 3D collagen matrices compared to 2D cell culture. However, simulated microgravity conditions attenuates these effects. Overall, our results suggest that the cellular microenvironment plays a role on lymphocyte behavior on Earth and in simulated microgravity."
62825419928
"Studies of cellular differentiation in simulated microgravity reveal an important role for β-actin in mechanosensing
Authors: Oscar Sapkota, Tomas Venit and Piergiorgio Percipalle
Science Division, Biology Program, New York University Abu Dhabi, Abu Dhabi, UAE
Introduction
Mechanotransduction is mediated by the actin cytoskeleton and it is important in helping stem cells determine their fate. Differentiating cells assess changes in mechanical stressors in the extracellular matrix (ECM) and transduce them into a reaction cascade to regulate genes involved in cellular lineage specification. During differentiation, the nuclear β-actin pool plays an important role in regulating gene expression as part of the chromatin remodeling complex BAF and bound to all three eukaryotic RNA polymerases. Based on these considerations, the aim of this study is to investigate whether the nuclear β-actin pool is involved in regulating gene expression that controls mechanosensory pathways in response to mechanical unloading in the ECM.
Methodology and results
Wild-type (WT) and β-actin knock-out (KO) mouse embryonic fibroblasts (MEFs) were subjected to simulated microgravity (sµG) for 72 hours using a Random Positioning Machine (RPM) to study changes in their global epigenetic landscape. This was achieved by staining MEFs with antibodies against active, repressive, and enhancer-associated histone marks and analyzing their intensity and distribution inside cell nuclei using the Cellomics CX7 Laser High Content Phenotypic Platform. Results show an increase in repressive chromatin marks (H3K9Me3, Hp1a) and dysregulated levels of enhancer-specific epigenetic marks (H3K27Ac, H3K4Me) in the β-actin KO but not in the WT cultured in sµG. This confirms a role of β-actin in regulating heterochromatin and hints to a novel role for the nuclear beta actin pool in the regulation of enhancers to transduce microgravity-induced changes. To examine this further, total RNA was extracted from WT and β-actin KO MEFs cultured in sµG for 24 and 72 hours and subjected to RNAseq. Analysis of the transcriptomic data obtained shows upregulation of genes involved in focal adhesion, osteogenesis, and axon guidance in the WT under sµG, but not in the KO.
Conclusion
Considering the observed epigenetic and transcriptomic changes observed upon β-actin depletion, we propose that β-actin regulates expression of genes involved in mechanosensing. We speculate that β-actin performs this novel function by controlling heterochromatin levels and potentially impacting on enhancers’ activity."
20941860328