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
"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. "
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.
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.
Introduction: Pancreatic cancer is a devastating malignancy, and treatment options are very limited . 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 . Elevated expression of KLK6 is associated with poor survival rates in pancreatic cancer, making it an attractive target for alternative treatment strategies . 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 .
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  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.
 Osuna de la Pena, D. et al., Nat Commun, 12, 5623, (2021).
 Below, C. R. et al., Nat Mater, 21, 110-119 (2022).
 Candido, J. B. et al., Cancers (Basel), 13, (2021).
 Mahajan, V. et al., Cancers (Basel), 13, (2021).
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.
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.
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)
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.
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)
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 . 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 . 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 . 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 . 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.
 Tomás-Bort E, Kieler M, Sharma S, et al. 3D approaches to model the tumor microenvironment of pancreatic cancer. Theranostics. 2020 10:5074–89.
 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.
 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.
 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.
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!
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.
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. Recently, we developed hexagonal 3D microfiber scaffolds with melt electrowriting (MEW). These scaffolds support contracting hiPSC-CMs and promote tissue organization and maturation. 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.
Hexagonal MEW meshes were fabricated with a 3DDiscovery (REGENHU). 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.
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.
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.
 Kristen & Ainsworth et al.(2019). Adv Healthcare Mater.
 Castilho & van Mil et al.(2018). Adv Func Mater.
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.
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 )). 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 . In previously developed beating heart-on-chip –, 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  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.
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 . 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.
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 . 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.
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.
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.
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.
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.
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.
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).
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.
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
"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 . In addition, BGs can induce immunomodulatory effects to trigger bone regeneration and wound healing . 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 , 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.
 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.
 K. Zheng, et al., Immunomodulatory bioactive glasses for tissue regeneration, Acta Biomater.133 (2021) 168-186.
 T. Reiter; et al. Bioactive glass based scaffolds coated with gelatin for the sustained release of icariin. Bioactive Mater. 4 (2019) 1-7. "
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 . 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. 
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.
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 . 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.
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.
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
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.
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.
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.
Since the discovery of bioactive glasses (BG) in the late 60s , 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 .
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.
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).
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.
 Hench L.L., J. Mater. Sci. Mater. Med. 17, 967–978 (2006)
 Hoppe, A., et al., Biomaterials. 32, 2757-2774 (2011)
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 . 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 . 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 . 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.
 S Veerachamy et al., Proceedings of the Institution of Mechanical Engineers. Part H, Journal of engineering in medicine 228 (10), 1083 (2014).
 SM Kurtz et al., The Journal of arthroplasty 27 (8 Suppl), 61 (2012).
 CL Ventola, P T 39 (10), 704 (2014).
 I Reigada et al., Microorganisms 8 (3) (2020).
 R Perez-Tanoira et al., J. Biomed. Mater. Res. Part A 105 (1), 62 (2017).
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.
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.
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.
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.
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)."
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.
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.
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.
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.
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.
"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."
"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."
"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.
"[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 . 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.
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. 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. 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. 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. 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).
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.
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. 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, 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.
Poly-e-caprolactone MEW scaffolds were fabricated using a 3DDiscovery printer (REGENHU), then activated using a computer-controlled APPJ device. 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.
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.
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.
 Castilho et al. 2019. Acta Biomaterialia.
 Wang, Rigueur & Lyons. 2014. Birth Defects Res C Embryo Today.
 Alavi et al. 2020. ACS Applied Materials & Interfaces.
"Quantum Sensing for measuring free radical generation in living cells
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. We were able to conduct a similar study also in primary cells which where harvested from donors. 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.
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.
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.
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 .
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.
Extracellular matrix communicates to the nuclear environment by external stimuli that affect Lamina organization and chromatin distribution . 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 . SUN proteins are supposed to link the nuclear Lamina , 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.
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.
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).
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 . 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.
 Remuzzi A et al. Cells. 2020;9(8):1873. doi:10.3390/cells9081873
 Donnaloja F, et al. Cells. 2020: 24;9(5):1306. doi: 10.3390/cells9051306
 Haque F, et al. J Biol Chem. 2010 29;285(5):3487-98. doi: 10.1074/jbc.M109.071910.
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.
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.
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.
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.
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.
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)
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.
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.
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).
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.
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)
This research was funded by Xunta de Galicia (ED481D-2021-014).
"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.
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."
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 . 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 . 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 . Therefore, in the present work, we investigated structure formation in bioprinted models of the TME both experimentally and computationally . 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 . 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.
 Bray, L.J., Hutmacher, D.W., Bock, N., Front. Bioeng. Biotechnol. 7, 217 (2019)
 Li, J., Parra-Cantu, C., Wang, Z., Zhang, Y.S., Trends Cancer 6, 745–756 (2020)
 Datta, P., Dey, M., Ataie, Z., Unutmaz, D., Ozbolat, I.T., Precision Oncology, 4, 18 (2020)
 Turley, S.J., Cremasco, V., Astarita, J.L., Nat. Rev. Immunol. 15, 669–682 (2015)
 Robu, A., Aldea, R. et al., BioSystems 109, 430–443 (2012)
 Bojin, F., et al., Micromachines 12, 535 (2021)
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.
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.
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.
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.
Taken together, we have proved that TDM bioinks could be used for bioprinting artificial breast tumors closely recreating the tumor ECM.
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).
1. Bahcecioglu, G. et al., Acta Biomater. 106, 1-21 (2020).
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
(1) Scott LE, Weinberg SH, Lemmon CA. Front Cell Dev Biol, (2019)
(2) Haugh MG et al., Tissue Eng - Part A,1201–8 (2011)
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.
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.
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.
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.
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.
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.
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).
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.
Chronic kidney diseases (CKD) affect approximately 10% of worldwide population . 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.
 Wilson S, et al., J Clin Hypertens (2021) 23:831–4.
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. 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.
 Bernal, P.N. et al., Adv. Mater. 1, 1904209 (2019).
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.
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.
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.
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.
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.
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).
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.
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.
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).
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.
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.
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.
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.
"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."
"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)."
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?
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.
"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."
"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. 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."
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.
"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.
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.
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.
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.
Funding: ReCAP: ERC Advanced Grant number 788753"
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.
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)
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.
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."
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.
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.
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.
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.
 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.
 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.
 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."
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. 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.
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.
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.
 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.
 Ryan et al. Electroconductive Biohybrid Collagen/Pristine Graphene Composite Biomaterials with Enhanced Biological Activity, Advanced Materials 30(15)(2018)(1706442).
Science Foundation Ireland AMBER Centre, IRFU Charitable Trust, and the Anatomical Society"
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.
"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."
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 .
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 .
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 . 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).
 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.
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.
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 .
Elastomeric cross-linked poly(ester-urethane-urea) (PEUU) scaffolds have been developed through an emulsion technique allowing to produce highly interconnected porous structure . 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.
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.
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.
1. Langueh, C. et al., Polym. Deg. Stab. 183, 109454 (2021).
2. Changotade, S. et al., Stem Cells Int. Article ID 283796 (2015).
The authors thank the Ministère de l’Enseignement Supérieur, de la Recherche et de l’Innovation for the MENRT scholarship granted to Credson Langueh."
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.
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.
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.
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."
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.
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).
Supported by an MRC-AMED Regenerative Medicine and Stem Cell Research Initiative (ref.MR/V00543X/1).
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 . Oxidation and reduction reactions take place, resulting in a constant exchange of electrons and ions between the metal and the surrounding fluid . The release of metal ions in different oxidation states can be promoted by the acidic pH caused by specific cells . This in turn may have direct effects on the surrounding tissue and its cells . 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 . 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).
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)
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.
"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."
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.
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.
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.
This study shows a valuable approach to optimize implant surfaces via Melt Electrowriting.
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.
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.
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.
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.
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.
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).
Cardiovascular disease is one of the major causes of death worldwide . 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  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 .
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 . 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.
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.
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.
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.
 H.H.G.Song et al. CellStem 22, 2018.  A.Hasan et al. Acta Biomater.10, 2014.  M.J.McClure et al. J.Drug Deliv.Sci.Technol. 21, 2011.
"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."
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.
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.
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.
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.
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.
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.
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.
"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)."
Osteoarthritis (OA) is the most prevalent degenerative joint disorder, but no reversing therapies are currently available . This is mainly due to the disease complexity, that involves a failure of the entire joint, and to the disease multifactorial etiology . 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 . 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 .
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 . 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 . Induction of synovial inflammation was then quantified upon valves opening through FACS analysis.
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.
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.
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).
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."
"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."
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.
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.
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."
"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."
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.
"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."
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.
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.
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.
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.
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.
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.
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."
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.
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.
"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."
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.
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).
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).
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.
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).
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.
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.
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.
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.
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.
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. 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.
 Petta et.al, 2018, ACS Biomater. Sci. Eng, DOI: 10.1021/acsbiomaterials.8b00416. </div>
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). 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 . 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.
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 . 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 . 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.
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.
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).
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.
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 . 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.
 Frangogiannis, N.G., J Clin Invest.127(5), 1600-1612 (2017)
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.
Overall, PEDOT:PSS scaffolds provided an asset for the production of versatile platforms for tissue engineering applications.
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.
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.
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.
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.
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.
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.
The human gut microbiota constitutes the most bountiful and divergent community of organisms compared to the other areas of the body . 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 . Meanwhile, predominant cell cultures and animal models encounter substantial limitations .
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.
"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."
"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
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.
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.
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.
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.
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.
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.
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.
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."
"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."
"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."
Patients with chronic kidney disease (CKD) experience multiple comorbidities, among which mineral/bone disorders (MBD) contribute to high mortality due to increased facture risk . 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 . 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.
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.
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.
This project received funding from the 3Rs stimulus Funds of the Utrecht University.
 Covic A, et al., Lancet Diabetes Endocrinol. 2018 6(4):319-331, PMID: 29050900
 Kamprom W, et al., Int J Med Sci. 2021 18(3):744-755. PMID: 33437209."
"Introduction: Folding is a crucial process that modulates the function of proteins: 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. 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.
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.
 Shental-Bechor, D. et al. PNAS. 24, 8256-8261 (2008).
 Xu, C. et al. Nat. Rev. Mol. 16, 742-752 (2015).
 Brito, A.B. et al. JACS, 143, 19703-19710 (2021)."
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.
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.
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.
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).
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).
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.
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.
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.
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."
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 . 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.
Scaffolds were harvested from bovine ears by punching 8mm diameter discs as described previously . 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.
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.
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.
1. S. Nürnberger et al., Acta Biomater. 86, 207–222 (2019)"
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.
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.
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.
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."
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.
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.
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.
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."
"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.
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.
"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.
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.
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.
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.
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, email@example.com
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.
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.
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.
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.
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.
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.
1. Gribova, V. et al., Sci. Rep. 11, 18702 (2021)"
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.
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.
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.
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.
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.
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.
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)."
"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."
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.
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.
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.
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.
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.  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.
 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).
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. Our research aims to highlight all the factors and thus should give new design directions to engineered organ designers.
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 was employed to analyze the data by distributing the interview’s material into groups such as design strategy or regulation.
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.
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.
 Daston L. The Moral Economy of Science. Osiris 10, 2, 1995
 Blanchet A., Gotman A. L’entretien. Broché. 2015
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 . 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.
The shell bioink consisted of 3 wt% alginate and 9 wt% methylcellulose dissolved in fresh frozen plasma (plasma-algMC) as described ; 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 ; 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.
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.
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.
1 Taymour, R. et al., Sci Rep, 2021, 11, 5130
2 Ahlfeld, T. et al., ACS Appl Mater Interfaces, 2020, 12, 12557
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.
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.
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 . 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).
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.
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 . Among other things, it has recently enabled realization of highly porous biodegradable microscaffolds capable of hosting individual cell spheroids . The resulting tissue units can be used for bottom-up self-assembly of larger tissue constructs with very high initial cell density . 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 .
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.
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.
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.
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>
We acknowledge funding from the United Kingdom Regenerative Medicine Platform 2 (UKRMP2) [MR/R015651/1] and Next Generation Biomaterials Discovery [EP/N006615/1].
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 . 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.
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.
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).
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.
1 Zhou, L. et al., Adv. Mat. 32, 2002183, 2020
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.
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.
Financial support from the Spanish Ministry of Science and Innovation (MCINN, AEI/FEDER funds) through the project RTI2018-097862-B-C21 is acknowledged.
 Langridge et al. Journ. Mat. Scie. 2021, 32, 15.
 Heang Oh et al. Biomaterials. 2007, 28, 1664.
 Siddiqui et al. Mol. Biotech. 2018, 60, 506.
 Chang et al. Biomacromolecules 2018, 19(6), 2302.
 Smith et al. Nano Mat. Sci. 2019, 1, 31.
 Palmieri et al. Front. Biotech. 2020, 8, 383.
 Patel et al. Ann. Biom. Eng. 2016, 44, 2036.
 Ramalingam et al. , J. Ind. Eng. Chem. 2020, 83, 315.
 Mnatsakanyan et al. Nature Sci. Rep. 2018, 8, 13642.
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.
"[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 .
[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.
Infection is the major cause of implant failure after breast reconstruction surgery . 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 . 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.
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.
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.
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.
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
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
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.
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.
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.
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).
"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 . 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 . 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.
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  and tissue mechanics replication which can involve biological (or biological-like) substrate engineering . In an approach to unify surface shape and mechanical properties, soft polymeric wrinkling  was utilized to form a topographical surface for LESC culture and fate control.
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).
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.
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.
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."
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.
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 .
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.
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.
This work was funded by In2Sight: Horizon 2020 GA: 964481
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.
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.
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.
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.
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.
In2Sight: Horizon 2020 GA: 964481
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).
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.
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.
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.
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.
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.
"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."
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.
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.
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.
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.
The results indicated that the BCPG3 bone paste can become a high-performance bone filler in the treatment of osteoporotic bone defects.
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"
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 . 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 , 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.
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.
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.
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.
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)
"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 . 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 . 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.
 Parmentier L, et al. Materials, 2020;
 Nguyen LH, et al. Tissue Eng Part B Rev, 2012;
 Hoyle C, Bowman C. Angew Chem Int Ed, 2010;
 Van Hoorick, J, et al., Macromol Rapid Commun, 2018."
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.
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.
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.
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."
"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 . 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 . 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:  Putri, et al., Journal of Biomedical Materials Research Part A, 108(3) (2020), 625-632.  Skibiński, et al., Ceramics International 47(3) (2021), 3876-3883."
"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.
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.
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).
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.
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.
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.
1. Gain, P. et al., JAMA Ophthalmol. 134(2):167-73 (2016).
2. Koivusalo, L. et al. Biomaterials. 225, 119516 (2019).
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, 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. 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.
Hibbitts (et al.), Matrix Biology, (2022)
Woods & O’Connor (et al.), Adv Healthcare Mat., (2021)
Introduction: The past few years saw an increasing trend in liver disease prevalence . 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 . 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 . hiPSCs-IH were transferred to Matrigel® 3D culture in hepatic organoid expansion medium to generate self-replicating hepatic organoids . To obtain decellularized scaffolds, mouse livers were cannulated via portal vein and decellularized via already established detergent-enzymatic treatment . 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 . 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.
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).
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.
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.
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.
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."
"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."
"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.
1. Barry et al, J Orthop Res 37, 1229-1235 (2019)
2. Rodriguez-Ruiz et al, Cell Tissue Res 386, 309-320 (2021)"
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.
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.
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).
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.
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.
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)"
Annually, millions of people die because of liver failure, while the waiting duration for a donor liver is around 12 months. 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) 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.
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).
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).
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) 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)"
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.
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.
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
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.
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)."
"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."
"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 . 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.
 Eppley BL et al. Plast Reconstr Surg. 114(6), 1502-8 (2004)
 Steinberg, et al., Photoacoustics. 14, 77–98 (2019)
 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)."
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.
1 - Geuens, T., et al, Biomaterials, 275, 120976.
"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.
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."
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.
"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.
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.
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.
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.
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.
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, 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).
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 . 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  and tissue-on-a-chip techniques .
In this work, we report the 3D bioprinting of avascular structures that recapitulate several features of the TME . 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.
 Turley, S.J., Cremasco, V., Astarita, J.L., Nat. Rev. Immunol. 15, 669–682 (2015)
 Shieh, A.C., Ann. Biomed. Eng. 39, 1379–1389 (2011)
 Privat-Maldonado, A., Bengtson, C., Razzokov, J., Smits, E., Bogaerts, A., Cancers 11, 1920 (2019)
 Li, J., Parra-Cantu, C., Wang, Z., Zhang, Y.S., Trends Cancer 6, 745–756 (2020)
 Shang, M., Soon, R.H., Lim, C.T., Khoo, B.L., Han, J., Lab Chip 19, 369–386 (2019)
 Bojin, F., et al., Micromachines 12, 535 (2021)
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
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.
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.
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.
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.
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.
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.
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.
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).
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.
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.
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 , we could see no differences between bladder and urethral epithelial cells in the flow experiments.
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
 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.
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.
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.
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.
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.
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.