Speaker
Description
"Introduction: Cardiac fibrosis is a common pathophysiologic mechanism of most myocardial diseases like myocardial infarction (MI), associated with impaired cardiac function and arrhythmias, preventing the heart from effectively satisfying the metabolic demand of the body[1]. Following cardiomyocytes injury, adverse fibrotic remodeling results from the altered activity of cardiac fibroblasts (CFs), which upon mechanical or biochemical stimuli (e.g., TGF-β profibrotic cytokine) undergo phenotypic changes toward fully differentiated myofibroblasts (MyoFs)[2]. MyoFs expresses high levels of contractile proteins such as alpha-smooth muscle actin and they markedly enhance the deposition of ECM components like fibronectin and collagens. The increase in ECM proteins amount is accompanied by progressive architecture disruption causing anisotropy loss. Due to these structural changes, myocardial stiffness increases from few kPa to 35-70 kPa. Heart transplantation remains the only regenerative medicine approach in case of severe cardiac fibrosis. For this reason, reversing cardiac fibrosis is still a key challenge for cardiac regenerative medicine research. In the context of the preclinical validation of new advanced therapies/drugs, the use of highly reliable human in vitro models could be useful to overcome the limitations of traditional cultures and animal in vivo models low predictability and ethical controversies. In this work, we propose the development of a biomimetic in vitro human model in which both morphological and biochemical cues are integrated to mimic specific native microenvironment features for drug testing purposes.
Methodology: Polycaprolactone (PCL) based scaffolds with random morphology nanostructure were fabricated by solution electrospinning technique in order to provide topographical and mechanical cues. Electrospun PCL scaffolds were surface grafted with human collagen type I and fibronectin (PCL/polyDOPA/C1F), exploiting a mussel inspired approach, in order to mimic the ECM protein microenvironment of fibrotic tissue and support human cardiac fibroblasts (HCFs) culture, which mainly populate native cardiac fibrosis. Lastly, biochemical profibrotic stimulus was integrated by TGF-β addition to culture medium in order to study fibroblasts activation under different culture conditions. Each step of modeling was thoroughly characterized by morphological (SEM, immunofluorescence), mechanical (wet and dry AFM tests), chemical (BCA colorimetric assay), physical (QCM-D) and biological analyses (viability/cytotoxicity assay, immunostaining, activation markers quantification).
Results: Morphological characterization demonstrated the collection of PCL scaffolds with homogeneous fibers and pores size distributions. Immunostaining for collagen type I and fibronectin coating on PCL membranes, demonstrated successful proteins grafting mimicking the main features of human fibrotic ECM. BCA assay confirmed biomimetic coating deposition and its stability during 7 days test. AFM mechanical characterization demonstrated that PCL/polyDOPA/C1F scaffolds, in wet conditions, resemble fibrotic tissue stiffness behavior. Biological validation showed that PCL/polyDOPA/C1F scaffolds support HCFs adhesion, proliferation and activation even without TGF-β biochemical stimulus.
Conclusions: In conclusion, this platform recapitulates fibrotic tissue random structure, stiffness and ECM composition. The proposed in vitro model of human cardiac fibrosis could be a promising biomimetic platform for preclinical testing of new advanced regenerative therapies thanks to its ability to replicate fibrosis hallmarks.
[1] N. G. Frangogiannis, Cardiovascular Research,117, 6, 2021, 1450–1488
[2] F. A. van Nieuwenhoven et al., Vascular Pharmacology, 58, 3, 2013,182-188"
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