3D In Vitro Modelling Of Human Cardiac Fibrotic Tissue Through Bio-hybrid Stretchable Scaffolds

Purpose/Objectives: Human cardiac fibrotic tissue is a pathological condition that arises after myocardial infarction. It is characterized by outnumber fibroblasts activated into myofibroblasts, increased stiffness and passive mechanical stress during heart functionality. New advanced regenerative approaches are currently under investigation to reduce or revert cardiac fibrosis and in vitro; models of human cardiac fibrotic tissue may improve their preclinical investigation. Herein, an in vitro model of human pathological cardiac tissue based on stretchable scaffolds, able to mimic the biophysical and biochemical properties of post-infarct fibrosis was developed. *Methodology: Scaffolds of polycaprolactone (PCL) with parallel-wavy fibers pattern were designed and fabricated by melt-extrusion additive manufacturing (MEAM). Gelatin methacrylate (GelMA) hydrogels with different concentrations (5, 7, 10% w/v) were prepared using lithium phenyl-2,4,6 trimethylbenzoylphosphinate (LAP) as photoinitiator. Photo-rheology allowed to define the optimal hydrogel curing protocol. Both PCL scaffolds and GelMA hydrogels were analysed by SEM imaging.PCL scaffold pores were filled with GelMA hydrogels, to mimic extracellular matrix (ECM)-like microenvironment. To improve interfacial adhesion between GelMA hydrogel and scaffold structure, PCL were surface functionalized with poly(4-Dihydroxy-DL-phenylalanine) (polyDOPA). Static and cyclic tensile tests were performed on PCL/GelMA scaffolds. Human Cardiac Fibroblasts (HCFs) were seeded in PCL/GelMA scaffolds (cell density: 5·106 cells/mL) and cultured for up to 2 weeks. HCFs viability was tested through Resazurin assay, while their morphology and distribution were analyzed through F-actin staining in order to identify optimal GelMA concentration for HCFs culture. Then, cellularized PCL/GelMA scaffolds were subjected to cyclic mechanical stimulation for 7 days. Fibroblast activation into myofibroblasts was analyzed through immunofluorescence of a-Smooth Muscle Actin (a-SMA). Results: Stretchable PCL scaffolds with high shape fidelity were obtained by optimizing MEAM processing parameters. Analytic and finite element analysis analyses allowed the validation of the experimental results. Mechanical properties of PCL/GelMA scaffolds were tailored by PCL scaffold thickness, mesh geometry, and GelMA hydrogel concentration in order to mimic the stiffness (1-9 MPa) and maximum elastic strain (15-22%) of human cardiac fibrotic tissue. GelMA hydrogel pore sizes increased by reducing GelMA concentration. PCL/GelMA scaffolds preserved stretchability and integrity after both static and cyclic tensile tests. HCFs viability after 7 and 14 days was significantly higher for cells cultured in PCL/GelMA 5% w/v and PCL/GelMA 7% w/v, compared to PCL/GelMA 10% w/v. F-actin staining showed homogeneous cell distribution inside GelMA hydrogels, while an elongated morphology was noted only on HCFs in PCL/GelMA 5%. Finally, HCFs demonstrated a-SMA expression only on samples treated with cyclic mechanical stimulation. Secretion of fibrotic ECM proteins such as Collagen I, Collagen III, and Fibronectin are under investigation. Conclusion/Significance: In this work, an in vitro model of human fibrotic cardiac tissue based on stretchable PCL/GelMA scaffolds was developed. The model demonstrated fibrotic hallmarks in a dynamic cyclic culture environment. In the future, this model will be validated for use in preclinical testing of new cardiac regenerative strategies, with the advantage to allow long-term dynamic testing. Acknowledgment: BIORECAR project has received funding from the European Research Council (ERC) under the H2020 research and innovation program (772168)