"Introduction: Cardiac fibrosis arises after myocardial infarction, causing progressive heart failure. New regenerative medicine approaches are under investigation to reduce or revert cardiac fibrosis.
In agreement with the 3Rs principle, testing in in vitro platforms mimicking pathological cardiac tissue could reduce in vivo animal trials.
Previous literature reports in vitro models mainly based on non-human cells and hydrogels, which lack structural biomimetic cues, adequate mechanical properties and have fast degradation rate.
The aim of this work was to design mechanically stretchable scaffolds with biomimetic composition for in vitro engineering of human cardiac fibrotic tissue, reproducing different pathological conditions and allowing long-term dynamic testing.
Methodology: Scaffolds were fabricated by additive-manufacturing from polycaprolactone (PCL) providing proper mechanical support and slow degradation rate. Scaffold pores were filled with gelatin methacrylate (GelMA) based hydrogels, mimicking extracellular matrix-like microenvironment1. PCL scaffolds with parallel wavy fibers were fabricated by melt-extrusion additive manufacturing (MEAM).
GelMA hydrogels with different concentrations (5, 7 and 10% w/v) were prepared, using lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) as photoinitiator. Photorheology allowed the definition of the optimal hydrogel curing preserving biocompatibility. Cell viability and protein expression were analysed after encapsulating human cardiac fibroblasts (HCFs) in the different GelMA hydrogels (cell density: 5×106 cells/ml).
PCL scaffolds were functionalized with poly(4-Dihydroxy-DL-phenylalanine) (polyDOPA) to improve interfacial adhesion with GelMA hydrogels. PCL/GelMA scaffolds were then obtained by filling PCL scaffolds with GelMA hydrogels. Static and cyclic tensile tests were then performed on such composite scaffolds. Further in vitro cell characterization is in progress.
Results: Highly stretchable PCL scaffolds were obtained by tailoring PCL fiber diameter and mesh geometry and size, with the aim to mimic cardiac fibrotic tissue stiffness (1-9 MPa) and its maximum elastic deformation (15-22%). PCL/GelMA scaffolds preserved stretchability and integrity after both static and cyclic tensile test. Cell viability tests on GelMA hydrogels showed that UV curing did not damage cells. HCFs were cultured up to 2 weeks in hydrogels and then stained for Phalloidin and α-SMA expression, showing homogeneous cell distribution, spreading and demonstrating HCFs activation into fibrotic phenotype. Collagen Type I and IV content in the cellularized hydrogels was also confirmed after 1 and 2 weeks, demonstrating fibrotic extracellular matrix deposition.
Conclusions: PCL/GelMA scaffolds are promising for cardiac cells culture, such as HCFs, to reproduce post-infarct human cardiac tissue at different severity degrees depending on the stiffness of GelMA hydrogels. Moreover, considering their stretchability, these platforms can be used in dynamic cyclic culture environment (such as bioreactor) to add more complex fibrotic-like stimulus to the model. In the future, such platforms will be also useful for in vitro drug screening and preclinical validation of cardiac regenerative therapies2, with the advantage to allow long-term dynamic testing.
1. Sadeghi, A. H. et al., Adv. Healthc. Mater. 6, 1–14 (2017).
2. Paoletti, C. et al., Front. Bioeng. Biotechnol. 8, 1–9 (2020)."