Myocardial infarction (MI) is the main cause of mortality and morbidity worldwide. MI initiates a wound healing process resulting in a fibrotic tissue, characterized by random morphology and increased stiffness . Currently, several advanced strategies based on regenerative medicine are under investigation and in vitro models of pathological cardiac tissue have attracted interest as predictive platforms for their preclinical validation. Previous models of cardiac fibrotic tissue were developed by tissue engineering or organ-on-chip approaches, mainly exploiting cellularized hydrogels[2,3]. However they did not faithfully reproduce the architecture and mechanical properties of cardiac fibrotic tissue. In this context, the aim of this research is to engineer 2D and 3D models of early-stage human fibrotic tissue through bioartificial scaffolds with biomimetic architecture, chemical composition and stiffness.
Materials and methods
Polycaprolactone (PCL) was used as scaffold bulk material; in order to resemble low and high thicknesses of fibrosis 2D random membranes were fabricated by solution electrospinning, while 3D scaffolds with 150 µm and 350 µm square-mesh were built up through melt-extrusion additive manufacturing. Exploiting 3,4-Dihydroxy-DL-phenylalanine polymerization (PolyDOPA), type A gelatin was grafted on PCL to obtain biomimetic surface composition. Scaffold physico-chemical properties were thoroughly investigated. Ventricular human cardiac fibroblasts were cultured on scaffolds up to 3 weeks, to mimic cellular fibrotic microenvironment. Immunofluorescence and two-photon microscopy were used to evaluate the activation of fibrotic cell phenotype and the deposition of pathological cardiac ECM on scaffold area.
Electrospun 2D scaffolds showed homogeneous and defect-free fibers with average diameter of 127 ± 33 nm and pores with lower size than 1 µm. 3D PCL scaffolds (0.7 mm thickness) with 150 and 350 µm square mesh size showed high shape fidelity as suggested by the high similarity between measured and theoretical porosity degree. The progressive formation of gelatin coating mediated by mussel inspired approach was monitored using QCM-D equipment. Immunostaining showed that bioartificial scaffold properties trigger the activation of myofibroblast phenotype and fibrotic-like ECM deposition. Moreover, scanning electron microscopy and two-photon excitation fluorescence show ECM homogeneous distribution on 2D and 3D scaffolds with smaller mesh (150 µm).
In this work, 2D and 3D bioartificial scaffolds, based on PCL and surface functionalized with polyDOPA/G were designed, provided with biomimetic (G coating, isotropic structure and high stiffness) resembling properties of the human cardiac fibrotic tissue, able to support long-term culture of human cardiac fibroblasts, favouring their adhesion, proliferation, differentiation into myofibroblasts and deposition of ECM on scaffolds. Our results suggest that such innovative in vitro models of human cardiac fibrosis, reproducing patient-specific pathological features, may allow a fine preclinical tuning of new regenerative therapies.
Acknowledgements. This project received funding from the European Research Council under the European Union's Horizon 2020 research and innovation programme grant agreement No-772168.
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