Among biomaterials tested so far in cardiac tissue engineering, the decellularized native ECM (d-ECM) appears by far the most promising and appealing. In order to obtain intact scaffold of d-ECM while preserving its composition, we compared the human cardiac d-ECM produced through four different protocols (named Pr1, Pr2, Pr3 and Pr4) in terms of efficiency of decellularization, composition of scaffolds and their suitability for repopulation with cardiac stem cells to develop myocardial patches. As regards efficiency of decellularization, DNA measurement revealed ineffectiveness of Pr1 and Pr3 in producing thoroughly acellular d-ECM, as they yielded scaffolds containing 46,33+5,3 and 96,7+12,2 ng of DNA/mg of tissue, respectively, while DNA content was as low as 9+2,1 in d-ECMs produced with Pr2, which includes DNase treatment, and 17,33+2.22 in the ones obtained with Pr4. Hematoxylin and Eosin staining confirmed the effectiveness of the decellularization procedure for Pr2 and Pr4, while Masson's staining revealed that Pr4 produced a d-ECM with more preserved architecture. Sirius Red, PAS and Gomori stainings documented the retention of collagen, non-collagenous proteins and elastic fibers in d-ECM from all protocols. Immunodetection of fibronectin, tenascin and laminin confirmed the retention of such ECM proteins in d-ECM, while assay for insoluble collagen and sulphated glycosaminoglycan confirmed the presence of collagen and GAGs in all protocols. Mostly due to ineffective decellularization and, as such, to mild treatment, ECM obtained through Pr3 retained more collagen and GAGs. Nevertheless, collagen content was only 1,5-fold higher than in d-ECM obtained with Pr4, which retained the highest amount of GAGs as well. Notably, GAGs protect growth factors against proteolysis, and protein array to investigate the retention of growth factors by d-ECM proved the highest efficiency of Pr4 in retaining growth factors as HGF, VEGF, TGF and IGF-1 in d-ECM. Finally, to assess suitability of d-ECM scaffolds for cell repopulation, we cultured cardiac stem cells (CSCs) on d-ECM to evaluate their viability and differentiation trend. Among all, Pr2 and Pr4 better supported cellular viability, but Pr4 provided the best environment to promote terminal differentiation towards myocardium, endothelium and smooth muscle cell lineages. Interestingly, we previously reported the inability of human cardiac stem cells to acquire fully differentiated state in vitro, but d-ECM supports stem cell terminal differentiation prompting progenitor cells to further proceed in differentiation towards precursor and mature phenotype. In conclusion, the composition and architecture of scaffolds of human cardiac d-ECM depend on decellularization protocol. We report here an adaptation of previously described protocol which yields highly preserved d-ECM, in terms of composition and architectures, that might be successfully be employed in regenerative medicine as biological scaffold.
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