The cardiomyogenic differentiation of rat mesenchymal stem cells on silk fibroin–polysaccharide cardiac patches in vitro

Polysaccharides and proteins profoundly impact the development and growth of tissues in the natural extra-cellular matrix (ECM). To mimic a natural ECM, polysaccharides were incorporated to/or co-sprayed with silk fibroin (SF) to produce SF/chitosan (CS) or SF/CS–hyaluronic acid (SF/CS–HA) microparticles that were further processed by mechanical pressing and genipin cross-linking to produce hybrid cardiac patches. The ATR–FTIR spectra confirm the co-existence of CS or CS–HA and SF in microparticles and patches. For evaluating the cellular responses of rMSCs to the SF/CS and SF/CS–HA cardiac patches, the growth of rMSCs and cardiomyogenic differentiation of 5-aza inducing rMSCs cultured on patches was examined. First, the isolated rMSCs were identified with various positive and negative surface markers such as CD 44 and CD 31 by a flow cytometric technique, respectively. For examining the growth of rMSCs on the patches, MTT viability assay was performed, and the results demonstrated that the growth of rMSCs on SF and SF-hybrid patches significantly exceeded (P < 0.001) that on culture wells after seven days of cultivation. Additionally, the relative growth rates of rMSCs on SF/CS and SF/CS–HA hybrid patches were significantly better (P < 0.01) than that on SF patches that were also observed by using vimentin stain to the cells. For instance, the relative cell growth rates (%) in cell culture wells, SF, SF/CS and SF/CS–HA patches were 100%, 282.9 ± 6.5%, 337.0 ± 8.0% and 332.6 ± 6.6% (n = 6, for all), respectively. For investigating the effects of the hybrid patches on cardiomyogenic differentiation of 5-aza inducing rMSCs, the expressions of specific cardiac genes of cells such as Gata4 and Nkx2.5 were examined by real-time quantitative polymerase chain reaction (real-time PCR) analysis. The results of cardiomyogenic differentiation of induced rMSCs on SF/CS and SF/CS–HA hybrid patches significantly improved the expressions of cardiac genes of Gata4, Nkx2.5, Tnnt2 and Actc1 genes (all, P < 0.01 or better, n = 3) than those on SF patches and culture wells. Interestingly, the results of cardiac gene expressions of the cells on the SF/CS–HA hybrid patches were the most pronounced in promoting cardiomyogenic differentiations in this investigation. Furthermore, immunofluorescence staining of cardiac proteins such as cardiotin and connexin 43 for induced rMSCs cultured on SF/CS and SF/CS–HA hybrid patches were much pronounced compared with SF patches, indicating the improvements of cardiomyogenic differentiation on the hybrid patches. The results of this study demonstrate that the SF/CS and SF/CS–HA hybrid patches may be promising biomaterials for regenerating infarcted cardiac tissues.