Silk fibroin-based patches on differentiation of mesenchymal stem cells into cardiomyogenic and regenerating myocardial

• Main Authors: Ming-Chia Yang, 楊明嘉
• Other Authors: 謝銘鈞
• Format: Others
• Language: en_US
• Published: 2010
• Online Access: http://ndltd.ncl.edu.tw/handle/11259383167949573796

博士 === 臺灣大學 === 醫學工程學研究所 === 98 === 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 into or co-sprayed with silk fibroin (SF) to produce SF/chitosan (CS), SF/hyaluronic acid (HA) or SF/CS-HA microparticles that were further processed by mechanical pressing and genipin crosslinking to produce hybrid cardiac patches. We examined the influence of a CD44-blockage treatment of rMSCs on the aforementioned issues on new SF-based hybrid cardiac patches after they were cultivated. The ATR-FTIR spectra and the contact angle confirmed the co-existence of CS, HA or CS-HA and SF in microparticles and patches. First, the isolated rMSCs were identified with various positive and negative surface markers such as CD44 and CD31, respectively, by a flow cytometric technique. To examine the growth of rMSCs on the patches, MTT viability assays were performed, and the results demonstrated that the growth of rMSCs on SF hybrid patches significantly exceeded (P<0.001) that on cultural wells after seven days of cultivation. This was also observed by adding vimentin to the cells. RMSCs cultured on cultural wells and SF/HA patches with a CD44-blockage treatment were 100%, 208.9 ± 7.1 (%) and 48.4 ± 6.0 (%) (n=3, for all), respectively, after five days of cultivation. Moreover, rMSCs grown on SF/HA patches highly promoted fibronectin expressions of the cells, while those with a CD44-blockage treatment markedly diminished the expression. To investigate 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 showed that cardiomyogenic differentiation of induced rMSCs on SF/CS-HA hybrid patches significantly improved the expressions of cardiac genes Gata4, Nkx2.5, Tnnt2 and Actc1 (all, P<0.01 or better, n=3) compared to those on SF and SF/CS patches and cultural wells. To investigate the interaction between CD44 of rMSCs and HA of SF/HA patches possibly modulated cardiomyogenic differentiation. Cardiomyogenic differentiation significantly promoted the expressions of cardiac genes Gata4, Nkx2.5, Tnnt2 and Actc1 (all, P<0.01 or better, n=3) on SF/HA patches compared with those expressions for the cells with CD44-blockage treatment. Furthermore, immunofluorescence staining of cardiac proteins such as cardiotin and connexin 43 for induced rMSCs cultured on SF/CS, SF/HA and SF/CS-HA hybrid patches were much more pronounced compared with SF patches, indicating the improvement of cardiomyogenic differentiation on the hybrid patches. In echocardiographic examinations, the SF/CS-HA and SF/CS patch groups effectively reduced progressive LV dilatation and preserved LV systolic function as compared to the cryoinjured group. The SF/CS-HA and SF/HA groups showed a significantly higher LVFS than the cryoinjured group. The results of this study demonstrate that the novel SF/HA and SF/CS-HA hybrid patches may be promising biomaterials for regenerating infarcted cardiac tissues. By examining the results of CD44-blockage treatment on rMSCs, we found that CD44 of rMSCs modulated the growth, fibronectin expression and cardiomyogenic differentiation of rMSCs cultured on a new cardiac SF/HA patch that may have great potential for regenerating cardiac tissue.