| Objective Myocardial infarction (MI) severely threatens peoples’ health and life, and places a big burden on patient’s family and society. Effective treatment for MI has not been found yet. Although stem cell therapy is a prospecting strategy in heart regeneration, some problems still exist, such as low cell survival and limited capacity for cardiomyocyte differentiation. These problems seriously influence the therapeutic effect of stem cell transplantation. Therefore, more and more attention has been paid in recent years on designing effective strategy for stem cells transplantation. In this study, nanomaterial fibers carrying MSCs or MSCs overexpressing Tβ4 (Tβ4-MSCs) were applied to treat MI. Furthermore, their effects on exogenous and endogenous cardiac repair were investigated. Our experimental results provide a novel strategy and feasible proof for further clinical application of stem cell transplantation.Methods Two high polymer materials, PCL and gelatin, were prepared PG nanofibers by electrospinning. Compatibility of nanofibers and cells was examined in 3-dimensional culture in vitro. To examine effect of PG loading MSCs on cell survival and viability under hypoxia condition, an in vitro hypoxia model was used. It mimics hypoxic-ischemic microenviroment in vivo. One week after female rat MI models were established, the nanofibrous patch seeded with male GFP-MSCs was transpanted onto the surface of heart. Four weeks later, cardiac functions were examined with echocardiography. Morphological changes of the left ventricular wall were examined by HE and Masson’s trichrome staining, then thickness of the wall was measured. To assess angiogenesis in infarcted region, vessels density was examined by immunostaining of CD31. To detect lymphangiogenesis in infarcted region, lymphatic vessels were examined by immunostaining of LYVE-1. Immunostaining of cTnT and Cx43 were performed to detect cardiomyocyte regeneration. Survival and distribution of transplanted cells were traced with Y chromosome and GFP. To evaluate effect of PG on cell survival, the copies of Siy gene were detected by PCR. In addition, to further confirm the differentiation of MSCs after transplantation, CD31 and cTnT immunostaining were used to detect whether MSCs differentiate into endothelial cells or cardiomyocytes. Distribution and differentiation of endogenous cardiac stem cells such as Wtl and c-Kit positive cells were detected. To confirm the effect of Tβ4 overexpressing on endogenous regeneration, MSCs were possessed of the capability to overexpress and secrete T(34 by tranfected with Tβ4 gene recombinant lentiviral. Nanofibrous patch seeded with Tβ4-MSCs was transplanted onto rat heart after MI. Cardiac functions were detected by echocardiography four weeks after transplantation. Morphological changes of hearts were examined by Masson’s trichrome staining four weeks after transplantation. Immunostaining was used to examine the survival and differentiation of Tβ4-MSCs after transplantation. To detect effect of Tβ4 on activating and mobilizating of endogenous cardiac stem cells, immunostaining of Wtl and c-Kit were performed.Results PG nanofibrous membrane displayed porous structures. The diameter of PG nanofibers was 244 ± 51 run. Porosity of PG nanofibers was 83.6 ± 0.8%, and pore size was 0.83 ±0.15 μm. The cells seeded on the PG nanofibers spread well, and contacted with each other. Cytoskeletal arranged orderly and parallelly within the cells. Under hypoxic condition, PG promoted cell survival and increased activity of the cells. After transplanted onto MI heart, PG seeded with MSCs obviously enhanced cardiac function, reduced scar size, thickened left ventricle wall, increased angiogenesis, decreased lymphangiogenesis and promoted heart tissue repair. PG significantly increased survival rate of the transplanted cells. The results of cell tracing by Y chromosome and GFP showed that PG stimulated MSCs to differentiate into endothelial cells and cardiomyocytes. After transplanted with PG seeded MSCs, the number of Wtl+ and c-Kit+ cells increased in infarcted region, and some of them expressed cTnT. These results indicated that PG seeded with MSCs play an important role in activating and mobilizing endogenous stem cell to repair cardiac tissue. After tranfected with the recombinant lentiviral, MSCs overexpressed Tβ4 and secreted it in culture medium. After transplanted with PG seeded Tβ4-MSCs, cardiac function improved well, and thickness of left ventricle enhanced obviously. More remarkably, Tβ4 overexpression significantly promoted Wtl+ cells and c-Kit+ cells to proliferate and migrate into infarcted region. It also facilitated these cells to differentiate into cardiomyocytes. This would be another important mechanism by which PG seeded Tβ4-MSCs promotes cardiac repair after transplantation.Conclusion Because of its good biological compatibility and similar stiffness with native myocardium, PG nanofibers are a kind of desired nanomaterial for cardiac tissue engineering. PG nanofibers protect cell from hypoxia damage, and enhance cell survival. Transplantation of PG seeded with MSCs obviously restricts the expansion of the left ventricle, and promotes cell survival and differentiation into cardiomyocyte and endothelial cells. Moreover, PG seeded with MSCs effectively stimulates EPDCs and recruits endogenous CSCs to repair cardiac tissue. Therefore, the effect of stem cell on cardiac repair is significantly improved. Tβ4 overexpression not only enhances cell survival, but also remarkably promotes endogenous regeneration of the myocardium by activating EPDCs and recruiting CSCs. These results are involved in double effect of exogenous and endogenous mechanisms for cardiac repair. Thus, it is a prospective strategy for optimizating of stem cell transplantation by PG and Tβ4 overexpression. |
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