| Objective:The ischemia and hypoxia of myocardium after myocardial infarction caused the decrease of survival and differentiation of transplanted stem cells. Therefore, to improve the survival and differentiation efficiency of transplanted stem cells is a pressing problem. Fibrin glue (FG) was prepared and a novel self-assemble peptide, which may form self-assemble peptide nanofibers (SPNF) in physiological conditions, was designed and synthesisd. Explore the effects and mechanism of FG and SPNF on MCSC transplantation for myocardial infarction. To provide novel strategy and experimental evidence for the clinical application of biomaterials in stem cell transplantation for treatment of ischemic heart disease such as myocardial infarction.Methods:MCSC with cardiac-specific differentiation potential were selected from MMSC with single-cell cloning culture and c-kit, Sca-1 and CD133 immunoflourence staining. Early cardiac transcription factors GATA-4, Nkx2.5, Mef2C and Tbx5 were detected by RT-PCR analysis. In vitro, survival and apoptosis of MCSC in FG were examined with AO/EB staining. Proliferation of MCSC in FG was detected by cell count. Wounding assay and migrating assay were performed to evaluate the migration of MCSC out from FG. After the model of cell ischemia and hypoxia was established, cytoprotective effect of FG for MCSC was detected by AO/EB staining and lactate dehydrogenase (LDH) release assay. The novel self-assemble peptide was designed and synthesised through the modification of the self-assemble peptide RADA16 with functional groups. Efficiency of self-assemble and structures of SPNF were observed by atomic force microscopy (AFM) and scanning electron microscope (SEM) after self-assemble. Immunogenicity of self-assemble peptide was detected by in vivo experiment and ELISA. The biocompatibility of SPNF and MCSC was examined through in vitro experiments. The degradation of SPNF in vitro and in vivo was determined through SEM and transmission electron microscope (TEM). Effect of SPNF on adhesion of MCSC was detected by cell counting. Cytoprotective effect of SPNF for MCSC was examined through AO/EB staining, LDH release assay and Annexin V/PI double-labeled flow cytometry. Effects of SPNF on differentiation of MCSC towards cardiomyocytes were examined by observation under light microscopy, immunocytochemical staining for cTnT and SEM, respectively. Thirty minutes after the establishment of acute myocardial infarction model of female rats, cell transplantation was performed to evaluate the effects of MCSC transplantation in FG and SPNF for the treatment of rat MI. The structures of FG were determined by SEM. The structure of SPNF was observed by SEM and TEM. At 4 weeks after transplantation, the changes of cardiac function after transplantation were detected by M-mode echocardiography. Heart tissue slices were prepared and stained with Masson and CD31. Scar area, collagen content, infarcted wall thickness and microvessel density were measured using image analysis system. Expression of cTnT and Cx-43 of Y chromosome positive cells were detected by fluorescence in situ hybridization and immunohistochemistry (IHC) staining. Y chromosome positive cells which expressed cTnT were counted. Results:c-kit+/Sca-1+/CD133+ MCSC selected from MMSC express early cardiac transcription factors GATA-4, Nkx2.5, Mef2C and Tbx5. MCSC can effectively differentiate into cardiomyocytes after induction with BMP-2. FG formed with 5 mg/ml fibrinogen has more flexibility and lower mechanical strength, which was conducive to the growth of MCSC. MCSC in FG maintained high viability and proliferation. MCSC could migrate out from FG. FG may protect MCSC against ischemia and hypoxia caused apoptosis and necrosis, reduce the release of LDH. HPLC detection showed that the purity of synthetic peptide was 96.23%. AFM and SEM demonstrated that the self-assemble peptide can self-assembled into nanofiber structure. Diameter of nanofiber was about 10 nm, length of nanofiber was 100-300 run and pore size was 5-200 nm. The anti-peptide antibody in the serum was not detected after injection of self-assemble peptide. In vitro experiments showed that MCSC may adhere on SPNF scaffold and full spread. At 2 weeks, the structures of SPNF almost keep integrity. At 4 weeks, most of SPNF has been degraded at 4 weeks, only a small amount of nanofiber fragments can be observed on the cells and between the cells. Cell count showed that SPNF enhanced the adhesion of MCSC. AO/EB staining and MTT showed that MCSC in SPNF has high viability, and could maintain good proliferation. At 12 h after ischemia and hypoxia, in SPNF group, the number of survived MCSC was more, the number of apoptotic and necrotic cells was less and the level of LDH in supernatant was significantly lower than MCSC group. At 2 weeks after induction, MCSC in SPNF was short column like, arranged in parallel, cell junctions appeared between adjacent cells, and the cells expressed cTnT. At 4 weeks after induction, cell junctions between the adjacent cells increased, cardiac cell-like structure was formed, and expression of cTnT was significantly increased. At 30 min after MCSC transplantation in FG, FG presented fiber bundles or fiber network structure, distributed between the host myocardial cells. The transplanted cells attached on the FG fiber. At 4 weeks after cell transplantation, compared with MCSC and FG alone, improvement of cardiac function was better, scar area was reduced, infarct area wall thickness was increased, and microvessel density in the infarction region and peri-infarction regions was enhanced in FG-MCSC group. There were more Y chromosome positive cells in FG-MCSC group and most of the cells expressed cTnT and Cx-43. The self-assemble peptide self-assembled into SPNF with 10 nm in diameter at the injection site at 30 min after transplantation. Transplanted cells attached on SPNF network. At 2 weeks after injection, SPNF almost keeped integrity structure and wrapping around the transplanted cells. At 4 weeks after injection, most of the SPNF have been degraded, only some SPNF fragments were observed among the cardiomyocytes. Compared with MCSC and SPNF alone group, cardiac function was markedly improved, scar area was significantly reduced, infarct area wall thickness was significantly increased, microvessel density was obviously echanced and the number of Y chromosome positive cells were significantly increased in SPNF-MCSC group, the differences were all statistically significant. IHC staining showed that most of the Y chromosome positive cells expressed cTnT and Cx-43, the Y-chromosome positive cells expressed of Cx-43 formed connections with host cardiomyoctyes.Conclusions:MCSC selected from MMSC express early cardiac transcription factor GATA4, Nkx2.5, Mef2C and Tbx5, and have a higher tendency of myocardial differentiation, can be used as a good source of seed cells for stem cell transplantation in myocardial infarction. FG as a natural biomaterial may provide a favorable microenvironment for the growth and differentiation of MCSC towards cardiomyocytes. Under ischemic hypoxia, FG may protect MCSC against damage caused by ischemia and hypoxia, promoting cell survival and proliferation. MCSC transplantation in FG after myocardial infarction can promote cardiac repair and improvement of cardiac function through maintaining the survival and migration of MCSC, enhancing the differentiation of MCSC towards cardiomyocytes and inducing angiogenesis. The self-assemble peptide, as a synthetic nanometer biomaterial, has low immunogenicity, good biocompatibility and biodegradability. The novel self-assemble peptide modified with functional groups can bind cells and release cytokines slowly. Therefore, MCSC transplantation in SPNF was more conducive to the survival and differentiation of transplanted cells, so was more useful to improve cardiac repair and cardiac function. Both FG and SPNF can be used as a delivery of stem cells and expected to apply in the clinical treatment using stem cell transplantation. However, because SPNF is a nanometer biomaterial which can be modified, it may be expected to an ideal source of biomaterials. |
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