| OBJECTIVE:Many previous researches reported stem cells transplantation can be an effective way to treat ischemic diseases. Most researches transplanted stem cells by injecting cells suspension to the ischemic tissue or injecting cells suspension in the endovascular treatment. We transformed the delivery method with tissue-engineered material——functionalized self-assembling peptide hydrogel, to investigate the therapeutic efficacy compared with cell suspension injection method. And hindlimb ischemic models were established for studies in vivo and provide a reliable basis of using functionalized self-assembling peptide hydrogel loaded with adipose derived mesenchymal stem cells to treat ischemic diseases.METHODS: In this study, adipose derived mesenchymal stem cells (ad-MSCs) were isolated with 0.1% collagenase, then cells culture and a series of tests were performed to study the cultured cells’growth characteristics. The obtained cells were identified by osteogenic and adipogenic induced differentiation, and immunophenotype CD29, CD31, CD34, CD44 and HLA-DR were analyzed with flow cytometry. The self-assembling peptide RADA16-I and containing VEGF mimicking sequence peptide KLT were synthesized and tested. After the peptide powders were dissolved in biological grade deionized ultrapure water respectively, they were mixed in a volume ratio of 1:1 (RADA16-1:KLT) to obtain a functionalized peptide mix (RADA/KLT), then the atomic force microscopy test was performed. PBS was used to induce the self-assemble process of the peptide solution in an insert. The formed hydrogel was observed and tested by scanning electron microscope. The PBS was replaced with two kinds of cell culture mediums for several times to penetrate the composition of the hydrogel. Thus the hydrogel could be used for three dimensional cell culture. Passage 3 ad-MSCs suspension was seeded into the hydrogel with different culture conditions, the seeded cells’ migration and morphology were observed every 2 days and stained with AO/EB to evaluate cells’ survival rate. Rabbit hindlimb ischemic models were prepared and assigned into three groups randomly. Then we transplanted containing ad-MSCs hydrogel for experimental group, the same number ad-MSCs for positive control group, and physiological saline for the negative control group. The general conditions were evaluated from postoperative to 30 days after the cells transplantation. Tissue samples were harvested from the muscles near the injection sites finally. And tissue sections were stained with Hematoxylin and Eosin and observed for morphological analysis. Immunostaining with anti-Ⅷ factor antibody was performed to identify endothelial cells and capillaries finally. The general conditions and histologic analysis were analyzed statistically.RESULTS:The adherent ad-MSCs presented a fibroblast-like morphology and showed higher proliferation capabilities. Flow cytometry of immunophenotype analysis showed that CD29 and CD44 expressed significantly, meanwhile distinctly negative for hematopoietic lineage marker CD34 and the endothelial cell marker CD31. It was also obtained a desired effect about the adipogenic and osteogenic induced differentiation. After the stock peptide solutions were prepared, the AFM photos obtained from the 0.01% peptide solution sample showed the peptide’s nanofiber, needle-like morphology. After the peptide solutions was induced by PBS, it can be seen that hydrogel formed in the transwell insert. The formed hydrogel has a transparent appearance, a certain strength and extremely rich moisture content. SEM micrographs showed the grid like internal structure and the pore was about 10-50μ m in diameter. The seeded ad-MSCs in the functionalized self-assembling peptide hydrogel group showed a better cellular growth behavior, cells stretched well and connected to each other. After the rabbits hindlimb ischemic model was successfully established, all rabbits showed ischemic symptoms and the DSA showed the operation achieved the expected effect.30 days after the ad-MSCs transplantation, the skin temperature difference, and histological analysis of both HE staining and immunostaining showed rabbits in hydrogel group had a certain tissue survival status, and the muscle fibers were arranged in order, and more factor Ⅷ stained cells. Sections in hydrogel group got a higher score than others statistically.CONCLUSION:The functionalized self-assembling peptide hydrogel loaded with ad-MSCs obtained better therapeutic efficacy compared with the single cell suspension injection group to treat rabbits hindlimb ischemic. |
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