缓释TGF-β3的去细胞软骨微丝/温敏型水凝胶复合支架的制备

目的：探讨一种可注射且能缓释TGF-β3的去细胞软骨微丝/温敏型水凝胶复合支架的制备方法。方法：使用去污剂-酶化学消化法和层层自组装技术制备缓释TGF-β3 的去细胞软骨微丝，混入温敏型水凝胶后制成缓释TGF-β3的去细胞软骨微丝/温敏型水凝胶复合支架，用ELISA试剂盒检测TGF-β3缓释情况；然后再将骨髓间充质干细胞（BMSCs）与复合支架共培养，显微镜下观察细胞生长情况，Western blot法检测Collagen II（Col II）、Aggrecan（AGG）和SOX9表达水平。结果：复合支架在低温状态下为溶胶状态，当温度升高至37 ℃时变为凝胶状态；复合支架中的TGF-β3 可有效缓释25 d左右；BMSCs在复合支架上生长情况良好，细胞呈现类似软骨细胞的形态，Col II、AGG和SOX9 的表达较对照组显著增加（P ＜0.05）。结论：成功制备了一种可注射且能缓释TGF-β3的去细胞软骨微丝/温敏型水凝胶复合支架，该支架能较好地诱导BMSCs成软骨分化。

Preparation of acellular cartilage microfilaments/thermosensitive hydrogel composite scaffold with sustained release of TGF-β3

Objective: To explore the methods for preparation of acellular cartilage microfilaments/thermosensitive hydrogel composite scaffold which is injectable and can release TGF-β3 sustainedly. Methods:Decellularized cartilage microfilaments with sustained release of TGF-β3 were prepared by the method of detergent-enzyme chemical digestion and layer-by-layer self-assembly technology. After being mixed with thermosensitive hydrogel, the acellular cartilage microfilaments/thermosensitive hydrogel composite scaffold was prepared. The sustained release of TGF-β3 was detected by ELISA kit. Then the bone marrow mesenchymal stem cells (BMSCs) were co-cultured with the composite scaffold and observed under microscope. The expression of Col II, AGG and Sox9 were detected by the method of Western blot. Results: The composite scaffold was in sol state at low temperature, and changed to gel state when the temperature increased to 37 ℃. The TGF-β3 in the composite scaffold could be released slowly and effectively for about 25 d. BMSCs grew well on the composite scaffold, and the cells were similar to chondrocytes. And the expression of Col II, AGG and SOX9 increased significantly (P<0.05). Conclusion: The acellular cartilage microfilaments/thermosensitive hydrogel composite scaffold, injectable and able to release TGF-β3 sustainedly, was successfully prepared, which can induce chondrogenic differentiation of BMSCs.