【摘要】 目的:实验合成载碱性成纤维细胞生长因子(bFGF)壳聚糖/β-甘油磷酸钠/明胶温敏水凝胶,研究其对骨髓间充质干细胞(BMMSCs)成骨分化的作用。方法:制备壳聚糖/β-甘油磷酸钠/明胶水凝胶,冻干后电子显微镜下观察其表征;检测水凝胶毒性;CCK-8法筛选出载bFGF温敏水凝胶促BMMSCs增殖最适浓度并用于后续研究;碱性磷酸酶染色、茜素红染色及钙结节半定量分析观察其对BMMSCs成骨向分化能力的作用;实时荧光定量PCR检测其对BMMSCs成骨相关基因表达的影响。结果:(1)成功制备壳聚糖/β-甘油磷酸钠/明胶温敏水凝胶,扫描电镜下冻干后的水凝胶孔径为20～80μm。(2)活/死细胞染色结果显示壳聚糖/β-甘油磷酸钠/明胶温敏水凝胶具有良好的生物相容性。(3)载bFGF温敏水凝胶能显著促进BMMSCs增殖,并呈剂量依赖性,最适浓度为100 ng/mL(P<0.01)。(4)载bFGF温敏水凝胶组碱性磷酸酶表达增多,钙结节形成增多(P<0.05)。(5)载bFGF温敏水凝胶组成骨相关基因(ALP、BMP-2、Runx2)表达增高。结论:载bFGF温敏水凝胶能促进大鼠BMMSCs成骨分化。更多还原

【Abstract】 Objective: To synthesize bFGF-loaded chitosan/β-glycerophosphate/gelatin thermosensitive hydrogel and study its effect on osteogenic differentiation of bone marrow mesenchymal stem cells(BMMSCs). Methods: The chitosan/β-glycerophosphate/gelatin thermosensitive hydrogel was prepared and characterized by electron microscope after freeze-drying. The toxicity of hydrogel was detected. The CCK-8 method was used to screen out the optimum concentration of thermosensitive hydrogel loaded with bFGF to promote BMMSCs proliferation. The effects of bFGF-loaded thermosensitive hydrogel on osteogenic differentiation of BMMSCs were confirmed by ALP staining, alizarin red staining, and semi-quantitative analysis of the calcium nodules. Real-time fluorescence quantitative PCR was used for detecting the effect of bFGF-loaded thermosensitive hydrogel on osteogenesis-related gene expression of BMMSCs. Results: The chitosan/β-glycerophosphate/gelatin thermosensitive hydrogel was successfully prepared, and the pore size of the freeze-drying hydrogel under scanning electron microscope was between 20 and 80 μm. The results of live/dead cells staining showed that the chitosan/β-glycerophosphate/gelatin thermosensitive hydrogel had good biocompatibility. The bFGF-loaded thermosensitive hydrogel could significantly promote the proliferation of BMMSCs in a dose-dependent manner, and the optimum concentration was 100 ng/mL. In the bFGF-loaded thermosensitive hydrogel group, ALP expression and calcium nodule formation(P<0.05)both increased. Also, the expression of osteogenesis-related genes ALP, BMP-2, and Runx2 increased in the bFGF-loaded thermosensitive hydrogel group. Conclusion: The bFGF-loaded thermosensitive hydrogel can promote the osteogenic differentiation of rat BMMSCs.更多还原