载碱性成纤维细胞生长因子温敏水凝胶对大鼠骨髓间充质干细胞体外成骨分化的作用研究

doi:10.13701/j.cnki.kqyxyj.2021.12.017

摘要/Abstract

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20~80 μm。(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。

关键词: 骨缺损, 水凝胶, 碱性成纤维细胞生长因子, 骨髓间充质干细胞

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.

Key words: bone defect, hydrogel, bFGF, bone marrow mesenchymal stem cells

司超, 邹馨颖, 刘悦, 张佩佩, 徐依山, 郝艺帆, 公柏娟. 载碱性成纤维细胞生长因子温敏水凝胶对大鼠骨髓间充质干细胞体外成骨分化的作用研究[J]. 口腔医学研究, 2021, 37(12): 1139-1144.

SI Chao, ZOU Xinying, LIU Yue, ZHANG Peipei, XU Yishan, HAO Yifan, GONG Baijuan. Effect of bFGF-loaded Thermosensitive Hydrogel on Osteogenic Differentiation of Rat Bone Marrow Mesenchymal Stem Cells in Vitro[J]. Journal of Oral Science Research, 2021, 37(12): 1139-1144.

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