丝素蛋白/羟基磷灰石/聚多巴胺/BMP-2多孔支架的构建及促进BMSCs成骨分化的研究

Construction of silk fibroin/hydroxyapatite/poly dopamine/BMP-2 porous scaffold and its effects of enhancing osteogenetic differentiation of BMSCs

目的构建丝素蛋白/羟基磷灰石/聚多巴胺/骨形成发生蛋白-2(SF/HA/PDA/BMP-2)多孔支架并评估其对骨髓间充质干细胞(BMSCs)体外成骨分化能力的影响。方法冷冻干燥法制备SF/HA支架,PDA涂层后复合BMP-2改性,构建SF/HA/PDA/BMP-2多孔支架。以SF/HA支架、SF/HA/PDA支架为对照组,将BMSCs作为种子细胞分别在上述支架上培养,评价3组支架的表面特征、理化性质及细胞生长状态,通过检测碱性磷酸酶(ALP)活性、茜素红染色比较3组支架上BMSCs的成骨分化能力。结果SF/HA支架、SF/HA/PDA支架、SF/HA/PDA/BMP-2支架接触角分别为(54.7±4.8)°、(43.7±3.7)°、(32.3±2.4)°,差异有统计学意义(P<0.05);在PDA涂层及接枝BMP-2后,支架表面粗糙度增加,细胞数量增多;BMSCs在SF/HA/PDA/BMP-2支架上的增殖能力优于其他两组支架(P<0.05)。培养2周后,SF/HA支架、SF/HA/PDA支架、SF/HA/PDA/BMP-2支架上细胞ALP水平分别为(2.9±0.4)、(3.4±0.7)、(6.8±0.8)U/mg,差异有统计学意义(P<0.05);茜素红染色结果提示3种支架均有不同程度的成骨诱导能力,但SF/HA/PDA/BMP-2支架最优。结论SF/HA/PDA/BMP-2多孔支架具有良好的生物相容性,能够促进BMSCs体外增殖及成骨分化,在组织工程骨领域具有广阔的应用前景。

Objective To construct the porous scaffold of silk fibroin(SF)/hydroxyapatite(HA)/poly dopamine(PDA)/bone morphogenetic protein(BMP)-2,and to assess its effects on the osteogenetic differentiation of bone marrow mesenchymal stem cells(BMSCs)in vitro.Methods SF/HA scaffold was prepared by freeze-drying method.By the surface modification of SF/HA scaffold with PDA coating and compositing with BMP-2,SF/HA/PDA/BMP-2 porous scaffold was constructed successfully.SF/HA scaffold and SF/HA/PDA scaffold were as controls,and three kinds of scaffolds were co-cultured with BMSCs respectively.The surface characterization,physicochemical property and cell growth morphology on three groups of scaffolds were observed,abilities of osteogenic differentiation of the cells were compared among 3 groups of scaffolds by alkaline phosphatase(ALP)activity and Alizarin red staining.Results The water contact angles were(54.7±4.8)degree in SF/HA scaffold,(43.7±3.7)degree in SF/HA/PDA scaffold,and(32.3±2.4)degree in SF/HA/PDA/BMP-2 scaffold respectively,the differences showed statistically significance(P<0.05).After PDA coating and immobilization of BMP-2,the surface roughness of the scaffold increased,and the number of cells on scaffold also increased.The proliferation of BMSCs on SF/HA/PDA/BMP-2 scaffold was stronger than those of the other two groups of scaffolds(P<0.05).After 2 weeks of co-culture,ALP levels of the cells on SF/HA scaffold,SF/HA/PDA scaffold,and SF/HA/PDA/BMP-2 scaffold were(2.9±0.4),(3.4±0.7),and(6.8±0.8)U/mg respectively,there were statistical differences among three groups(P<0.05).Alizarin red staining results indicated different degrees of osteogenic differentiation in three groups of scaffolds,in which the ability of SF/HA/PDA/BMP-2 scaffold showed strongest.Conclusion SF/HA/PDA/BMP-2 porous scaffold can effectively promote the proliferation and osteogenic differentiation of BMSCs in vitro with good biocompatibility,and has broad application prospects in the field of bone tissue engineering.