骨形态发生蛋白7复合国产多孔钽对骨髓间充质干细胞向软骨分化及功能的影响

文题释义：骨形态发生蛋白7：又称成骨蛋白1，是骨形态发生蛋白家族中的一员，可促进骨髓间充质干细胞向软骨细胞分化；体外可促进软骨细胞增殖及软骨细胞标志因子分泌，体内可协同骨髓间充质干细胞修复兔膝关节软骨损伤。国产多孔钽：实验中应用的多孔钽具有自主知识产权，采用高温煅烧技术制备，具有立体三维空间结构，与人体骨组织的力学强度、弹性模量相似，有较好的生物相容性及骨传导能力。植入体内时可使其周围骨组织黏附并向孔隙内生长，正逐渐替代自体骨、同种异体骨、金属钛和不锈钢等传统医用生物材料，成为骨缺损修复的新型修复材料。背景：结合物理因素与支架材料建立共培养体系并采用细胞因子诱导，成为骨髓间充质干细胞成软骨分化的热点。目的：观察骨形态发生蛋白7复合国产多孔钽对骨髓间充质干细胞软骨向分化的影响。方法：分离培养SD大鼠(由北京华阜康生物提供)骨髓间充质干细胞，分组干预：①实验组加入多孔钽片，对照组不加多孔钽片，培养第5天，鬼笔环肽染色观察多孔钽片表面的细胞生长情况；培养1，3，5，7 d，CCK-8 法检测细胞增殖；②A组加入软骨细胞诱导液，B组加入软骨细胞诱导液与骨形态发生蛋白7，C组加入国产多孔钽材料与软骨细胞诱导液，D组加入国产多孔钽材料与软骨细胞诱导液和骨形态发生蛋白7，培养第 7，14，21天，采用 ELISA 法检测细胞分泌Ⅱ型胶原、SRY型高迁移率族蛋白、基质金属蛋白酶13的水平，Western-blot法检测细胞中Ⅱ型胶原、SRY型高迁移率族蛋白、基质金属蛋白酶13的表达。动物实验获得华北理工大学动物实验伦理委员会批准。结果与结论：①鬼笔环肽染色显示，骨髓间充质干细胞在多孔钽表面及周围生长及增殖良好；②实验组培养3，5 d的增殖慢于对照组(P < 0.05)，培养1，7 d的增殖与对照组无差异(P > 0.05)；③培养第 7，14，21天时，A-D组Ⅱ型胶原、SRY型高迁移率族蛋白质量浓度逐渐升高(P < 0.05)。培养第7天时，A-D组基质金属蛋白酶13质量浓度逐渐减少(P < 0.05)；培养第14天时，A组高于其余3组(P < 0.05)，B、C、D组间比较差异无显著性意义(P > 0.05)；培养第21天时，4组间比较差异无显著性意义(P > 0.05)；④Western-blot检测显示培养第 7，14，21天时，A-D组Ⅱ型胶原、SRY型高迁移率族蛋白表达逐渐升高(P < 0.05)。培养第7天时，A-D组基质金属蛋白酶13蛋白表达逐渐减少(P < 0.05)；培养第14天时，A组高于C、D组(P < 0.05)，B、C组高于D组(P < 0.05)；培养第21天时，A组高于其余3组(P < 0.05)，其余组间比较差异无显著性意义(P > 0.05)；⑤结果表明，骨形态发生蛋白7复合国产多孔钽可诱导骨髓间充质干细胞软骨向分化，可促进Ⅱ型胶原、SRY型高迁移率族蛋白的表达，抑制基质金属蛋白酶13的表达。ORCID: 0000-0002-5869-6982(崔逸爽)中国组织工程研究杂志出版内容重点：生物材料；骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性；组织工程

Effect of bone morphogenetic protein-7 combined with porous tantalum on chondrogenic differentiation and function of bone marrow mesenchymal stem cells

BACKGROUND: The establishment of coculture system combined with physical factors and scaffold materials and the induction of cytokines have become the focus of chondrogenic differentiation of bone marrow mesenchymal stem cells. OBJECTIVE: To observe the effect of bone morphogenetic protein 7 combined with porous tantalum on chondrogenic differentiation of bone marrow mesenchymal stem cells. METHODS: Bone marrow mesenchymal stem cells of Sprague-Dawley rats(provided by Beijing Huafukang Biology) were isolated and cultured. Group intervention:(1) in the experimental group, porous tantalum tablet was added, while in the control group, porous tantalum tablet was not added. At 5 days after culture, cell growth on the surface of porous tantalum tablet was observed by phalloidin staining. At 1, 3, 5, and 7 days after culture, CCK-8 method was used to detect cell proliferation.(2) Group A was added with chondrocyte inducer;group B with chondrocyte inducer and bone morphogenetic protein 7;group C with domestic porous tantalum material and chondrocyte inducer;group D with domestic porous tantalum material and chondrocyte inducer and bone morphogenetic protein 7. At 7, 14 and 21 days after culture, the levels of type II collagen, SRY type high mobility group protein and matrix metalloproteinase-13 secreted by cells in each group was detected by ELISA. Western blot assay was used to detect the expression of type II collagen, SRY type high mobility group protein and matrix metalloproteinase-13. This study was approved by the Animal Experimental Ethics Committee of North China University of Science and Technology. RESULTS AND CONCLUSION:(1) The phalloidin staining results showed that bone marrow mesenchymal stem cells grew well on and around the porous tantalum surface.(2) At 3 and 5 days after culture, the proliferation of bone marrow mesenchymal stem cells was slower in the experimental group than in the control group(P < 0.05). There was no statistical difference in cell proliferation between the two groups at 1 and 7 days(P > 0.05).(3) At 7, 14 and 21 days, the expression of type II collagen and SRY high mobility group protein increased gradually among groups A, B, C and D(P < 0.05). At 7 days, the expression of matrix metalloproteinase-13 decreased gradually among groups A, B, C and D(P < 0.05). At 14 days, matrix metalloproteinase-13 secretion of matrix in group A was highest compared with that in group B, group C and group D(P < 0.05), but there was no significant difference between groups B, C and D(P > 0.05). At 21 days, there was no significant difference among groups A, B, C and D(P > 0.05).(4) Western blot assay showed that at 7, 14 and 21 days after culture, the expression level of type II collagen and SRY high mobility group protein increased gradually in groups A, B, C and D(P < 0.05). At 7 days, the expression of matrix metalloproteinase-13 decreased gradually in groups A, B, C and D(P < 0.05). At 14 days, the expression of matrix metalloproteinase-13 was higher in group A than in groups C and D(P < 0.05), and higher in groups B and C than in group D(P < 0.05). At 21 days, the expression of matrix metalloproteinase-13 was higher in group A than in groups B, C and D(P < 0.05). No significant difference was found among groups B, C and D(P > 0.05).(5) The results showed that bone morphogenetic protein-7 combined with domestic porous tantalum could induce cartilage differentiation of bone marrow mesenchymal stem cells, facilitate the expression of type II collagen and SRY high mobility group protein, and inhibit the expression of matrix metalloproteinase-13.