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大鼠BMSCs成骨诱导及复合支架材料构建组织工程骨组织的研究*
Research of rat BMSCs osteogenic and composite scaffolds for tissue engineering bone
  
DOI:
中文关键词:  骨髓基质干细胞,成骨诱导分化,支架,组织工程骨
英文关键词:Bonemarrow stromal stem cells  Osteogenic differentiation  Scaffolds  Tissue engineering bone
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作者单位
年争好1,2,3 孙凯1,2,3 李晖1* 李东1,2,3 徐成3 张新昌3 张西正3 李瑞欣3* 作者单位:1天津医科大学总医院骨科天津3000522 天津医科大学天津3000703 军事医学科学院卫生装备研究所天津300161 
通讯作者:年争好1,2,3 孙凯1,2,3 李晖1* 李东1,2,3 徐成3 张新昌3 张西正3 李瑞欣3*  作者单位:1天津医科大学总医院骨科,天津,300052;2 天津医科大学,天津,300070;3 军事医学科学院卫生装备研究所,天津,300161  
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中文摘要:
      [摘要]目的 利用诱导成骨分化的骨髓间充质干细胞(bone marrowmesenchymal stem cells, BMSCs)复合生物支架材料构建组织工程骨组织。方法 采用密度梯度离心法获取大鼠骨髓间充质干细胞,原代培养扩增后,条件培养基诱导成骨分化作为实验组,并设非条件培养基培养为对照组。诱导培养后,通过碱性磷酸酶、钙结节染色;I型胶原、骨钙素检测鉴定成骨性。将诱导的BMSCs利用滴加法种入自制组织工程生物支架复合培养,采取扫描电镜、HE切片染色观察培养8天时细胞在支架内部的生长情况。结果 密度梯度离心法获取培养的原代骨髓间充质干细胞呈梭形或三角形贴壁生长,以梭形为主;经成骨诱导剂诱导后细胞呈多角形贴壁生长,碱性磷酸酶染色呈阳性、茜素红染色出现阳性的钙化结节;Western blotting检测Ⅰ型胶原蛋白表达较对照组明显增加(P<0.05);ELISA法检测骨钙素结果较对照组明显升高(P<0.01)。HE切片染色可见支架内部有细胞长入,细胞呈圆形或椭圆形。扫描电镜可见支架内部有大量细胞长入,细胞粘附、生长良好,呈现完全伸展状态,细胞-支架-细胞之间有基质连接。结论 本实验获取的原代细胞为骨髓间充质干细胞,诱导剂诱导后成功分化为成骨细胞。采用经诱导成骨后的细胞作为组织工程骨构建的种子细胞,与三维支架材料复合后共培养,使构建的组织复合物更接近骨组织,为临床大段骨缺损的修复增加可能性。
英文摘要:
      [Abstract ]Objective To work out tissue engineering bone tissue by combining induce osteogenic differentiation of mesenchymal stem cells with biological scaffolds. Methods Primary culture rat bone marrow mesenchymal stem cells get bythe method of density gradient centrifugationafter amplification. The experimental group was induced osteogenic differentiation with conditioned medium, and set unconditioned culture medium as a control group. To identifythe osteogenic by alkaline phosphatase, calcium nodules stain, detection of collagen I and osteocalcin after induction. Plantthe induced BMSCsinto tissue engineering scaffold made by ourselves bydropping infiltration method, then culturethe composite. Observe thecells growth inside thescaffold bytaking a scanning electron microscope, and HE staining in slices. Results Primary bone mesenchymal stem cells get by density gradient centrifugation were spindle or triangular adherent growth; Induced cells were polygonal adherent growth , alkaline phosphatase staining was positive, positive alizarin red staining appeared calcified nodules ; The expression of collagenⅠ protein by Western blotting was significantly increased compared with the control group (P <0.05); The results of osteocalcin by ELISA assay significantly higher than the control group (P <0.01). Scanning electron microscopy showed a large number cells, the length inner frame into the cell adhesion, growth good, showing a fully extended state of the cells - at the connection between the support matrixes-cells.HE staining sections have cell ingrowth internal scaffold, which were round or oval. Cell activity is good,its core is dyed purple, red cell matrix. Conclusion This experiment shows that the cells were bone marrow mesenchymal stem cells and successfully differentiate into bone cellsafter induced.The composite built by combining the induced cells with the three-dimensional scaffold was closer bone tissue, whichincrease possibilities for repairing large bone defectsclinically.
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