骨髓间充质干细胞与同种异体脱钙骨基质组合生长因子诱导向软骨细胞分化

背景：用组织工程学方法高质量修复关节软骨缺损并达到很好的远期疗效目前尚无定论。鉴于此,课题组提出＂同种异体骨髓间充质干细胞关节腔内定向培养组织工程软骨＂的实验设技。目的：同种异体脱钙骨基质组合转化生长因子β1和胰岛素样生长因子Ⅰ诱导骨髓间充质干细胞向软骨分化的能力,同时探索其在关节腔内培养促进向关节软骨定向分化的方法。方法：分离兔骨髓间充质干细胞并行体外培养,分为两组：实验组DMEM培养液中加入转化生长因子β1和胰岛素样生长因子Ⅰ,对照组中未加入诱导因子。比较两组细胞的增殖情况和成软骨分化情况。制备同种异体脱钙骨基质支架材料,实验组骨髓间充质干细胞负载到同种异体脱钙骨基质中,构建组织工程软骨复合体,取另2只兔的腰背筋膜包裹后缝合固定到30只兔膝关节腔内行腔内培养。分别于植入后4,8,12周各取10只标本进行组织学切片观察及Ⅱ型胶原免疫组织化学观测,并对结果进行分析。结果与结论：实验组中骨髓间充质干细胞集落形成效率明显高于对照组（u=3.326,P〈0.01）。实验组细胞爬片做Ⅱ型胶原免疫组化检测呈阳性,对照组未见阳性细胞。组织工程复合体在腔内培养12周后,苏木精-伊红染色见大量软骨细胞增生,胞核染色呈蓝色;甲苯胺蓝染色见软骨细胞成串排列,大量软骨陷窝形成,周围大量基质包绕;Ⅱ型胶原免疫组织化学反应见细胞外基质中出现大量棕黄色颗粒,Ⅱ型胶原染色强阳性。提示转化生长因子β1和胰岛素样生长因子Ⅰ可显著促进骨髓间充质干细胞增殖和成软骨分化,骨髓间充质干细胞与同种异体脱钙骨基质结合后可在关节腔内成功培养出组织工程软骨。同种异体脱钙骨基质符合组织工程软骨支架材料的基本要求。

Bone marrow mesenchymal stem cells differentiation into chondrocytes under growth factor composing allogeneic decalcified bone matrix

BACKGROUND：Effective repair of articular cartilage defects and achieving a good long-term efficacy using tissue engineering methods remain unclear.The research group proposed an experimental hypothesis that＂allogeneic bone marrow mesenchymal stem cells in articular cavity culture tissue engineered cartilage＂.OBJECTIVE：To demonstrate the role of allogeneic decalcified bone matrix combined transforming growth factor β1 and insulin-like growth factorⅠto differentiate bone marrow mesenchymal stem cells（MSCs） into cartilage,and to investigate the method of promoting differentiation into articular cartilage by the culture in articular cavity.METHODS：Bone marrow MSCs were separated and cultivated in vitro.In experimental group,transforming growth factor β1 and insulin-like growth factorⅠwere applied into DMEM to induce proliferation and chondrogenic transformation,while in control group only DMEM was used.The proliferation and chondrogenic transformation between two groups were compared.Allogeneic decalcified bone matrix was prepared,bone marrow MSCs in the experimental group were seeded into the decalcified bone matrix,to construct tissue engineered cartilage complex,which was then place into knee cavity of 30 rabbits after parceled with fascia.At 4,8,12 weeks following implantation,ten specimens were selected for histological section observation and type Ⅱ collagen immunohistochemistry staining.RESULTS AND CONCLUSION：Colony forming efficiency in experimental group was significantly higher than that of control group（u=3.326,P 0.01）.Immunohistochemical identification of typeⅡcollagen was positive in experimental group,while negative in control group.At 12 weeks after the tissue engineered complex were cultured,hematoxylin-eosin staining showed that a large amount of chondrocytes began to proliferate,caryon was stained blue.Toluidine blue staining showed that the chondrocytes aligned,there were plenty of cartilage lacunas,and were surrounded by lots of extracellular matrix.Immunohistochemical identification of typeⅡcollagen was strongly positive,lots of brown-yellow stained particles could be discerned in extracellular matrix.Bone marrow MSCs proliferation and differentiation into chondrocyte can be significantly promoted by the synergistic action of transforming growth factorβ1 and insulin-like growth factor Ⅰ.MSCs combine with decalcified bone matrix can successfully cultivate tissue engineered cartilage in articular cavity.Allogeneic decalcified bone matrix may satisfy the demands of tissue engineered cartilage scaffold.