膝关节腔内培养骨髓间充质干细胞复合脱钙骨的组织工程软骨

背景：如何更好地以组织工程学方法修复关节软骨缺损并达到良好的远期疗效目前尚无公识。目的：创新性地在膝关节腔内培养兔骨髓间充质干细胞复合同种异体脱钙骨的组织工程软骨。方法：采用全骨髓贴壁筛选法分离培养兔骨髓间充质干细胞，DMEM／F12完全培养基培养，成软骨诱导条件培养基诱导分化。取同种异体兔的髂骨和椎体骨制作成脱钙骨支架，诱导后的骨髓间充质干细胞种植于脱钙骨支架上，培养1d后将细胞支架复合物用筋膜包裹置于兔左膝关节腔内培养，单纯脱钙骨支架筋膜包裹置入右膝关节腔。于培养第4，8，12周分别取材，行大体观察并制成石蜡切片，采用苏木精伊红染色、甲苯胺蓝染色，Ⅱ型胶原免疫组化染色方法进行组织学观察。结果与结论：培养4，8周，细胞一支架组标本Ⅱ型胶原免疫组化的平均吸光度值（A）分别为0．263±0．031，0．340±0．052，单纯支架组标本分别为0．147±0．027，0．165±0．030，两组比较差异有显著性意义（P〈0．05）；培养12周细胞支架组标本Ⅱ型胶原免疫组化A值平均为0．362±0．037，标本类似正常软骨外观，Ⅱ型胶原免疫组化反应呈阳性；而单纯支架组脱钙骨支架降解。培养12周细胞一支架组苏木精一伊红染色结果显示细胞数量多，脱钙骨支架基本被吸收；而甲苯胺蓝染色结果显示有被染成紫红色的异染性基质形成。结果提示兔骨髓间充质干细胞复合同种异体脱钙骨可在兔膝关节腔内培养出组织工程软骨。

Culture of bone marrow mesenchymal stem cells plus allogenic decalcified bone matrix in the knee cavity of rabbits for tissue engineered cartilage

BACKGROUND： It remains controversial how to repair articular cartilage defects and achieve a good long-term efficacy using tissue engineering methods. OBJECTIVE： To investigate the feasibility of culturing rabbit bone marrow mesenchymal stem cells in combination with allogenic decalcified bone matrix in the knee cavity of rabbits for constructing tissue engineered cartilage. METHODS： Bone marrow mesenchymal stem cells were isolated using adherence screening method and cultured in complete Dulbecco＇s modified Eagle＇s medium/Ham＇s nutrient mixture F-12. The third generation was induced to chondrocytes. Allogenic decalcified bone matrix, made from rabbit＇s ilium and vertebral bone, was combined with the induced bone marrow mesenchymal stem cells in vitro for 1 day. The cell-scaffold composites wrapped by fascia were cultured in the left knee cavities of adult rabbits （cell-scaffold group, n=1 5） while simple homologous decalcified bone matrix wrapped by fascia were cultured in the right knee cavities （scaffold group, n=15）. The specimens were harvested at 4, 8, and 12 weeks after culture for gross and histological observations. Histological observation was conducted using hematoxylin-eosin staining, toluidine blue staining and immunohistochemical staining. RESULTS AND CONCLUSION： At 4 and 8 weeks after culture, the mean absorbance value was respectively 0.263±0.031 and 0.340±0.052 for the cell-scaffold group and 0.147±0.027 and 0.165±0.030 for the scaffold group, with significant differences between two groups （P 〈 0.05）. At 12 weeks, the mean absorbance value of specimens in the cell-scaffold group was 0.362±0.037, and the samples exhibited similar appearance of normal cartilage and positive immunohistochemical reaction of type Ⅱ collagen. However, the scaffold degraded in the scaffold group. At 12 weeks, hematoxylin-eosin staining revealed increased chondrocytes and mostly absorbed decalcified bone matrix scaffold in the cell-scaffold group while toluidine blue staining showed metachromasia matrix dyed red or purple. Results indicate that tissue engineered cartilage can be induced by culturing rabbit bone marrow-derived mesenchymal stem cells combined with allograft decalcified bone matrix in the knee cavity of rabbit.