同种异体脱钙骨与骨髓间充质干细胞关节腔内共培养：与同腔软骨性状的对比

同种异体脱钙骨与骨髓间充质干细胞关节腔内共培养：与同腔软骨性 状的对比 徐 斌，周 亮，王英明，钱三祥 安徽医科大学第一附属医院骨四科，安徽省合肥市 230022 Chondrogenic co-culture of allogenic decalcified bone matrix and bone marrow mesenchymal stem cells in the joint cavity: comparison of cartilage traits in the same joint cavity Xu Bin, Zhou Liang, Wang Ying-ming, Qian San-xiang Fourth Department of Orthopaedics Surgery, the First Affiliated Hospital of Anhui Medical University, Hefei 230022, Anhui Province, China 摘要 背景：临床发现膝关节内游离体能长期存在于关节腔内并能保持一定的软骨组织学特性和生 理学特性，因此大胆提出假设：关节腔环境可能是软骨细胞生长、发育的较佳环境并提出“腔 内培养，腔内移植”的理念。 目的：观察兔骨髓间充质干细胞复合同种异体脱钙骨基质体外培养或关节腔内培养组织工程 软骨与同腔软骨的性状差异。 方法：实验分 3 组进行，体外培养组将经成软骨诱导的乳兔骨髓间充质干细胞与成年兔脱钙 骨基质体外复合培养；腔内培养组将经成软骨诱导的乳兔骨髓间充质干细胞与成年兔脱钙骨 基质以筋膜包裹，复合培养于成年新西兰兔膝关节腔内，以同腔内正常软骨为对照。 结果与结论：培养 12 周后：体外培养组苏木精-伊红染色见软骨细胞少量增生，胞核蓝染； 甲苯胺蓝染色见软骨细胞排列无序，少量周围基质包绕；Masson 染色阳性区域小，细胞排列 无序；型胶原免疫组织化学见软骨细胞胞浆及胞外基质少量黄色颗粒。腔内培养组苏木 精-伊红染色见软骨细胞增生，胞核蓝染；甲苯胺蓝染色见软骨细胞成串排列，软骨陷窝形成， 周围基质包绕；Masson 染色阳性，软骨细胞多，基质蓝染，按一定应力方向排列；型胶原 免疫组织化学见细胞外基质中出现较多棕黄色颗粒，型胶原染色阳性。说明骨髓间充质干 细胞与同种异体脱钙骨基质复合物可在体外及膝关节腔内培养出组织工程软骨，关节腔内培 养的软骨比体外培养的软骨更接近正常软骨。 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 全文链接： 关键词 ： 生物材料； 软骨生物材料； 骨髓间充质干细胞； 同种异体脱钙骨； 组织工程软骨； 关 节腔； Abstract： BACKGROUND: Loose bodies in the knee are found to survive for a long term and maintain certain histophysiological properties of cartilage tissue. Therefore, a bold hypothesis is proposed that the joint cavity may be a preferred environment for chondrocyte growth and development, supporting the concept of “intracavitary culture and intracavitary transplantation”. OBJECTIVE: To observe the trait difference of chondrogenic culture with allogenic decalcified bone matrix and bone marrow mesenchymal stem cells in the joint cavity or in vitro versus cartilage in the same cavity. METHODS: There were three groups in this experiment: in in vitro culture group, bone marrow mesenchymal stem cells from newborn rabbits undergoing chondrogenic culture were co-cultured with decalcified bone matrix from adult rabbits in vitro; in intracavitary culture group, bone marrow mesenchymal stem cells from newborn rabbits undergoing chondrogenic culture were co-cultured with decalcified bone matrix from adult rabbits in the joint cavity; normal cartilage in the same cavity served as control group. RESULTS AND CONCLUSION: (1) After 12 weeks of culture, in the in vitro culture group, hematoxylin-eosin staining showed a small amount of chondrocytes proliferated, with blue-stained nuclei; toluidine blue staining showed chondrocytes arranged disorderly, surrounded by a small amount of matrix; Masson staining showed a small positive area and irregular cell arrangement; type II collagen immunohistochemistry staining showed a few of yellow particles in the cytoplasm and extracellular matrix. (2) After 12 weeks of culture, in the intracavitary culture group, hematoxylin-eosin staining showed proliferation of chondrocytes with blue-stained nuclei; toluidine blue staining showed cluster-shaped arrangement of chondrocytes surrounded by the matrix with lacuna formation; Masson staining showed there were many positive cells with blue-stained matrix that arranged in a certain stress direction; immunohistochemical identification of type II collagen was positive, and brown- yellow stained particles could be discerned in the extracellular matrix. These findings indicate that tissue-engineered cartilage can be generated by co-culture of allogenic decalcified bone matrix and bone marrow mesenchymal stem cells in the joint cavity or in vitro, and the cartilage cultured in the joint cavity is more close to normal cartilage than that cultured in vitro. 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 全文链接： Key words： biocompatible materials; tissue engineering; cartilage; stem cells; 收稿日期: 2013-11-25 中图分类号: R318