骨髓间充质干细胞构建人工软骨的实验研究

Chondrogenesis of rabbit bone marrow-derived mesenchymal stem cells and construction of tissue-engineered cartilage

目的以骨髓间充质干细胞作为种子细胞，探索组织工程化软骨的构建。方法分离、获取、扩增骨髓间充质干细胞，通过将扩增的骨髓间充质干细胞接种于聚羟基乙酸（PGA）上，在成软骨诱导因子作用下作体外诱导培养3周，将实验组和对照组的标本转植入自体兔腹腔内，进行体内培养6～12周后，再进行检测，评估工程化软骨的形成。结果骨髓间充质干细胞接种于PGA支架上，经过体外诱导培养和体内培养后，实验组获得的标本出现软骨组织样外观，进行组织学切片可见软骨陷窝，进行Ⅱ型胶原蛋白的免疫组化、Ⅱ型胶原mRNA原位杂交均为阳性。结论兔骨髓间充质干细胞在成软骨诱导剂作用下，经体外和体内培养，可生成组织工程化类软骨。

Objective To explore the feasibility of constructing tissue--engineered cartilage by inducing rabbit bone marrow-derived mesenchymal stem cells（MSCs）. Methods By Percoll density gradient centrifuge in vitro MSCs were isolated from rabbit bone marrow and purified in monolaryer culture. Then, MSCs were seeded onto polyglycolic acid（PGA） scaffolds in the experimental group to form cells-PGA composites which were subsequently cultured in a chemically defined medium mainly including transforming growth factor-1 （10ng/ml）, dexamethasone （10^-2 mmol/L）, ascorbic acid（50μg/ml）, 1% insulin,1% selenium and 1% transferring for three weeks in vitro. After that, cells-PGA composites in experimental group were covered by greater omentum and implanted into belly of adult New Zealand white rabbits for 6-12 weeks. At same time, PGA scaffolds without MSCs in control groups were implanted in the same measure. Finally, morphological obser.vation, hematoxylin-eosin （HE） staining, immunohistochemistry test and hybridization test in situ were conducted to assess cartilage regeneration after 6-12 weeks. Results MSCs were harvested from rabbit bone marrow and equally attached to the surface of porous PGA scaffolds in experimental group. Tissue-engineered specimens presented to be similar to cartilage in gross shape were successfully harvested in experimental group. Their specific markers were positive for immunohistochemistry of type II collagen and in situ hybridization of collagen type Ⅱ mRNA. ConClusion Tissue-engineered cartilage could be harvested by inducing MSCs-PGA composites.