摘要
目的比较同种异体脱钙骨和细胞因子诱导骨髓间充质干细胞（BMSC）制成细胞-支架复合物在兔膝关节腔内环境较体外培养出组织工程软骨的优点。方法BMSC向软骨细胞诱导后与同种异体脱钙骨支架复合分别在体外培养（A组）和成年雄性新西兰白兔（15只）左侧膝关节腔内（B组）培养，右侧膝关节腔内（C组）培养单纯脱钙骨支架做空白对照。每4、8、12周各组标本分别取材，制石蜡切片行苏木素-伊红（HE）染色、甲苯胺蓝染色、Ⅱ型胶原免疫组织化学等组织学观察，并通过形态学分析软件计算免疫组织化学平均光度（A）值。结果标本石蜡切片HE染色：4周时A组标本见软骨细胞散在分布于支架表面，内部基本未观察到细胞。B组标本支架内软骨细胞数量明显较多，软骨陷窝形成，陷窝周围基质深染，可见由单个细胞分裂形成的同源细胞群。8周时B组标本以成熟软骨细胞为主，细胞数量多，出现柱状排列的同源细胞群，细胞周围分泌着色均一的玻璃样基质，且渗入支架结构内部，脱钙骨支架有部分被吸收。12周时各组支架结构明显被吸收，A组支架内填满软骨细胞，部分已纤维化，但结构排列紊乱。B组支架呈透明软骨样，软骨细胞充分渗透进入支架内，呈一定应力方向排列。II型胶原免疫组织化学A值统计学分析比较发现：第4，8，12周B组Ⅱ型胶原免疫组织化学的A值均高于A组，差异有统计学意义（P〈0．01）。两组间A值随时间的变化趋势差异有统计学意义（P〈0．01）。结论同种异体脱钙骨支架复合经细胞因子诱导的BMSC，在膝关节腔内进行培养，利用关节腔内低氧、多种生长因子的微环境以及关节活动时的应力刺激等优势，较体外培养可以培养出组织学特点更好的工程软骨。
Objective To compare the superiority of cultivating tissue engineered cartilage by homologous decalcified bone matrix combined with bone-marrow mesenchymal stem cells （BMSCs） in rabbits＇ knee cavity with culture in vitro. Methods Archiogeneration BMSCs were isolated and purified by adhering to the culture glassware wall from neonatal New Zealand white rabbits. The third generation BMSCs were induced to chondrocytes by transforming growth factor TGF-β1, insulin-like growth factor IGF-1 and vitamin C. Seven days later, the cells were bond to homologous decalcified bone matrix and cultured in vitro （group A）, cultured in rabbits＇ left knee cavity （group B）, or bond to homologous decalcified bone matrix and cultured in rabbits＇ right knee cavity （ group C）. Every 4 weeks after cellular transplant, all rabbits in groups B and C were sacrificed and paraffin-embedded sections were made from the specimens. All the section were subjected to H-E stain, toluidine blue stain and type I[ collagen immunohistochemical staining with chromogen diaminobenzidine （DAB）. Immunohistochemical absorbance （A） values were calculated by morphology analysis software. Measurement data were expressed as mean ± standard, and satistical analysis was performed by t test, one-way ANOVA using SPSS 15.0 software. Results After cultivation for 4 weeks, H-E stain showed diffuse distribution of chondrocytes on scaffold＇ s surface in group A. In group B, there were a great quantity of chondrocytes in the scaffold, and cartilage lacuna, matrix anachromasis and isogenous group were group. At the 8th week, in group B maturated chondrocytes and isogenous groups arranged as column, surrounded by a pond of cellular matrix infiltrating into the scaffolds. The decalcified bone matrix was absorbed partly. At the 12th week, all the scaffolds were absorbed obviously. Chondrocytes filled into the scaffolds in group A, but arranged irregularly. In group B chondrocytes ar ranged in the stress orientation. At 4th, 8th, and 12th week, A values of type I1 collagen in group B was significantly higher than in group A with the different being significant. Conclusion Compound of Homol- ogous decalcified bone matrix-induced BMSCs cultured in rabbits＇ knee cavity could yield better histological feature tissue engineered cartilage.
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2011年第6期979-982,共4页
Chinese Journal of Experimental Surgery
关键词
组织工程软骨
骨髓间充质干细胞
关节腔
Tissue engineered bone
Bone marrow mesenchymal stem cell
Joint cavity
