摘要
目的:探讨正常关节腔环境对骨髓基质干细胞-磷酸三钙复合体的诱导作用,分析正常关节腔能否诱导骨髓基质干细胞向软骨细胞分化。方法:实验于2004-01/10在解放军第四军医大学组织工程中心实验室完成。磷酸三钙多孔陶瓷材料由法国地中海大学研制并提供,平均孔径为100～300μm,孔隙率为52%。选取中国美利奴绵羊12只,均从髂骨抽取骨髓10mL,分离培养骨髓基质干细胞,然后以1011L-1细胞密度接种2mm×2mm×2mm磷酸三钙多孔陶瓷材料,体外培养24h后移植到自体绵羊膝关节腔。分别在复合体移植后4周和8周取材,6只/次,常规制备石蜡切片,分别进行甲苯胺蓝染色、苏木精-亮绿-番红花-“O”染色、免疫组化染色,镜下观察阳性染色分布和强度。结果:实验选取中国美利奴绵羊12只,全部进入结果分析。①术后骨髓基质干细胞-磷酸三钙复合体大体观察结果:复合体移植后4周从关节腔中取出的骨髓基质干细胞-磷酸三钙复合体为白色,质地较硬;8周时取出,复合体为白色半透明,质地比较有弹性,形似软骨。②骨髓基质干细胞-磷酸三钙复合体软骨基质特染观察结果:甲苯胺蓝染色发现,植入关节腔的骨髓基质干细胞-磷酸三钙复合体有明显阳性染色,表明有糖蛋白基质形成。番红-亮绿染色发现,关节腔骨髓基质干细胞-磷酸三钙复合体有砖红色异染,在小孔内壁有明显软骨形成。Ⅱ型胶原免疫组化染色发现,复合体移植后4周标本有Ⅱ型胶原阳性表达,8周时表达增强,但骨钙素免疫组化染色没有发现阳性反应。结论:正常关节腔环境能够诱导骨髓基质干细胞-磷酸三钙复合体中的干细胞发生软骨细胞表型分化,表现出软骨基质特染强阳性。
AIM： To explore the induction of normal joint cavity environment on the marrow-derived mesenchymal stem cells-tricalcium phosphate （TCP） complex, and analyze whether the marrow-derived mesenchymal stem cells can he induced to chondrocytic differentiation by normal joint cavity environment.
METHODS： The experiment was conducted in the Central Laboratory of Tissue Engineering, Fourth Military University of Chinese PLA from January to October 2004. Ceramic porpus scaffold of TCP was manufactured and provided by Mediterranean University of Franece with an average pore size of 100-300 μm and the interval porosity was 52%. Twelve Chinese merino sheep were selected, 10 mL marror from iliac bones in sheep were sucked and marrow-derived mesenchymal stem cells were isolatedly cultured and then innoculated of 2 mm×2 mm×2 mm TCP ceramic porous scaffold at the density of 10μL^-1, and then transplanted into the knee joint cavity of autoallergic sheep after cultivated in vitro for 24 hours. Materials were drew 4 and 8 weeks after the complex transplantation respectively with 6 sheep each time, and then routinely made into paraffin sections, after that, toluidine blue （TB） staining, campechy-bright greensafranin-O staining and immunostaining were performed, and then the distribution and density of positive staining were observed under the microscope. RESULTS： A total of 12 included Chinese merino sheep were involved in the analysis of results. ①General observation on the complex of marrowderived mesenchymal stem cells-TCP： TCP complex was hard and in white 4 weeks after the transplantation; The complex was took out at the 8^th week, which was much flexible and in half-white, and was similar to cartilage. ②Staining observation on complex mesochondrium of marrow- derived mesenchymal stem cells-TCP： It was found with TB staining that the complex of marrow-derived mesenchymal stem cells-TCP inside the joint cavity obviously had positive staining, which indicated that there were glycosidoprotein base formed. It was found with safranine-light green staining that there were brick-red metachromia in the complex of marrow- derived mesenchymal stem cells inside the joint cavity, and there were obvious cartilage formed on the inner wall of stoma. It was found with type Ⅱ collagen immunostaining that there were positive expression of type Ⅱcollagen in samples 4 weeks after the transplantation of complex, which was reinforced at the 8^th week, while there were no positive reaction in the osteocalcin irnrnunochernical staining.
CONCLUSION： Normal joint cavity envirornent can induce the phenotypic differentiation of stern cells in the complex of marrow-derived rnesenchyrnal stern cells-TCP towards chondrocytes, which represents a strong positive staining of mesochondriurn.
出处
《中国临床康复》
CSCD
北大核心
2006年第17期33-35,i0002,共4页
Chinese Journal of Clinical Rehabilitation
基金
国家自然科学基金资助(30400503)~~
