永生化hBMSCs复合异种骨异位成骨  

ECTOPIC OSTEOGENESIS OF IMMORTALIZED HUMAN BONE MARROW MESENCHYMAL STEM CELLS AND HETEROGENEOUS BONE

作　　者：滕勇[1] 胡蕴玉[2] 李旭升[1] 王臻[2] 白建萍[2] 关玉成[1] 

机构地区：[1]兰州军区乌鲁木齐总医院骨一科,乌鲁木齐830000 [2]第四军医大学西京医院骨科研究所

出　　处：《中国修复重建外科杂志》2010年第12期1515-1519,共5页Chinese Journal of Reparative and Reconstructive Surgery

基　　金：兰州军区医药卫生科技计划项目(LXH2007014)~~

摘　　要：目的为解决组织工程中种子细胞数量不足及为组织工程种子细胞研究提供标准细胞,建立永生化hBMSCs(MSCxj)系,进一步探讨MSCxj的异位成骨能力。方法取冻存复苏后生长良好的第35代和第128代MSCxj,扩增培养至2109个,分别与牛松质骨复合培养,作为A、B组(n=12),C组为单纯牛松质骨(n=12)。成骨诱导培养48h和18d时行扫描电镜观察细胞在材料上的生长情况。于18只4～6周龄裸鼠背部两侧皮下各作一3mm小切口,将3组共36个标本采用组内随机、组间两两配对分别植入切口内。处死动物前分别行四环素荧光标记。于术后4、8、12周各组分别取4个标本行HE、丽春红三色、四环素荧光染色、鼠抗人骨钙素单克隆抗体免疫组织化学染色观察,并行形态学定量分析。结果细胞-异种骨复合培养48h,扫描电镜观察见A、B组细胞贴附生长良好;18d可见大量合成的丝状细胞外基质及细小颗粒样分泌物,并覆盖细胞。裸鼠皮下埋植实验:HE、丽春红三色、四环素荧光染色观察示,术后4周A、B组标本成骨不明显,8周A、B组标本周边及异种骨内部孔隙有类骨基质沉积,12周A、B组标本成骨增加,A、B组间成骨情况比较无明显差异;C组各时间点均未见骨质形成。鼠抗人骨钙素单克隆抗体免疫组织化学染色显示,A、B组8周及12周骨陷窝内骨钙素染色呈阳性。形态学定量分析显示,术后8周A、B组骨长入率分别为5.64%±2.68%和4.92%±2.95%,术后12周分别为13.94%±2.21%和14.34%±3.46%,两组间比较差异均无统计学意义(P>0.05);组内术后12周骨长入率均高于术后8周,差异有统计学意义(P<0.05)。结论 MSCxj与原代培养hBMSCs一样具有良好的异位成骨能力,可作为骨组织工程的种子细胞进行相关实验及临床研究。Objective To provide the seed cells for bone tissue engineering, to establish immortalized human bone marrow mesenchymal stem cells （MSCxj） and to investigate the ectopic osteogenesis of MSCxj. Methods MSCxjs of the 35th and 128th generations were maintained and harvested when the cell density reached 2 109. Then, these cells were co-cultured with heterogeneous bone scaffold in groups A （the 35th generation, n=12） and group B （the 128th generation, n=12）; heterogeneous bone alone was used in group C （n=12）. The cell proliferation was observed by scanning electron microscopy （SEM） after 48 hours and 18 days of osteogenic induction culture. The complex was implanted subcutaneouly through a 3-mm-incision at both sides of the back in 18 nude mice. Tetracycline labeling was performed before the animals were sacrificed. Tetracycline fluorescence staining, HE staining, ponceau staining, and immunohistochemistry staining for osteocalcin were performed at 4, 8, and 12 weeks after transplantation; the morphologic quantitative analysis was made. Results After 48 hours, SEM showed that MSCxjs adhered to heterogeneous bone and grew well; after 18 days, a large number of new filamentous extracellular matrix and small granules were found to cover the cells. The results of tetracycline fluorescence staining, HE staining, and ponceau staining in groups A and B showed that the osteogenesis was not obvious at 4 weeks after transplantation; osteoid matrix deposition was noted around and in the heterogeneous bone at 8 weeks; and osteogenesis was increased at 12 weeks. There was no significant difference in bone formation between groups A and B. Osteogenesis was not observed in group C. The osteocalcin expressions were positive in groups A and B. The bone ingrow percentages of groups A and B were 5.64% ± 2.68% and 4.92% ± 2.95% at 8 weeks, and 13.94% ± 2.21% and 14.34% ± 3.46% at 12 weeks, showing significant differences between 8 weeks and 12 weeks at the same group （P 0.05） and no significant difference b

关 键 词：骨组织工程 永生化hBMSCs 异种骨 异位成骨 裸鼠 

分 类 号：R68[医药卫生—骨科学]