组织工程化骨膜的构建及其在体外的成骨活性检测  

Constructing tissue-engineered periosteum and investigating its biological characteristics and osteogenic potential in vitro

作　　者：张苍宇[1] 汪新柱[2] 任广铁[1] 拓振合[1] 庾佳佳[1] 孙瑞[1] 汪静[1] 赵琳[1] 

机构地区：[1]兰州大学第二医院骨科甘肃省骨与关节疾病研究重点实验室,兰州730030 [2]甘肃省中医院放射科,兰州市730050

出　　处：《中国矫形外科杂志》2014年第3期256-261,共6页Orthopedic Journal of China

基　　金：国家自然科学基金资助项目(编号:30973064);国家留学回国人员优选资助课题(甘人社通【2010】343号)

摘　　要：[目的]利用兔骨髓间充质干细胞与猪小肠粘膜下层复合构建组织工程骨膜,体外观察其生物学特性并检测成骨活性,为将来体内移植提供实验依据。[方法]以猪小肠粘膜下层基质作为支架材料,与新西兰大白兔骨髓间充质干细胞(BMSCs)或体外成骨诱导培养后的BMSCs,采用沉淀法复合构建两种组织工程化骨膜(M2及M1),用MTT法检测种子细胞在支架材料上的生长情况;扫描电镜(SEM)观察种子细胞在支架材料上的生长、粘附状态;通过ELISA检测碱性磷酸酶活性和骨钙素分泌水平。[结果]种子细胞在SIS上生长良好,M2及M1在含有成骨诱导剂的培养基中培养可分泌一定量骨钙素(11 d达峰值),并表达碱性磷酸酶活性(7 d达峰值)。而且,Ml所分泌骨钙素的量与碱性磷酸酶活性高于M2,差异有统计学意义(P<0.05)。[结论]利用兔骨髓间充质干细胞与猪小肠粘膜下层复合构建组织工程化骨膜能够保持较稳定的生物活性,种子细胞能良好地在支架上附着、生长增殖,并稳定地分泌一定量的骨钙素和碱性磷酸酶,在体外表达较强的成骨活性。[ Objective] To investigate the biological characteristics and osteogenic potential of tissue -engineered perioste- urn, which was constructed by bone marrow mesenchymal stem cells （BMSCs） and porcine small intestinal submucosa （SIS） . [Methods] Porcine SIS was cocultivated with New Zealand rabbit BMSCs or osteogenically induced BMSCs in vitro to construct two kinds of tissue - engineered periosteum （ M1 and M2） . The growth curves of the seeding cells in the M1 and M2 groups were detected by MTT assay, and the ultrastructure of tissue - engineered periosteum was observed by electronic microscopy. Meanwhile, the osteocalcin content and alkaline phosphatase （ALP） activity of the ceils in the M1 and M2 groups were analyzed by ELISA. [ Results ] The growth status of seeding cells was consistently good on tissue - engineered periosteum. Osteocalcin quantity and ALP activity were significantly higher in the M1 group than in the M2 group （P 〈 0.05 ） . ALP activity expression reached its peak on the 7th day, while osteocalcin quantity peaked on the 11 th day. [ Conclusion] The biological characteristics of tissue - engineered periosteum, which was constructed with BMSCs and SIS, remained stable. The growth tendency of the BMSCs improves consistently in the SIS. Osteocalcin and ALP were secreted at a certain level. The tissue engineered periosteum thus showed a highly osteogenetic activity in vitro.

关 键 词：骨髓间充质干细胞 猪小肠粘膜下层 组织工程骨膜 骨钙素 碱性磷酸酶 

分 类 号：R318.08[医药卫生—生物医学工程]