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目的:探讨犬骨髓基质细胞(bone marrow stromal cells,BMSCs)细胞片层在构建组织工程骨中的价值。方法:制备犬同种异体脱钙骨基质(dermineralized bone matrix,DBM)。将人重组骨形态发生蛋白-2(rhBMP-2)复合到DBM上。抽取犬髂骨骨髓,采用密度梯度离心法分离犬骨髓基质细胞(BMSCs)。将经成骨诱导的第3代细胞接种于温度反应性培养皿中,制备BMSCs细胞片层。用得到的BMSCs细胞片层包裹DBM/rhBMP-2/BMSCs复合体,植入犬背阔肌血运丰富的肌筋膜下为实验侧,以无BMSCs细胞片层包裹的DBM/rhBMP-2/BMSCs复合体为对照侧。术后4、8、12周取材,行组织学观察,评价体内异位成骨的情况。采用SPSS13.0软件包对数据进行两样本均数差别的t检验。结果:实验侧成骨面积大于对照侧,2组差异有显著性(P<0.05)。术后12周,实验侧生成大量板层骨,有哈弗系统形成,骨髓腔内有红骨髓。对照侧有板层骨形成,无哈弗系统形成,骨髓腔内无红骨髓。结论:BMSCs细胞片层可促进具有致密板层骨和哈弗系统的组织工程骨的形成。
Objective: To investigate the value of bone marrow stromal cells (BMSCs) cell sheets in the construction of tissue engineering bone. Methods: Preparation of dog bone allograft demineralized bone matrix (dermineralized bone matrix, DBM). Human recombinant bone morphogenetic protein-2 (rhBMP-2) complexed to DBM. Canine bone marrow was isolated and canine bone marrow stromal cells (BMSCs) were isolated using density gradient centrifugation. The osteoblast-induced third generation cells were seeded into temperature-responsive culture dishes to prepare BMSCs cell sheets. The resulting BMSCs were encapsulated in the DBM / rhBMP-2 / BMSCs complex and implanted under the myofascial muscle of the latissimus dorsi in the experimental side. The DBM / rhBMP-2 / BMSCs complex as the control side. At 4, 8, and 12 weeks after operation, we observed the histopathology and evaluated the ectopic osteogenesis in vivo. SPSS 13.0 software package was used to test the data for the difference between the two sample means. Results: The osteogenic area of the experimental side was larger than that of the control side. There was significant difference between the two groups (P <0.05). Twelve weeks after operation, a large amount of lamellar bone was formed on the experimental side with the formation of a Haval system and red marrow in the marrow cavity. The control side of lamellar bone formation, no Haval system formation, bone marrow cavity without red bone marrow. CONCLUSIONS: BMSCs cell sheets promote the formation of tissue engineered bone with dense lamina and Haval systems.