应用细胞片层技术构建功能性组织工程骨的动物实验研究

摘要

目的 细胞片层技术与传统的骨组织工程方法相结合构建功能性组织工程骨.方法 密度梯度离心法分离培养犬骨髓基质干细胞(BMSCs);将向成骨细胞诱导后的BMSCs接种至温度反应性培养皿中,37 ℃、5%CO<,2>饱和湿度培养,然后降温至20℃制备BMSCs细胞片层;制备犬脱钙骨基质(DBM)及富血小板血浆(PRP);将DBM/PRP/BMSCs细胞片层/BMSCs植入犬左侧背阔肌深面、右侧相应部位植入DBM/PRP/BMSCs,观察其成骨效果.结果 当温度降至20℃时,BMSCs从温度反应性培养皿上完全脱落,形成细胞片层,将其覆盖于DBM/PRP/BMSCs,其成骨效果优于不加细胞片层的组织工程骨.结论 细胞片层技术与传统的骨组织工程方法相结合可构建出较理想的功能性组织工程骨.%Objective To construct functional tissue-engineered bone with cell sheet technology and method of traditional bone tissue engineering. Methods Canine bone marrow mesenchymal stem cells(BMSCs) were isolated with the method of density gradient centrifugatiun and cultured. BMSCs were induced to differentiate into osteoblasts and cultured in temperature-responsive culture dishes at 37 ℃, 5％CO2 and saturated humidity. BMSCs cell sheet was prepared when temperature was changed to 20 ℃. Demineralized bone matrix(DBM) and platelet-rich plasma(PRP) were prepared, and complex of DBM/PRP/BMSCs cell sheet/BMSCs was construsted and implanted under the left latissimus dorsi muscle. Complex of DBM/PRP/BMSCs was implanted under the right latissimus dorsi muscle. Results When temperature dropped at 20 ℃, BMSCs detached automatically from the temperature-respensive culture dishes and formed an intact cell sheet. The osteogenesis of the DBM/PRP/BMSCs cell sheet/BMSCs group was better than that of the DBM/PRP/BMSCs group. Conclusion Cell sheet technology combined with traditional bone tissue provides a new way for constroction of ideal functional tissue-engineered bone.