不同冻存方法对组织工程骨生物活性的影响

Effect of various cryopreservation methods on bioactivity of tissue engineered bone

目的:了解不同冻存方法对组织工程骨生物活性的影响。方法:以骨髓基质干细胞(marrow stromal cells,MSCs)复合部分脱蛋白骨培养制备组织工程骨,实验分为A组:组织工程骨用添加冻存保护剂的保存液保存;B组:组织工程骨用不添加冻存保护剂的保存液保存;C组:组织工程骨未行低温保存;D组:单纯MSCs培养。A组和B组的组织工程骨于-196℃的液氮中冻存3个月,3个月后复温冻存的组织工程骨。用倒置相差显微镜和扫描电镜观察MSCs的粘附和分布情况、检测细胞活力和碱性磷酸酶(alkaline phosphatase,ALP)活性、流式细胞仪分析细胞周期。结果:MSCs在材料表面和孔隙内均可粘附和分布,粘附于材料的细胞活力大小依次为:C组>A组>B组(P<0.01,P<0.05),粘附于材料的细胞ALP活性大小依次为:C组>A组>B组(P<0.01)。各组细胞周期未见明显变化,未见异倍体细胞。结论:选择适宜的冻存保护剂对组织工程骨的生物活性有一定的保护作用。

Objective To study the effect of various cryopreservation methods on bioactivity of tissue engineered bone.Methods MSCs were cocultured with partialy deproteinized bone to produce tissue engineered bone.The experiment was divided into A,B,C and D group.Group A:Tissue engineered bone had been stored in preservation solution with cryopreservation medium.Group B:Tissue engineered bone had been stored in preservation solution without cryopreservation medium.Group C:Tissue engineered bone without cryopreservation.Group D:Simple MSCs were cultured.The tissue engineered bone of group A and B had been stored in liquid nitrogen at-196℃ for three months and thawed three months later.The cell-material complex was observed under phase microscope and electronic scanning microscope in order to evaluate the adhesion and distribution of MSCs,cell viability and ALP activity were measured,cell cycle was analysed by flow cytometer.Results MSCs adhered to the surface of material and distributed in the hole of material.The cell viability of MSCs adhered to material was C>A >B group(P<0.01,P<0.05).The ALP activity of MSCs adhered to material was C>A >B group(P<0.01).The cell cycle of different groups did not change significantly,the abnormal cells were not seen.Conclusion The choice of proper cryopreservation solution could optimize the bioactivity of tissue engineered bone.