组织工程骨-80℃低温保存的初步研究

Preliminary study on cryopreservation of tissue engineered bone at -80 ℃

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(marrowstromal 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-80℃深低温保存3个月,3个月后复温冻存的组织工程骨。扫描电镜观察MSCs的黏附和分布情况,MTT法检测细胞活力,对硝基苯磷酸法检测碱性磷酸酶(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黏附和分布,黏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),黏附于材料的细胞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 effects of various methods of cryopreservation on the bioactivity of tissue engineered bone. Methods： MSCs were cocultured with partialy deproteinised bone to produce tissue engineered bone. The experiment was divided into A, B, C and D group. Group A：Tissue engineered bone was stored in preservation solution with cryopreservation medium. Group B：Tissue engineered bone was stored in preservation solution without cryopreservation medium. Group C ：Tissue engineered bone was stored without cryopreservation. Group D：MSCs were cultured without cryopreservation. The tissue engineered bone of group A and B had been cryopreserved at -80℃ for three months and thawed three months later. The electronic scanning microscope was used to evaluate the adhesion and distribution of MSCs,cell viability was measured by MTT, ALP activity was detected by p-nitrophosphate, cell cycle was analysed by flow cytometry. Results：MSCs could adhere to the surface of the material and distribute in the hole of material. The cell viability of MSCs adhered to the 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 cycles of different groups did not change significantly ; the abnormal cells were not observed. Conclusion： The choice of proper cryopreservative solution could optimize the bioactivity of tissue engineered bone.