复合水凝胶三维培养骨髓间充质干细胞的矿化及异位成骨研究  

Study on mineralization and ectopic osteogenesis of bone marrow mesenchymal stem cells cultured in three-dimensional composite hydrogel

作　　者：苗胜 周津如 刘斌 雷星[3] 吴昊 高祎 宋岳 毕龙 裴国献 Miao Sheng;Zhou Jinru;Liu Bin;Lei Xing;Wu Hao;Gao Yi;Song Yue;Bi Long;Pei Guoxian(Department of Orthopaedics,Xijing Hospital,Air Force Military Medical University,Xi’an 710032,China;Department of Radiology,Luoyang Central Hospital Affiliated to Zhengzhou University,Luoyang 471000,China;Department of Orthopaedics,Linyi people’s Hospital,Linyi 276002,China;Research Institute of Orthopedics of PLA,Air Force Military Medical University,Xi’an 710032,China)

机构地区：[1]空军军医大学西京医院骨科,西安710032 [2]郑州大学附属洛阳中心医院影像科,471000 [3]临沂市人民医院骨科,276002 [4]空军军医大学全军骨科研究所,西安710032

出　　处：《中华实验外科杂志》2020年第11期1988-1992,共5页Chinese Journal of Experimental Surgery

基　　金：国家重点研发计划(2016YFC1100300);国家自然科学基金重点项目(81672189)。

摘　　要：目的构建负载骨髓间充质干细胞(BMSCs)的明胶/海藻酸钠/硅酸镁锂水凝胶膜板,研究其体外矿化性能及体内异位成骨能力。方法从2周龄SD大鼠(SD大鼠均购自空军军医大学实验动物中心)骨髓中提取BMSCs并用流式细胞术鉴定,设置实验组T1、T2、T3水凝胶分别混合P3代BMSCs,终细胞浓度分别为1×10^8/L、1×10^9/L、1×10^10/L,对照组C不混合细胞。在1、3、7 d用钙黄绿素-AM/碘化丙锭染色检测细胞的存活率,7、14、21 d茜素红染色观察各组的矿化以及实时荧光定量聚合酶链反应检测成骨相关基因的表达。体内实验将4组水凝胶分别植入18只8周龄SD大鼠臀后侧肌袋内,术后1、2、4周取材,组织学观察成骨效果。两组数据组间比较采用t检验,多组数据组间比较采用方差分析。结果流式鉴定细胞是BMSCs,并且在水凝胶中7 d存活率可达(92.43±0.73)%。体外培养14 d后实验组矿化结节的数量逐渐增多且T3[(211.33±17.16)个]高于T2[(56.67±7.64)个]和T1[(28.33±3.51)个],差异有统计学意义(F=239.234,P<0.05),而对照组并无矿化结节的生成;7 d各组较高表达成骨相关转录因子基因而14 d高表达碱性磷酸酶(ALP)和骨钙素(OCN)基因。体内研究结果显示实验组2周开始形成软骨细胞囊泡、成骨细胞等,而对照组无成骨分化倾向。结论明胶/海藻酸钠/硅酸镁锂水凝胶具有良好的细胞相容性,可作为BMSCs三维培养的载体材料,复合水凝胶混合的BMSCs具有良好的矿化能力,在体内具有较好的异位成骨能力。Objective Bone marrow mesenchymal stem cells(BMSCs)were cultured in hydrogel membrane compounded with gelatin/sodium alginate/laponite,and their mineralization in vitro and ectopic osteogenesis in vivo were studied.Methods BMSCs was extracted from bone marrow of 2-week-old SD rat(purchased from the Experimental Animal Center of Air Force Military Medical University)and identified by flow cytometry.The experimental groups T1/T2/T3 were mixed with BMSCs in P3 generation with the final concentration of 1×10^8/L,1×10^9/L and 1×10^10/L,respectively,while the control group C did not mix cells.The cell survival rates were detected by Calcein-AM/Propidium lodide staining on the 1st,3rd and 7th day,and on the 7th,14th and 21st day the mineralization was observed by alizarin red staining,and the expression of osteogenesis-related genes was detected by real-time fluorescent quantitative polymerase chain reaction.In vivo experiment the four groups of hydrogels were implanted into the posterior gluteal muscle pouches of 188-week-old SD rats in vivo.The samples were taken at 1,2 and 4 weeks after operation,and the osteogenic effect was observed histologically.T-test was used for comparison between the two groups,and analysis of variance was used for comparison between multiple groups.Results The cells were identified as BMSCs by flow cytometry and the survival rate in hydrogel was(92.43±0.73)%on the 7th day.After 14 days of culture in vitro,the number of mineralized nodules increased gradually in the experimental groups,which in T3(211.33±17.16)was higher than that in T2(56.67±7.64)and T1(28.33±3.51),and difference was statistically significant(F=239.234,P<0.05),but there was no mineralized nodule in the control group.Osterix genes were highly expressed in 7 days and alkaline phosphatase and osteocalcin genes were highly expressed in 14 days.In vivo,it was found that chondrocytes,vesicles,osteoblasts and other osteoblasts began to form in the experimental groups in 2 weeks,while there was no tendency of osteogenic diffe

关 键 词：骨髓间充质干细胞 三维培养 复合水凝胶 成骨分化 

分 类 号：R681[医药卫生—骨科学]