间充质干细胞/内皮祖细胞基质依赖型组织工程骨修复大鼠股骨缺损  

Mesenchymal stem cell/endothelial progenitor cell extracellular matrix-based tissue engineering bone for the repair of rat femoral defects

作　　者：董睿 白赟 许建中 董世武 Dong Rui;Bai Yun;Xu Jianzhong;Dong Shiwu(Department of Biomedical Materials,Army Medical University,Chongqing 400038,China;Department of Logistics Support,948th Hospital of PLA,Wusu 833000,China;Department of Orthopedics,Southwest Hospital,Army Medical University,Chongqing 400038,China)

机构地区：[1]陆军军医大学生物医学材料学教研室,重庆400038 [2]解放军第九四八医院后勤保障部,乌苏833000 [3]陆军军医大学第一附属医院骨科,重庆400038

出　　处：《中华创伤杂志》2020年第9期837-846,共10页Chinese Journal of Trauma

基　　金：全军后勤保障研究计划重点项目(AWS17J004-02-06);全军医学科技青年培育(拔尖)计划(20QNPY022);陆军军医大学军事医学预研基金(2018XYY05)。

摘　　要：目的探讨大鼠骨髓间充质干细胞(MSC)/内皮祖细胞(EPC)作为种子细胞构建的基质依赖型组织工程骨(ECM-TEB)修复大鼠股骨缺损的效果。方法分离培养骨髓来源的MSC和EPC并进行功能鉴定,种植上架于纳米晶胶原基人工骨颗粒,培养14 d后冻干,获得MSC/EPC ECM-TEB和MSC ECM-TEB。扫描电镜观察MSC和EPC在支架表面的生长形态。分别提取MSC ECM-TEB蛋白浸提液(对照组)和MSC/EPC ECM-TEB蛋白浸提液(实验组),加入EPC培养系统中进行迁移、划痕修复、管腔形成检测;另加入MSC培养系统中进行茜素红染色和碱性磷酸酶(ALP)染色检测,观察两组细胞募集、血管生成和成骨分化情况。选取12只SD大鼠,建立股骨缺损模型,按照随机数字表法分为:(1)假手术组:仅在缺损处进行清创处理;(2)MSC ECM-TEB组:在缺损处植入MSC ECM-TEB;(3)MSC/EPC ECM-TEB组:在缺损处植入MSC/EPC ECM-TEB,每组4只大鼠。2个月后行Micro-CT检查和缺损区Masson三色染色观察骨缺损修复情况。冻干后低温保存3个月,采取同位素相对标记与绝对定量技术(iTRAQ)标记蛋白质谱检测MSC ECM-TEB和MSC/EPC ECM-TEB在成血管方面的差异,并采用基因本体论/京都基因与基因组百科全书技术(GO/KEGG)功能富集方法分析其原因。结果MSC和EPC在支架表面生长、增殖良好,形成光滑的细胞层状结构。实验组在细胞迁移数、划痕修复比例和管腔形成长度分别为(121.6±8.3)个、(61.5±5.9)%、(11.3±0.6)mm,较对照组显著增多[(85.0±6.7)个、(39.3±3.6)%、(5.9±0.4)mm](P均<0.01)。茜素红染色和ALP染色结果显示,实验组钙结节矿化面积比例显著增加[(38.8±3.3)%∶(49.9±3.0)%、(38.8±2.4)%∶(45.3±3.3)%](P均<0.05)。Micro-CT和Masson染色结果表明,MSC/EPC ECM-TEB组骨缺损修复良好,MSC ECM-TEB组仅形成少量新骨,假手术组几乎没有新骨形成;骨体积分数、骨小梁数量和骨小梁厚度差异均有统计学意义(P<0.05)。蛋白质谱分析结果Objective To investigate the treatment effect of marrow mesenchymal stem cells(MSC)/endothelial progenitor cells(EPC)extracellular matrix-based tissue engineering bone(ECM-TEB)in repair of femoral defects in rats.Methods Bone marrow-derived MSC and EPC were isolated and cultured for functional identification,and planted on the nanocrystalline collagen-based artificial bone particles.After culturing for 14 days,the cells were lyophilized to obtain MSC/EPC ECM-TEB and MSC ECM-TEB.A scanning electron microscope was used to observe the morphology of MSC and EPC on the surface of the scaffold.The protein extracts of MSC ECM-TEBs(control group)and the protein extracts of MSC/EPC ECM-TEBs(experimental group)were added to the EPC culture system for migration test,scratch repair assay,and tube formation detection;and to the MSC culture system for alizarin red staining and alkaline phosphatase?(ALP)staining detection.The cell recruitment,angiogenesis and osteogenic differentiation were observed.A total of 12 SD rats were selected to establish a femoral defect model.According to the random number table,the rats were divided into:(1)sham group:debridement treatment was performed only at the defect;(2)MSC ECM-TEB group:MSC ECM-TEB was implanted at the defect;(3)MSC/EPC ECM-TEB group:MSC/EPC ECM-TEB was implanted at the defect,with 4 rats per group.Two months later,micro-computed tomography(Micro-CT)and Masson's tricolor staining were performed to observe the treatment effect of the bone defect.When the cells were stored at low temperature for three months after lyophilization,the different protein profile between MSC ECM-TEB and MSC/EPC ECM-TEB in vascularization was detected by isotope relative labeling and absolute quantification technology(iTRAQ).The gene ontology/Kyoto Encyclopedia of Gene and Genome Technology(GO/KEGG)function enrichment was used to analyze the key differences.Results MSC and EPC grew well and formed a smooth cell layered structure on the surface of the scaffold.The number of cell migration,ratio of scra

关 键 词：细胞外基质蛋白质类 骨再生 新血管形成 组织工程 

分 类 号：R683[医药卫生—骨科学]