羟基磷灰石-磷酸三钙支架复合骨髓间充质干细胞和人脐静脉内皮细胞对大鼠颅骨缺损修复早期成血管的影响  

Effects of bone marrow mesenchymal stem cells and human umbilical vein endothelial cells combined with hydroxyapatite-tricalcium phosphate scaffolds on early angiogenesis in skull defect repair in rats

作　　者：陈思奇 先德彬 徐荣胜 覃中杰 张磊 夏德林 Chen Siqi;Xian Debin;Xu Rongsheng;Qin Zhongjie;Zhang Lei;Xia Delin(Plastic Surgery,Affiliated Hospital of Southwest Medical University,Luzhou 646000,Sichuan Province,China;Hospital of Traditional Chinese Medicine Affiliated to Southwest Medical University,Luzhou 646000,Sichuan Province,China;Department of Oral and Maxillofacial Surgery,Affiliated Stomatological Hospital of Southwest Medical University,Luzhou 646000,Sichuan Province,China)

机构地区：[1]西南医科大学附属医院整形外科,四川省泸州市646000 [2]西南医科大学附属中医医院,四川省泸州市646000 [3]西南医科大学附属口腔医院口腔颌面外科,四川省泸州市646000

出　　处：《中国组织工程研究》2021年第22期3458-3465,共8页Chinese Journal of Tissue Engineering Research

基　　金：泸州市科技局项目[2013-S-48(8/30)],项目参与者:夏德林;四川省科技厅应用基础研究资助项目(2008JY0014),项目负责人:夏德林。

摘　　要：背景:组织工程骨为大型骨缺损修复提供了新方向,但只有早期形成完善的毛细血管网才能保证移植物在体内的长期存活,因此早期组织工程骨的血管化是其临床应用的关键所在。目的:探索骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)与人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVECs)复合羟基磷灰石-磷酸三钙(hydroxyapatite tertiary calcium phosphate,HA-TCP)支架对大鼠颅骨缺损修复早期血管生成的影响。方法:①将HA-TCP支架分4组处理:A组加入体积比1∶1的DMEM培养液与细胞外基质培养液;B组加入体积比1∶1的BMSCs悬液与HUVECs悬液;C组加入体积比1∶1的DMEM培养液与HUVECs悬液,D组加入体积比1∶1的BMSCs悬液与细胞外基质培养液,培养21 d后用于体内植入实验。②在成年SD大鼠颅骨制作直径1 cm骨缺损,分别植入A、B、C、D组材料,术后4,8,12周,分别进行颅骨Micro-CT及颅骨支架复合物大体、组织形态学、CD31/CD34免疫组化染色、CD31/CD34分子含量、血管内皮生长因子A蛋白检测。动物实验已获得西南医科大学动物伦理委员会批准。结果与结论:①Micro-CT检测:术后8周时,B组新生骨质量高于其他3组,C、D组高于A组;术后12周时,新生骨质量由高到低依次为:B组>C组>D组>A组;②大体观察:B组支架上骨质爬行面积高于其他3组,C、D组高于A组;相同时间点下血性渗液量由多到少依次为:B组>C组>D组>A组;③组织学观察:术后4周时,骨质新生量由高到低依次为:B组>C组>D组>A组,其余时间点骨质新生量由高到低依次为:B组>D组>C组>A组;④免疫组化染色:相同时间点下CD31/CD34微血管密度由高到低依次为:B组>C组>D组>A组,除C、D组无差异无外(P>0.05),其余组间比较差异有显著性意义(P<0.05);⑤Western Blot检测:相同时间点下血管内皮生长因子A蛋白表达由高到低依次为:B组>C组>D组>A组,除C、D组无差异外(P>0.05),其余组间�BACKGROUND:Tissue-engineered bone provides a new direction for the repair of large bone defects,but the survival of tissue-engineered bone grafts in vivo can be guaranteed only if the capillary network is formed in the early stage.Therefore,vascularization of early tissue-engineered bone is the key to its clinical application.OBJECTIVE:To explore the effect of bone marrow mesenchymal stem cells and human umbilical vein endothelial cells combined with hydroxyapatite-tricalcium phosphate scaffolds on early angiogenesis in skull defect repair in rats.METHODS:(1)The hydroxyapatite-tricalcium phosphate scaffold was divided into four groups:Group A was added with DMEM culture medium and extracellular matrix culture medium with volume ratio of 1:1.Group B was added with bone marrow mesenchymal stem cells suspension and human umbilical vein endothelial cells suspension with a volume ratio of 1:1.Group C was cultured with DMEM culture medium and human umbilical vein endothelial cells suspension with a volume ratio of 1:1.Group D was cultured with bone marrow mesenchymal stem cells suspension and extracellular matrix medium with a volume ratio of 1:1 for 21 days for in vivo implantation experiment.(2)Bone defects with a diameter of 1 cm were made in the skull of adult SD rats,and materials of groups A,B,C and D were implanted respectively.Skull micro-CT and skull scaffold complex gross,histomorphology,CD31/CD34 immunohistochemical staining,CD31/CD34 molecular content,and vascular endothelial growth factor A protein were detected respectively 4,8 and 12 weeks after the operation.Animal experiment was approved by the Animal Ethics Committee of Southwest Medical University.RESULTS AND CONCLUSION:(1)Micro-CT showed that 8 weeks after operation,the mass of new bone in group B was higher than that in the other three groups,and the mass of new bone in groups C and D was higher than that in group A.At 12 weeks after surgery,the new bone mass was from high to low:group B>group C>group D>group A.(2)Gross observation showed that the

关 键 词：骨 材料 支架 组织工程骨 骨缺损修复 干细胞 人脐静脉内皮细胞 早期血管生成 

分 类 号：R459.9[医药卫生—治疗学] R318[医药卫生—临床医学]