血管化人工骨的体外构建和体内异位成骨

Construction of vascularization artificial bone in vitro and ectopic osteogenesis in vivo

背景:血管化在骨形成和改建中起重要作用,目前大的组织块难以获得充足的氧气和营养供应,因此组织工程中就出现了需要适当血管化的问题。目的:建立体外血管化人工骨模型并且体内异位成骨的实验体系。方法:分离培养鼠骨髓间充质干细胞和肾血管内皮细胞,体外构建鼠骨髓间充质干细胞和鼠肾血管内皮细胞直接接触培养血管化人工骨组,以间接接触三维培养体系和两种细胞单独培养体系作为对照。体外通过测定蛋白质含量,碱性磷酸酶活性和骨钙素,分析骨髓间充质干细胞在不同混合培养模型中的成骨能力。建立鼠骨髓间充质干细胞和肾血管内皮细胞的三维培养体系,再将两种细胞直接接触组材料和骨髓间充质干细胞单独培养组的材料分别植入鼠左右腿肌肉内,通过软 X 射线摄影和苏木精-伊红染色检测分析不同植入方法的血管形成和成骨能力。结果与结论:当两种细胞混合培养时,具有很好的细胞兼容性。两种细胞单独培养碱性磷酸酶活性和骨钙素较间接接触混合培养降低,而两种细胞混合培养时,体系中碱性磷酸酶活性和骨钙素水平增加。动物实验结果显示在混合培养体系中的骨髓间充质干细胞的成骨能力高于单独培养(P<0.05)。体内实验表明在血管化人工骨中的软 X 射线骨密度,血管数量和新形成的骨量均高于对照组(P<0.05)。提示在两种细胞混合培养时骨髓间充质干细胞的成骨能力可以被细胞因子和细胞膜蛋白质调节,和传统的人工骨比较,血管化人工骨有加速骨髓间充质干细胞分化,增加了局部的血管微循环生存率,以及加速成骨和更好的抗感染能力。

BACKGROUND：Vascularization plays an important role in bone formation and remodeling,however,the oxygen and nutrition are insufficient for large tissue block;therefore,it is necessary to solve the problem of vascularization in tissue engineering field.OBJECTIVE：To investigate the method of constructing vascularization artificial bone in vitro and ectopic osteogenesis in vivo.METHODS：Rat bone marrow mesenchymal stem cells （BMSCs） and kidney vascular endothelial cells were isolated and cultured,and osteogenesis ability of BMSCs in different co-culture models （co-culture with or without direct contact） were analysed by measuring the quantity of protein and the activity of alkaline phosphatase and osteocalcin.By establishing the three-dimensional co-culture model of rat BMSCs and kidney vascular endothelial cells,vascularized artificial bone was constructed,which was implanted into the muscle of rats.The angiopoiesis and osteogenetic ability of the implants were observed by soft X-ray examination and hematoxylin-eosin staining.RESULTS AND CONCLUSION：There was good cell compatibility when rat BMSCs and kidney vascular endothelial cells were co-cultured.The ALP activity and osteocalcin was decreased in the simple culture groups,but increased in the direct and indirect co-culture groups.Statistical analysis showed that the osteogenetic ability of BMSCs co-cultured group was higher than that of simple cultured （P〈0.05）.The in vivo experiment manifested that the intensity of soft X-ray and the quantity of vascular and new formed bone in the vascularized artificial bone group were higher than those in the control group （P〈0.05）.The osteogenetic ability of BMSCs can be regulated by cytokines and cell membrane proteins when co-cultured with endothelial cells.Compared with traditional artificial bone,vascularized artificial bones has lots of advantages,such as accelerating the differentiation and proliferation of BMSCs,increasing local blood circulation and survive ratio,as well as accelerating osteogenesis and having more powerful anti-infect ability.