三维打印组织工程骨支架的生物相容性实验研究

Experimental study of biocompatibility of three-dimensional printing tissue engineering bone scaffolds

目的探讨以煅烧羊椎骨松质双相陶瓷粉末/聚乙烯醇(polyvinyl alcohol vinylalcohol polymer,PVA)生物高分子聚合物凝胶为原材料的新型三维打印组织工程骨支架成型方法,及支架的生物相容性。方法取健康、清洁级新疆阿勒泰大尾羊骨髓液培养羊骨髓间充质干细胞;取新鲜市售羊椎骨,去皮质骨,脱蛋白质、脱脂、煅烧后松质双相陶瓷精细研磨,钴60放射灭菌,与体积分数15%PVA溶剂体积比1∶2混合,制备成型材料。将建立好的STL格式文件导入固高GXYZ303010-XYLE三维打印系统,装配快速成型材料,采用熔融堆积成型法完成三维打印组织工程骨支架的成型。将羊骨髓间充质干细胞与灭菌三维打印组织工程骨支架在体外复合培养,扫描电镜观察羊骨髓间充质干细胞在三维打印组织工程骨支架中的生长情况;制备支架材料浸提液,采用MTT法检测三维打印组织工程骨支架对羊骨髓间充质干细胞增殖的影响,采用皮内刺激试验评价三维打印组织工程骨支架材料的生物相容性。结果三维打印法建立的组织工程骨支架具有良好的通透性,孔隙率为68.3%,孔隙间连通率为84.0%,具备一定的力学性能;三维打印组织工程骨支架浸提液对羊骨髓间充质干细胞的细胞毒性为0~1级,无明显细胞毒性;羊骨髓间充质干细胞可紧密附着于支架材料表面,黏附生长、增殖,并有突起向支架内部空隙伸展;皮内刺激试验结果表明三维打印组织工程骨支架对新疆阿勒泰大尾羊无刺激性。结论利用羊椎骨松质煅烧双相陶瓷粉末/PVA生物高分子聚合物成型的三维打印组织工程骨支架无刺激性、无明显细胞毒性,具有良好的生物学功能,有望应用于骨组织工程学。

Objective To explore a new method of three-dimensional(3D)printing tissue engineering bone scaffold from sheep vertebrae cancellous ceramic powder and polyvinyl alcohol vinylalcohol polymer(PVA)biological polymer gel and to observe its biocompatibility.Methods Bone marrow mesenchymal stem cells(BMSCs)were cultured in bone marrow fluid of clean healthy Altay big-tailed sheep.Fresh sheep vertebral bone without cortical bone was obtained and was finely grinded after deproteinization,degreasing and calcining with cancellous biphasic ceramics,and mixed with 15% PVA at the ratio of 1∶2to prepare molding materials.The established STL format file was imported to Gugao GXYZ303010-XYLE 3Dprinting system to assemble material for rapid prototyping,melting and stacking method was used to complete3 Dprinting tissue engineering bone scaffold.Sheep BMSCs and sterilized 3Dprinting scaffolds were cultivated in vitro.The growth of the cells in 3Dtissue engineering bone scaffolds was observed by scanning electron microscopy,and the scaffold material extract was prepared.MTT assay was used to assess the effect of 3Dtissue engineered bone scaffolds on stem cell proliferation.The biocompatibility of 3D tissue engineering bone scaffolds was evaluated by intradermal stimulation test.Results The tissue engineering bone scaffolds established by the 3D printing method had good permeability,the porosity was about 68.3% and the connectivity between the pores was 84.0%,showing certain mechanical properties.The cytotoxicity of 3Dbone tissue engineering scaffold extract was 0to 1.Sheep BMSCs were closely attached to the surface of the scaffold,growing and proliferating,and extending into the scaffold.Intradermal stimulation test showed that the scaffold had not irritation to the sheep.Conclusion 3D printing of bone tissue engineering scaffold made by sheep vertebral cancellous calcined biphasic ceramic powder/PVA biopolymer has good biological function with no stimulation or obvious cytotoxicity,and and can be expected to be used in bone tissue engineering.