多孔钽材料细胞毒性、生物相容性及体内成骨性研究

摘要

目的 探讨国产多孔钽材料的细胞毒性和生物相容性,并通过兔骨内植入成骨示踪观察其成骨作用.方法 扫描电镜观察并测量多孔钽的形态学特征.来源于新西兰胎兔颅盖骨的成骨细胞以多孔钽浸提液(实验组)及完全培养基(对照组)培养,MTT法检测多孔钽的细胞毒性及其对增殖的影响.成骨细胞与多孔钽体外复合培养,观察成骨细胞的黏附、生长及增殖.雄性新西兰大白兔24只,制备股骨髁上多孔钽棒植入模型;4只动物于术后第5天和第19天分别肌肉注射荧光素钙黄绿素和茜素红,术后10周取材,于488 nm(钙黄绿素激发光)和543 nm(茜素红激发光)波长处,激光扫描共聚焦显微镜观察多孔钽-骨界面成骨;20只动物于术后2、4、8和12周取材行大体及硬组织切片观察.结果 多孔钽表面及断面可见均匀分布的三维立体连通孔隙结构.MTT法检测显示实验组与对照组细胞随培养时间的延长,其OD值的差异均无统计学意义.扫描电镜显示复合培养早期,细胞在多孔支架表面和孔壁上黏附,相互连接;晚期汇合成片并分泌细胞外基质覆盖材料表面.体内成骨实验显示植入的多孔钽棒与宿主骨结合紧密.硬组织切片显示术后2、4周时多孔钽-骨界面已出现新生骨及小血管,并向孔隙内生长;8、12周时多孔钽表面和孔隙内已长满新生骨组织,新生骨小梁已发育成熟并与材料直接接触.激光共聚焦扫描显微镜显示多孔钽-骨界面及孔隙内可见绿色荧光(钙黄绿素)和红色荧光(茜素红)标记的新生骨组织,红色荧光带位于绿色荧光带周围,早期均呈不连续性,晚期则融为一体.结论 国产多孔钽材料无细胞毒性,具有良好的生物相容性,多孔钽-骨界面为接触及传导性成骨并呈时间依赖性.%Objective To investigate cytotoxicity,biocompatibility and new bone formation traced of Chinese porous tantalum,and provide experimental strategies for further clinical application.Methods The physical properties of the porous tantalum were observed by the SEM.The osteoblasts were isolated from rabbit embryo.The extract fluid from tantalum was made.The cytotoxicity and proliferation of osteoblasts compounded with porous tantalum in vitro were detected by the MTT assay.The osteoblasts were co-cultured with extract of tantalum in vitro and the morphological changes,proliferation and adhesion were observed under SEM.A total of 24 New Zealand rabbits were used to establish the model of femoral condyles with porous tantalum bars implanted.Among which 4 of them was injected with calcein and alizarin on the 5th day and the 19th day and sacrificed at 10 week postoperatively.The specimens were observed with LSCM at 488 nm and 543 nm wavelength respectively.The remained 20 animals were sacrificed successively at 2,4,8,12 weeks of implantation,then were examined by histological observation.Results The SEM showed that the pore of porous tantalum were three-dimensional connected morphology.MTT assay showed that the osteoblasts grew well in extract and no significant difference between experimental and control groups.The osteoblasts grew and spread extensively on porous tantalum.Early on co-culture,the osteoblasts attached to the surface and inner walls of material,in the later stage,the osteoblasts excreted bone matrix over the surface of porous tantalum.The animal model showed that porous tantalum was bonded closely with host bone.Hard slicing showed that new bone and capillary regenerated on tantalum-bone interface at 2,4 weeks postoperatively.The pores were full with bone tissue at 8,12 weeks.The LSCM indicated that the green and red fluorescence-labeled new bone was displayed on tantalum-bone interface,while the red zone located around the green zones.They appeared to be discontinuous at early stage,but connected with each other at the end.Conclusion The Chinese porous tantalum has good biocompatibility and no cytotoxicity.The contact osteogenesis and bone conduction exist in tantalum-bone interface,and in a time-dependent manner.