兔骨髓基质细胞与陶瓷样异种骨的体外培养实验兔骨髓基质细胞形态、生长、附着及增殖性能观察

背景天然异种骨采用适当的理化方法可将其抗原性削弱或消除,且能保留其天然的网状孔隙系统,从而使其结构和组成符合生理要求,可望作为一种骨移植替代材料.目的采用传代的骨髓基质细胞与陶瓷样异种骨体外复合培养,以了解陶瓷样异种骨对兔骨髓基质细胞形态、生长、附着及增殖的影响.设计空白对照实验.单位昆明医学院第一附属医院中心实验室.材料3个月龄日本大耳白兔5只,体质量1.5～2.0kg,雌雄不限.方法实验于2003-06/10在昆明医学院第一附属医院中心实验室完成.取市售新鲜猪肋骨经理化处理制备陶瓷样异种骨,采用X射线衍射仪分析证实,其主要成分为羟基磷灰石;经扫描电镜观察证实该材料具有原骨组织的网状孔隙结构系统.取日本大耳白兔5只各抽取骨髓1 mL,经PRMI 1640培养液稀释制成骨髓基质细胞悬液,取上层细胞培养.以细胞1×106个/瓶,共接种25 mL培养瓶8瓶,按11比例传代,并将准备好的陶瓷样异种骨置入培养瓶内,每瓶置5块,共置5瓶,作为实验组,另3瓶(不置陶瓷样异种骨)作为对照组.2周后终止培养,取部分置人材料用于相差显微镜和扫描电镜观察.主要观察指标相差显微镜和扫描电镜观察陶瓷样异种骨对骨髓基质细胞的形态特征、生长、附着及增殖的影响.结果①兔骨髓基质细胞活体相差显微观察结果原代培养接种后10～14 d细胞增殖连接成单层网状结构.传代培养24 h后完全贴壁,实验组与对照组的细胞皆生长良好.实验组陶瓷样异种骨周围的成纤维样细胞排列更密集.②兔骨髓基质细胞扫描电镜观察结果实验组传代培养2周后可见一些连接呈网状的成纤维样细胞附于陶瓷样异种骨网状孔隙的内壁、孔底及骨小梁表面,细胞多呈梭形、三角形或多角形细胞.结论陶瓷样异种骨主要成分为羟基磷灰石,且有原骨组织的网状孔隙结构系统,对骨髓基质细胞的形态、生长、附着及增殖均无不良影响,可望与骨髓复合移植修复骨缺损.

In vitro culture of bone marrow stromal cell and ceramic xenogenic bone: Observation of bone marrow stromal cell on morphology property, growth, adhesion and proliferation in rabbits

BACKGROUND: Proper physiochemical method can alleviate or eliminate the antigenicity of natural xenogenic bone and preserve its natural porous reticular system so that its structure and components are in conformity with physiological requirements. It is expected that xenogenic bone becomes a kind of substitutive material in bone transplantation.OBJECTIVE: It was to adopt generative culture of marrow stromal cell and complex culture of ceramic xenogenic bone in vitro to understand the influence of ceramic xenogenic bone on morphology, growth, adhesion and proliferation of marrow stromal cell in rabbits.DESIGN: Blank, control experiment was designed.SETTING: Center Laboratory of First Hospital Affiliated to Kunming Medical College.MATERIALS: Five Japanese big ear white rabbits of 3-month old were employed, body mass varied from 1.5 to 2.0 kg, of either sex.INTERVENTIONS: The experiment was performed in Center Laboratory of First Hospital Affiliated to Kunming Medical College from June to October 2003. With physiochemical management, the fresh pig rib bought from market was prepared into ceramic xenogenic bone and was verified with Xray diffractometry to be mainly composed of hydroxyapatite. It was verified with scanning electron microscopic observation that such material presented porous meshwork system as osteogenie tissue. Five Japanese big ear white rabbits were employed to collect 1mL marrow for each. PRMI 1640culture solution was used to dilute it into suspension of marrow stromal cell and the cells of upper layer were collected for culture. With 1×106 cells/bottle, they were vaccinated to 8 culture bottles of 25 mL and generated at the ratio of 1:1. The prepared ceramic xenogenic bone was placed into culture bottles, 5 pieces in each bottle, totally for 5 bottles, taken as experimental group and the other 3 bottles (without xenogenic bone ) were taken as the control. The culture ended in 2 weeks and a part of cultured material was used for observation with phase microscope and scanning electron microscope.MAIN OUTCOME MEASURES: Influence of ceramic xenogenie bone on morphology, growth, adhesion and proliferation of marrow stromal cell in rabbits were observed with phase microscope and scanning electron microscope.to 14 days after vaccination, cell proliferation was connected to be monolayer meshwork. Generative culture: in 24 hours, adhesion was accomplished and the cells grew well in both experimental group and the cortrol.In the experimental group, fibroblastic cells were arranged more closely rabbits with scanning electron microscope: in experimental group, it was visible in 2 weeks of generative culture that some counected reticular fibroblastic cells adhered with the inner wall of reticular pores and porous base of ceramic xenogenic bone as well as the surface of bone trabecula.The cells were mostly fusiform, triangular or multiangular.CONCLUSION: Ceramic xenogenic bone is mainly composed of hydroxyapatite and presents reticular porous structural system of osteogenic tissue, which will not produce harmful effects on morphology, growth, adhesion and proliferation of marrow stromal cell and it is expected to be used in complex graft for repair of bone defects with bone marrow.