脱细胞基质骨与成骨细胞和血管内皮细胞的生物相容性

Biocompatibility of acellular matrix bone with osteoblast and vascular endothelial cells

目的:采用自行研制的脱细胞基质猪肋骨作为支架材料,复合兔成骨细胞和血管内皮细胞,对其生物相容性、抗原性和细胞毒性进行观察。方法:实验于2003-04/10在四川大学华西医院人类疾病生物治疗教育部重点实验室完成。用物理、化学方法制备脱细胞基质猪肋骨。体外培养兔成骨细胞、血管内皮细胞。而后将试验分成3组进行。①成骨细胞组:将成骨细胞与脱细胞基质骨材料复合。②血管内皮细胞组:血管内皮细胞与脱细胞基质骨材料复合。③成骨细胞+血管内皮细胞组:成骨细胞+血管内皮细胞与脱细胞基质骨材料复合。④对照组:单纯成骨细胞。于培养1,3,5,7d分别用倒置相差显微镜、组织学切片和扫描电镜观察脱细胞基质异种骨的生物相容性、抗原性;用流式细胞仪检测材料对细胞周期和DNA含量的影响,了解材料对细胞有无毒性的影响。结果:①脱细胞基质骨扫描电镜观察:脱细胞基质骨的骨小梁结构完整,骨陷窝空虚,未见细胞成分残留。②各组细胞的生长、分化和增殖情况倒置相差显微镜观察:3组细胞与脱细胞基质骨材料复合培养12h后,细胞在各组材料表面和孔隙内均可黏附和生长。③组织学观察:MASSON染色显示3组细胞在材料表面和孔隙内大量增殖,并分泌细胞外基质。以成骨细胞+血管内皮细胞组材料表面黏附的细胞数量最多。组织学切片染色显示,各组细胞沿材料边缘紧密附着,细胞与材料的组织相容性良好。④各组细胞黏附、生长、增殖和基质分泌情况扫描电镜观察:3组细胞与材料复合培养5d后,细胞紧密黏附在材料表面。成骨细胞呈梭形或多角形,相邻细胞间有突起相互连接。血管内皮细胞呈椭圆形,有角状突起,与材料附着紧密。其中,成骨细胞+血管内皮细胞组材料表面黏附大量细胞,并有较多胶原形成。血管内皮细胞组材料表面胶原形成很少。⑤细胞周期和DNA含量:成骨细胞+血管内皮细胞组1d,3dDNA合成期高于单独细胞组,无异倍体细胞。结论:脱细胞基质骨具有良好的生物相容性、极低的抗原性、无细胞毒性和致瘤性。成骨细胞与血管内皮细胞联合培养细胞在脱细胞基质异种骨中有很强的增殖潜力。

AIM： To observe the biocompatibility, antigenicity and cytotoxicity of acellular matrix porcine ribs as the scaffold material combined with rabbit osteoblast and vascular endothelial cells （VECs）. METHODS： The experiment was conducted in the Key Laboratory for Biotherapy of Human Diseases of Ministry of Education, West China Hospital of Sichuan University from April to October 2003. The acellular matrix porcine ribs were prepared by physical and chemical methods. Rabbit osteoblast and VECs were cultured in vitro. There were three groups in this study. ①Osteoblast group, which compounded the osteoblast with acellular matrix bone. ②VECs group, which compounded VECs with acellular matrix bone material. ③Osteoblast＋VECs group, which compounded the osteoblast＋ VECs with acellular bone material. ④Control group, which was the simple osteoblast. On days 1, 3, 5 and 7, the biocompatibility and antigenicity of acellular matrix xenograft were observed by inverted phase contrast microscope, histological sections and scanning electron microscope; the effects of the materials on cell cycle and DNA content were detected by flow cytometry to identify if the materials had toxic effect on cells. RESULTS： ①Scanning electron microscope observation of acellular matrix bone suggested that the trabecular bone structure of acellular matrix bone was complete with empty bone lacuna, and no cell components were found. ②The observation of the growth, differentiation and proliferation of cells under inverted phase contrast microscope appealed that when co-cultured with the acellular matrix bone matedal for 12 hours, the cells of three groups attached and grew on the material surface and in pore. ③Histological observation： MASSON straining showed that the cell of each group massively proliferated on the matedal surface and in pore, and secreted extracellular matrix. The number of attached cells was the most in the osteoblast＋VECs group. Histological stained sections appealed the cells could attach the material border tightly with good histocompatibility. ④The ob.servation of the growth, differentiation and proliferation of cells under scanning electron microscope found that when co-cultured with the material for 5 days, the cells of each group attached the material surface. Osteoblasts were fusiform or polygon, and the cells were connected by ecphyma. VECs were ellipse with cornua, and adhered with material closely. A plenty of cells in the osteoblast＋VECs group were attached the material surface, and lots of collagen was found. Little collagen was observed in the VECs group. ⑤On days 1 and 3, the DNA synthesis period of osteoblast＋VECs group was longer than the osteoblast group and VECs group, and no heteroploid cells were found. CONCLUSION： Acellular matrix bone displays good biocompatibility, low antigenicity, and no cytotoxicity or oncogenicity. Osteoblast co-cultured with VECs has strong proliferative potential in acellular matrix heterogenous bone.