改良法制备异种脱蛋白骨体内植入修复骨缺损的免疫学分析

摘要 背景：在支架材料选择中，目前尚未完全解决异种骨的免疫原性问题。 目的：观察改良法制备异种脱蛋白骨作为组织工程支架材料修复骨缺损后机体的免疫学改变。 方法：SPF级青山羊18只，随机分为3组，正常骨组不截骨，自体骨组、异种脱蛋白骨组在羊右侧胫骨中下段造成胫骨总长度20%骨膜和骨缺损。自体骨组在缺损处植入羊自体骨；异种脱蛋白骨组培养收集羊自体骨髓间充质干细胞，调整细胞数为1×109 L-1，将2 mL细胞悬液接种于以猪股骨为原料改良制备的猪脱蛋白骨上，再加入重组人骨形成蛋白2，半环槽外固定。采用流式细胞仪进行CD4+及CD8+ T淋巴细胞、血清抗体IgG免疫学检测，以双能X射线测量仪分析骨缺损修复效果。 结果与结论：异种脱蛋白骨组植入后3，7，14，28 d外周血CD4+和CD8+ T淋巴细胞的含量基本正常(P > 0.05)，血清抗体IgG水平略高于自体骨组(P > 0.05)；植入后24周骨密度、骨矿物含量与自体骨组基本相似(P > 0.05)，表现为骨缺损区两断端之间高密度钙化影。3组抗压缩压强及极限压强、抗弯曲载荷及极限载荷、抗扭转转矩及极限转矩均基本相似(P > 0.05)。植入后24周与自体骨组比较，异种脱蛋白骨组的成骨能力无明显差异(P > 0.05)。提示改良法制备的异种脱蛋白骨不引起明显的细胞和体液免疫反应，有良好的组织相容性，不影响自体骨髓间充质干细胞成骨，新骨生物力学性能与自体骨相当。 关键词：脱蛋白骨；异种；猪；山羊；重组人骨形成蛋白2；骨缺损；骨组织构建 doi:10.3969/j.issn.1673-8225.2010.42.004

Immunological analysis of improved heterogeneous deproteinized bone for bone defects repair

Abstract BACKGROUND: The immunogenicity of heterogeneous bone remains unsolved in the choice of scaffold materials. OBJECTIVE: To observe the immunological properties of heterogeneous deproteinized bone prepared by modified method. METHODS: Eighteen goats of SPF grade were randomly divided into three groups, normal bone group without osteotomy, autologous bone group and heterogeneous deproteinized bone group were produced a 20% length of periosteum and bone defects in right tibial middle and inferior segments. Autologous bone group was implanted with autologous bone; in heterogeneous deproteinized bone group, goat autologous bone marrow mesenchymal stem cells were collected and adjusted into a density of 1×109/L, 2 mL cell suspension was incubated onto porcine deproteinized bone modified with porcine femoral bone, adding recombinant human bone morphogenetic protein 2, followed by semi-ring trough external fixation. The immunological detection of CD4+ and CD8+ T lymphocytes, serum antibodies IgG was performed using flow cytometry, bone defects repair was determined with dual-energy X-ray measurement. RESULTS AND CONCLUSION: After heterogeneous deproteinized bone was implanted 3, 7, 14, 28 days, the levels of CD4+ and CD8+ T lymphocytes in peripheral blood were normal (P > 0.05), serum IgG antibody level was slightly higher than the autologous bone group (P > 0.05); at 24 weeks after implantation, bone mineral density and bone mineral content were similar to autologous bone group (P > 0.05), showed high-density calcification between the two ends of bone defect area. The pressure resistance and ultimate pressure, resistance to bending loads and limit load, torsion torque and ultimate torque were similar in three groups (P > 0.05). At 24 weeks after implantation, there was no significant difference in osteogenic potential between autologous bone group and heterogeneous deproteinized bone group (P > 0.05). The improved heterogeneous deproteinized bone does not cause significant cellular and humoral immune response, has good biocompatibility, does not affect the osteogenesis of autologous bone marrow mesenchymal stem cells, biomechanical properties of new bone are equal to autologous bone.