硫酸葡聚糖／重组人骨形态发生蛋白-2／壳聚糖纳米微球联合珊瑚羟基磷灰石修复大段骨缺损的成骨化研究

Osteogenesis of dextran sulfate/recombinant human bone morphogenetic protein-2/chitosan nano microspheres combined with coralline hydroxyapafite in repair of large segmental bone defects

目的探讨硫酸葡聚糖／重组人骨形态发生蛋白-2／壳聚糖纳米微球联合珊瑚羟基磷灰石（Ds／rhBMP2／CS／CHA）在修复大段骨缺损中成骨化能力。方法采用离子交联法制备DS／rhBMP2／CS缓释微球，并经冻干吸附到CHA中，将72只新西兰兔随机分为4组，行右桡骨骨缺损的造模，分别将CHA、rhBMP2／CHA、DS／rhBMP2／CS／CHA植入骨缺损区域中并依次命名为CHA组、rhBMP2／CHA组、DS／rhBMP2／CS／CHA组，另设18只动物为空白对照组（只造模，不植入材料）。术后4、8、12周抽血并取材，行血清骨钙素检测，移植骨标本行Micro—CT扫描并计算新生骨体积比，脱钙后行苏木精-伊红染色观察。结果72只动物术后恢复良好，术口未发生感染或移植物暴露。术后12周大体观察DS／rhBMP2／CS／CHA组骨组织的新生骨含量及质量最优，硬度最强，骨缺损区愈合较其他组好。血清学表明各时期DS／rhBMP2／CS／CHA组的血清骨钙素水平明显高于CHA组、rhBMP2／CHA组，差异均有统计学意义（P〈0．05）；Micro-CT结果表明DS／rhBMP2／CS／CHA组各时期均可观察到明显的成骨进程，明显的骨皮质和新生的骨髓腔，骨的重建进程最快，材料的降解伴随着骨的重建，DS／rhBMP2／CS／CHA组的新生骨体积水平高于其他组，差异均有统计学意义（P〈0．05）；组织学表明各时期DS／rhBMP2／CS／CHA组的组织学形态良好，可见成熟骨皮质和新生骨髓腔，组织学形态好于CHA组和rhBMP2／CHA组。结论DS／rhBMP2／CS／CHA的人造骨材料具备良好的缓释rhBMP2的骨诱导机制，在参与修复大段骨缺损中展现良好的骨修复能力，明显优于单纯CHA和rhBMP2／CHA人造骨材料。

Objective To investigate the osteogenic ability of dextran sulfate/recombinant human bone morphogenetic protein-2/chitosan （DS/rhBMP2/CS） nano microspheres combined with coralline hy- droxyapatite （CHA） in repair of segmental bone defects. Methods DS/rhBMP2/CS microspheres pre- pared by ionic crosslinking method were adsorbed into CHA by lyophilization. Seventy-two New Zealand rabbits were randomly divided into 3 equal groups after they had been made into models of bone defect at the right radius. The defects in the 3 groups were implanted respectively with CHA, rhBMP2/CHA and DS/rhBMP2/ CS/CHA. Another 18 animals served as a blank control group. Blood and bone samples were obtained at 4, 8 and 12 weeks after implantation. The serum BGP was detected, and the bone grafts were scanned by micro-CT for calculation of volume ratio of the new bone. Hematoxylin and eosin （HE） staining was performed after bone decalcification. Results All the 72 animals recovered well without any infection or graft exposure. Gross observation at postoperative 12 weeks showed that the DS/rhBMP2/CS/CHA group was the best in the quality, quantity and strength of the new bone, as well as in the healing of bone defects. The serum levels of bone gamma-carboxyglutamic-acid-eontaining protein at all time points in the DS/rhBMP2/CS/CHA group were significantly higher than those in the CHA and rhBMP2/CHA groups （P 〈 0. 05）. Miero-CT scanning demonstrated obvious progress in bone formation, cortical bone and marrow cavity at all time points in the DS/ rhBMP2/CS/CHA group which showed significantly faster bone reconstruction synchronized with material degradation and significantly higher volume ratio of the new bone than the other 2 groups （ P 〈 0. 05）. His- tological examinations showed better morphology of mature cortical bone and new marrow cavity at all time points in the DS/rhBMP2/CS/CHA group than in the other 2 groups. Conclusion Since DS/rhBMP2/ CS/CHA possesses a better mechanism of sustained-releasing rhBMP2 to induce bone formation because of its reticular and hole-hole-connected structure, it may perform better in repairing segmental bone defects than simple CHA or rhBMP2/CHA.