羧甲基壳聚糖/纳米胶原复合支架修复兔腓骨损伤

Carboxymethyl chitosan/collagen nanofibers composite scaffolds for repairing rabbit fibula defect

背景:研究证实,壳聚糖及其衍生物可作为骨损伤的填充材料以及骨组织工程支架材料,但壳聚糖降解缓慢且不可控制的特性限制了其在骨组织工程中广泛应用。目的:评估羧甲基壳聚糖/纳米胶原纤维复合支架对兔腓骨损伤的修复作用。设计、时间及地点:随机分组设计、对照动物实验,于2007-06/2008-03在清华大学生物科学与技术系生物膜与膜生物工程国家重点实验室完成。材料:以羧甲基壳聚糖和纳米胶原纤维为基础材料,模拟骨的结构设计制备了双层羧甲基壳聚糖/纳米胶原纤维复合支架。方法:选择成年新西兰大白兔10只,按随机数字表法分为3组,阴性对照组2只,壳聚糖支架组4只,复合支架组4只。制备兔腓骨10mm的损伤,分别以旷置缝合、植入壳聚糖支架、植入双层羧甲基壳聚糖/纳米胶原纤维复合支架处理各组。主要观察指标:扫描电镜观察支架微观形貌。术后12周,检测损伤部位的再生情况,以四环素荧光和Von Kossa染色检测实验动物骨损伤部位新生骨钙化情况。结果:10只兔均进入结果分析。①扫描电镜观察到外层致密光滑的壳聚糖膜和多孔的中心区域,多孔区域有大量纳米胶原纤维网络结构填充。其孔径分布的峰值为60～100μm,孔隙率大于85%。②手术后12周,不脱钙切片的四环素染色,可发现阴性对照组两个断端间还没有连上,依旧以游离端存在。复合支架组移植物中形成多个大的钙化岛,其中心网状材料已经大部分降解,钙化岛分布在整个支架的内部区域;而壳聚糖支架组,材料基本没有降解,未见钙化区域。③Von Kossa银染色显示,复合支架组动物骨损伤的中心区域,出现染成黑色的钙化区,中间夹杂着中性红复染的骨组织细胞。壳聚糖支架组动物没有明显的钙化区,多为组织细胞充斥在材料的孔隙中,可见材料基本没有降解。结论:双层羧甲基壳聚糖/纳米胶原纤维复合支架植入动物体内12周后,未见明显的炎症反应和坏死,降解明显,能够促进骨修复,是很有前景的骨组织工程用支架材料。

BACKGROUND： Studies have proved that, chitosan and its derivates can be used as filling material of bone injury and bone tissue engineering scaffolds, but chitosan degrades so slowly and unconlroLLablly as to limit its application in field of bone tissue engineering. LBJECTIVE： To assess the repair of rabbit fibula defect with carboxymethyl chitosan （CMC）/collagen nanofibers （CN） composite scaffolds. DESIGN,TIME AND SETTING： Randomized control animal experiments were completed in Department of Biological Sciences and Biotechnology, State Key Laboratory of Biomembrane and Membrane Biotechnology, Tsinghua University from Jane 2007 to March 2008. MATERIALS： Based on the preparation of CMC and CN, a bilayer CMC/CN composite scaffold was fabricated. . METHODS： Ten New Zealand white rabbits were divided into 3 groups： 2 for the negative control, 4 for chitosan scaffolds, and 4 for CMC/CN scaffolds. AFTer a 10-mm bone defect was made on each rabbit fibula, the CMC/CN scaffold or chitosan scaffold was implanted into the defect site. The rabbits without any implant were as the control. MAIN OUTCOME MEASURES： The micro-morphology of the scaffold was observed under scanning electron microscope, At 12 weeks after operation, the calcification of newly formed bone in the defect site was analyzed with the assessment of tetracycline fluorescence and Von Kossa staining. RESULTS： Ten rabbits were included in the final analysis. Compact chitosan membrane and the porous CMC/CN core were observed under scanning electron microscope with a porosityporsity over 85%. Pore size peak was 60-100 μm. Aa large amount of collagen nanofibers network structure filled the core. 12 weeks after surgery, the results of tetracycline fluorescence analysis of an-decalcified slices showed that no fluorescence was detected in the area between the two ends of negative control group, while no calcified areas were detected in the chitosan scaffold-implanted rabbits and chitosan were barely degraded. In the defect sites of CMC/CN scaffold-implanted groups many ＂calcified islands＂ appeared in a trend of connecting with each other. The Von Kossa staining of un-decalcified slices showed that in the center of bone defect of CMC/CN scaffold-implanted rabbits, black calcified area appeared in the middle of cells stained by neutral red. However, in chitosan scaffold-implanted group, cells were distributed in the pores of the scaffold and no calcified areas were detected. CONCLUSION： The bilayer CMC/CN composite scaffolds degrade obviously 12 weeks aider implantation and facilitate the bone regeneration of rabbit fibula defect, without obvious inflammatory reaction and necrosis. This kind of composite scaffold is a potential scaffold material of bone tissue engineering.