载药组织工程支架的构建及其治疗兔慢性骨髓炎的疗效

摘要

目的 构建含有万古霉素和骨形态发生蛋白-2(BMP-2)的微球组织工程支架,并用于治疗兔慢性骨髓炎,观察其治疗慢性骨髓炎的效果.方法 (1)将壳聚糖(CS)溶液、纳米羟基磷灰石(n-HAP)按1∶1混合,通过冻干技术制备成固体材料,并浸入BMP-2/聚乳酸(PLA)和万古霉素/PLA缓释微球溶液中,构建载药组织工程支架,并检测其释放效率.(2)构建骨髓炎模型,将60只健康新西兰大白兔按随机数字表法分为清创后不做任何处理(Ⅰ组)、单纯n-HAP/CS支架材料(Ⅱ组)、n-HAP/CS载万古霉素/PLA(Ⅲ组)、n-HAP/CS载万古霉素/PLA+ BMP-2/PLA(Ⅳ组),每组15只.8周后各组行胫骨X线片检查并行Norden评分、胫骨HE染色及骨髓细菌培养,评价各组抗感染能力及修复骨缺损能力.结果 构建的n-HAP/CS支架孔径大小为80 ～ 270 μm,平均为146.53 μm.生物力学测试显示,支架材料的弹性模量为(27.50±1.58) MPa,压缩强度为(0.75 ±0.04) MPa.BMP-2/PLA和万古霉素/PLA缓释微球成团状均匀分布于载药支架的孔隙中;载药支架能够较稳定地释放万古霉素和BMP-2,持续时间约18 d,随着时间的延长释放量逐渐减少.植入载药支架材料8周后,X线片示Ⅲ、Ⅳ组可见骨痂形成,Ⅰ、Ⅱ组仍可见骨膜反应伴局部低密度影.Ⅰ、Ⅱ、Ⅲ及Ⅳ组Norden评分分别为(2.64±0.33)分、(2.63 ±0.36)分、(1.14±0.29)分、(0.89±0.42)分,Ⅳ组明显低于其余三组(P＜0.05);Ⅲ组与Ⅰ、Ⅱ组比较差异有统计学意义(P＜0.05).Ⅰ、Ⅱ组胫骨组织HE染色显示有大量炎性细胞浸润,骨小梁明显破坏;Ⅲ、Ⅳ组骨小梁排列整齐,且Ⅳ组骨小梁较Ⅲ组粗大.Ⅰ、Ⅱ组骨髓培养见明显淡黄色菌落形成;Ⅲ、Ⅳ组未见菌落形成.结论 成功构建载万古霉素和BMP-2的组织工程支架材料,并证实载药n-HAP/CS联合BMP-2是治疗兔慢性骨髓炎较为理想的植入材料.%Objective To investigate the anti-infection and bone repair effects of composite scaffolds encapsulated with vancomycin and bone morphogenetic protein-2 (BMP-2).Methods Chitosan (CS) solution and nano-hydroxyapatite (n-HAP) mixed in a ratio of 1∶1 were made into solid materials by freeze drrying technology,and immerged into BMP-2/polylactic acid (PLA) and vancomycin/PLA microsphere solution to fabricate drug-loaded scaffolds.Efficiency of drug release was measured.After the induction of chronic infectious tibial bone defect,60 New Zealand white rabbits were assigned to isolate debridement group (Group Ⅰ),n-HAP/CS scaffold group (Group Ⅱ),n-HAP/CS + vancomycin/PLA group (Group Ⅲ) and n-HAP/CS + vancomycin/PLA + BMP-2/PLA group (Group Ⅳ) according to the random number table,with 15 rabbits per group.After 8 weeks of treatment,X-ray filming,Norden score,HE staining and narrow culture were performed to evaluate the anti-infection ability and bone repair ability in each group.Results Pore size of the n-HAP/CS scaffold constructed in this study ranged from 80 to 270 μm (mean,146.53 μm).Biomechanical test showed the elastic modulus of the scaffold material was (27.50 ± 1.58)MPa and the compressive strength was (0.75 ± 0.04)MPa.BMP-2/PLA and vancomycin/PLA microspheres into ball shape uniformly distributed on the scaffold pores.Drug-loaded scaffold showed relatively stable release of vancomycin and BMP-2 in a period of 18 days.With the extended time amount of the drug release gradually reduced.On the X-ray films 8 weeks after the treatment,sinus formation was visible in groups Ⅲ and Ⅳ,and emergence of periosteal reaction with focal low density shadow was demonstrated in groups Ⅰ and Ⅱ.Norden scores of groups Ⅰ,Ⅱ,Ⅲ and Ⅳ were (2.64 ±0.33) points,(2.63 ±0.36) points,(1.14 ±0.29) points and (0.89 ±0.42) points respectively,indicating group Ⅳ ranked the lowest compared with other three groups (P＜0.05) and group Ⅲ showed significant difference in comparison with groups Ⅰ and Ⅱ (P ＜0.05).HE staining showed massive infiltration of inflammatory cells and destroyed bone trabeculas in groups Ⅰ and Ⅱ,trabecular bone arranged neatly in groups Ⅲ and Ⅳ,and thicker trabecular bone in group Ⅳ than in group Ⅲ.Marrow culture showed obvious pale yellow colony formation in groups Ⅰ and Ⅱ,but no colony formation in groups Ⅲ and Ⅳ.Conclusions Vancomycin/BMP-2-loaded tissue engineering scaffolds are constructed successfully.Drug-loaded n-HAP/CS composite scaffold is identified as an ideal implant material for treatment of chronic osteomyelitis in rabbits