骨形态发生蛋白对骨膜移植诱导再生关节软骨的影响

Influence of bone morphogenetic protein on articular cartilage regeneration after periosteal grafting

目的：研究证实，骨形态发生蛋白是多效性的形态发生素，在一定环境下，可诱导骨与软骨的形成。实验拟进一步验证骨形态发生蛋白对骨膜移植诱导再生关节软骨的影响。方法：实验于2006-09／2007-01在潍坊市人民医院动物实验室（absl-3）完成。①实验分组：选择健康成年新西兰大白兔16只（共32膝），体质量2．5～3．0kg，随机抽签法分为实验组和对照组，每组8只（16膝）。②实验方法：所有兔子均造成股骨髁间窝3．5mm全层软骨缺损，然后取同侧胫骨上段前内侧3．5mm骨膜，待用。实验组：软骨缺损区注入20μg骨形态发生蛋白和20％Pluronic凝胶，然后覆盖骨膜，缝合固定。对照组：软骨缺损区注入生理盐水20μg和相同剂量的20％Pluronic凝胶，然后覆盖骨膜，缝合固定。③实验评估：术后4，8，12周麻醉并处死动物，肉眼观察膝关节活动度及修复组织与周边组织的结合情况；苏木精-伊红染色和甲苯胺蓝染色观察修复组织的性质；根据Wakitani评分标准测定各组不同时间点标本光镜组织学评分；扫描电镜观察移植组织的超微结构。结果：纳入大白兔16只，均进入结果分析。①肉眼观察：术后4周，实验组关节软骨缺损区被类软骨组织替代；对照组关节软骨缺损区仍被骨膜充填。术后8周，实验组表面光滑，与周围正常软骨分界线模糊；对照组表面不光滑，与周围正常软骨分界线清楚。术后12周，实验组与周围软骨性质近似：对照组表面趋于光滑。②苏木精-伊红染色和甲苯胺蓝染色观察：第4周，实验组软骨缺损区完全充填细胞和基质：对照组骨膜生发层成纤维细胞仅少许增生分化。第8周，实验组骨膜增厚：对照组骨膜无增厚，形成少量纤维软骨。第12周，实验组缺损区接近正常软骨：对照组形成大量的纤维瘢痕组织。③织学评分：实验组关节软骨缺损的再生修复明显优于对照组，差异显著（P〈0．05）。④扫描电镜观察：实验组：4周时修复组织中除纤维组织外，细胞略呈圆形：8周细胞核凹陷；12周核膜清晰可见，染色质分布均匀。对照组：4周时修复组织为层状排列的纤维组织：8，12周修复组织多为层状排列的纤维组织，软骨细胞少（主要为纤维软骨），且不活跃。结论：骨形态发生蛋白对骨膜移植诱导再生关节软骨具有明显的促进作用。

Abstract AIM： Bone morphogenetic protein （BMP） as polyphenic morphogen can induce the formation of bone and cartilage. This study investigates the effect of BMP on articular cartilage regeneration after periosteal graft. METHODS： Experiments were performed at the Animal Laboratory （absl-3） of Weifang People＇s Hospital from September 2006 to January 2007. Sixteen New Zealand white rabbits （32 knees） （2.5-3.0 kg） were divided into experimental and control groups randomly each 8 rabbits （16 knees）. The 3.5 mm in diameter of full-thickness articular cartilage defect was made on femoral intercondylar fossa in all rabbits, and 3.5 mm in diameter of periosteum was cut out from the anteromedial part of the upper tibial bone. In the experimental group, the cartilage defect was covered with periosteum, into which 20 μg BMP and 20% Pluronic were injected. In the control group, the cartilage defect was covered with periosteum, into which the same dosages of 9 g/L saline and 20% Pluronic were injected. All the rabbits were sacrificed in 4, 8 and 12 weeks postoperatively. Motion of joint, conjunction of repair tissue and perienchyma were examined macroscopically. Haematoxylin-eosin staining and toluidine blue staining were used to observe the characteristics of repair tissues. Histological scores on samples in each group were measured by Wakitani score standard at different time points with light microscope. Ultramicrostructure of transplanted tissues was observed with scanning electron microscopy （SEM）. RESULTS： Sixteen rabbits were included in the final analysis. Macroscopic observation： 4 weeks after the surgery, the defect was covered with tissue like cartage in the experimental group, and with periosteum in the control group. 8 weeks after the surgery, the surface of the defect was smooth, with boundary unclear in the experimental group. In the control group, the outcome was the opposite In 12 weeks, cartilage had formed in the experimental group, and tissue like cartilage began to happen in control one. Histological observation： 4 weeks after the surgery, the defect was filled with cells and matrix with abundant proliferation of periosteal cambium layer in the experimental group, and slight proliferation in the control group. 8 weeks after the surgery, the periosteum in the experimental group became fibrocartilage with little hyaline cartilage. Just litde fibrocartilage with on hyaline one was detected in the control group. In 12 weeks, the repair tissue in the experimental group approached to normal cartilage. Just fibrous tissue with little fibrocartilage was detected in the control group. Regenerative repair of cartilage defect was better in the experimental group than in the control group （P 〈 0,05）. Electronmicroscopical observation： In the experimental group, 4 weeks after the surgery, the cells were round with spherical nucleus and irregular process; 8 weeks postoperatively, the nucleus was depressed; In 12 weeks, the repair tissue appeared to be normal cartilage. In the control group, only lamellar disposed fibrous tissue emerged in 4 weeks and fibrous tissue and fibrocartilage in 8 and 12 weeks postoperatively. CONCLUSION： BMP plays an effective role in articular cartilage regeneration after periosteal graft.