聚乳酸/聚羟基乙酸复合骨形成蛋白修复兔关节软骨缺损

目的以聚乳酸/聚羟基乙酸(poly-lactide-co-glycolide,PLGA)为载体,探讨重组人骨形成蛋白2(recombinant human bone morphogenetic protein2,rhBMP-2)在关节软骨修复方面的作用及可行性。方法将PLGA制成直径4mm,厚3mm的圆柱形,与rhBMP-2复合(0.5mg/块),制备PLGA-rhBMP-2复合物。选取2月龄新西兰兔,抽取骨髓行原代及传代培养,调整细胞密度为2×107/ml,与PLGA共培养24h,制备PLGA-细胞复合物。另取2月龄新西兰兔72只,于双侧髌股关节股骨髁部制备直径4mm、深达髓腔的缺损。其中36只兔右侧缺损处植入PLGA-rhBMP-2复合物,为实验组;左侧植入PLGA,为单纯载体组;另36只兔左侧缺损不作任何处理,为空白对照组,右侧缺损处植入PLGA-细胞复合物,作为细胞组。术后4、8、12、24、36和48周取材,行大体、组织学观察以及组织学评分。结果术后动物均存活。术后4周,实验组和细胞组缺损内被半透明组织填充,触之柔软,表面较光滑,软骨细胞周围基质异染弱,新生软骨厚度较正常软骨厚;空白对照组和单纯载体组未见明显组织形成。8周,实验组和细胞组内新生组织呈白色,半透明,质较韧,表面平整,与周围正常软骨界限模糊;新生软骨细胞分布均一,但仍较正常软骨厚,PLGA已大部分降解,仅遗留少量颗粒;单纯载体组和空白对照组缺损明显,底部形成少量白色膜状组织。12、24周,实验组和细胞组缺损内完全充填白色半透明新生软骨组织,质韧,表面平整,与正常软骨界限消失,厚度接近正常软骨,与正常软骨连接良好,表面细胞平行排列,深层有纵向排列的倾向,呈团状,陷窝形成,但有别于正常软骨细胞;单纯载体组和空白对照组缺损边缘及底部形成少量软骨细胞,大部分为纤维组织。36、48周,实验组和细胞组新生软骨组织色稍发白,表面连续,欠平整,与正常软骨界限消失,未见滑膜增生;新生软骨厚度较正常软骨薄,软骨细胞周围基质异染较弱;单纯载体组及空白对照组缺损仍存在,但较以前缩小,基底形成纤维组织,髁部可见部分软骨面不平整、剥脱,部分软骨下骨外露,滑膜增厚。组织学评分,实验组和细胞组术后12、24周分别与4、8和48周比较,差异均有统计学意义(P<0.01);各时间点实验组、细胞组分别与单纯载体组和空白对照组比较,差异均有统计学意义(P<0.01),实验组与细胞组在各时间点比较差异均无统计学意义(P>0.05)。结论PLGA-rhBMP-2复合物在降解过程中释放出rhBMP-2,rhBMP-2作用于缺损局部的骨髓基质细胞,诱导其向软骨细胞分化,从而修复软骨缺损;此方法简便易行,有实用价值,有望成为治疗软骨缺损的一种新方法。

REPAIR OF ARTICULAR CARTILAGE DEFECT WITH POLY-LACTIDE-CO-GLYCOLIDE LOADED WITH RECOMBINANT HUMAN BONE MORPHOGENETIC PROTEIN IN RABBITS

OBJECTIVE: To study the effect and feasibility of poly-lactide-co-glycolide (PLGA) loaded with recombinant human bone morphogenetic protein 2 (rhBMP-2) on repairing articular cartilage defect in rabbits. METHODS: PLGA was made into cylinders which were 4 mm in diameter and 3 mm in thickness. rhBMP-2 was fully homogenated before used. PLGA combined with 0.5 mg rhBMP-2 under the condition of vacuum (700 mmHg), and then lyophilized, packed, sterilized with ethylene oxide and reserved. Defects of 4 mm in diameter and reaching medullary cavity were made in femoral condyles of 72 two-month-old New Zealand white rabbits. The 36 right defects were repaired with PLGA-rhBMP-2 composites as the experimental group, the 36 left defects with PLGA only as PLGA group, the other 36 left defects were left untreated as control group, and the other 36 right defects with PLGA-MSCs composites as cell group. At 4, 8, 12, 24, 36 and 48 weeks after operation, macroscopical and microscopical observations were made, and the histological grade was done. RESULTS: After 4 weeks of operation: In the experimental group and cell group, defects were filled with white translucent tissue which appeared smooth and soft; the matrix around chondrocytes was weakly metachromatic, the newly formed cartilage tissue was thicker than normal cartilage tissue; there was no formed tissue in the PLGA group and the blank control group. After 8 weeks of operation: In the experimental group and cell group, the new tissue was white, translucent, tenacious and smooth. The boundary with normal cartilage became vague. New cartilage cells distributed evenly. The cells of the surface layer paralleled, but the deeper layer lost directivity. The matrix dyed weakly. The new cartilage gradually became thinner, but it still thicker than the normal cartilage ones. The PLGA degraded besides some drops. In the blank control group and PLGA group, a little white membrane formed at the bottom of the defect. After 12-24 weeks of operation: In the experimental group and cell group, defects were filled with new tissues which were white, translucent, tenacious and smooth. The boundary disappeared. The thickness of the new cartilage was similar to that of the normal ones. The cells of the surface layer paralleled to each other,but the cells of the deeper layer tended to arrange vertically. The matrix around chondrocytes was metachromatic, but the color was lighter than that of the normal cartilage. Bone under the cartilage and the tide mark recovered. The new cartilage linked with nomal cartilage finely. In the blank control group and PLGA group, there was a little fibrous tissue at the bottom of the defect withe obvious boundary. After 36 weeks and 48 weeks of operation: in the experimental group and the cell group, the new cartilage was slightly white, continuous and less smooth. The boundary disappeared. There was no proliferated synovial membrane. The thickenss of the new cartilage was thinner than that of the normal ones. The matrix around chondrocytes was weakly metachromatic. In the blank control group and PLGA group, the defect still existed, but became smaller. At the bottom of the defect, fibrous tissues formed. Some cartilage denudated and became less smooth. Some bone under cartilage exposed,and the synovial membrane became thick. The histologic grade of the repair tissue at 12 weeks and 24 weeks of operation in experimental group and cell group was significantly different from that at 4, 8 and 48 weeks of operation (P<0.01). There was also significant difference in the experimental group and cell group compared with the blank control group and PLGA group at each time after operation (P<0.01). But there was no significant difference between the experimental group and the cell group. CONCLUSION: In the course of degradation, PLGA-rhBMP-2 composites releas rhBMP-2, which then act an effect on MSCs around defect and induced them to differentiate for chondrocytes, and finally the defect is repaired. This simple and easy method may be used clinically in the future.