Repair of sciatic nerve defect in rats by polyethylene glycol modified polylactic acid nerve conduit scaffold
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摘要:
背景 组织工程有望替代自体神经移植，成为修复周围神经缺损的新途径，而组织支架神经导管正是组织工程三要素之一，针对组织支架神经导管的改进方法是目前的研究热点。 目的 制备用于神经修复及再生的聚乙二醇(polyethylene glycol，PEG)改性聚乳酸(poly lactic acid，PLA)神经导管支架并用其修复大鼠坐骨神经缺损。 方法 利用溶剂挥发法制备PLA神经导管支架，用丙烯酰氯与PEG发生酰氯酯化反应合成聚乙二醇双丙烯酸酯(PEGDA)，用PEGDA通过化学交联的方式对导管内壁进行改性。电镜观察其微观形貌，利用CCK-8实验检测大鼠神经施万细胞RSC96在聚乙二醇改性的聚乳酸神经导管(PEG-PLA)预处理培养基中的增殖情况。取SD大鼠30只，随机分为自体神经移植组(ANG)、聚乙二醇改性聚乳酸神经导管组(PEG-PLA)、单纯聚乳酸神经导管组(PLA)，每组10只。制作大鼠坐骨神经10 mm缺损模型，分别采用自体神经移植、PEG-PLA导管和PLA导管桥接修复。术后行大体观察，术后2周行移植段神经再生速度评价；术后4周、8周、12周行步态分析，测定坐骨神经功能指数；术后12周取材行移植段再生神经组织学评价、腓肠肌湿重恢复率测量和组织学检查。 结果 扫描电镜可见制备的PEG-PLA神经导管内壁质地疏松，有取向性排列整齐的纹路；各组神经导管的浸提液与RSC96细胞共培养后，RSC96细胞增殖未受到抑制，提示各组神经导管的生物相容性良好；术后2周移植段神经免疫荧光染色结果可见PEG-PLA组轴突再生情况优于PLA组，低于ANG组；术后12周，PEG-PLA组的步态分析、腓肠肌肌肉湿重恢复率以及Masson染色等结果均明显优于PLA神经导管组，更接近自体神经移植的修复效果。 结论 聚乙二醇改性聚乳酸神经导管具有良好的组织相容性，神经修复效果良好。 Abstract:Background Tissue engineering is promising to replace autogenous nerve transplantation and has become a new method to repair peripheral nerve defects. Tissue scaffold nerve conduit is one of the three elements of tissue engineering, and the improved method of tissue scaffold nerve conduit is the current research hotspot. Objective To prepare poly (ethylene glycol) (polyethylene glycol, PEG) modified polylactic acid (poly lactic acid, PLA) nerve conduit scaffold for nerve repair and regeneration and evaluate its biological characteristics. Methods PLA nerve conduit was prepared by solvent volatilization method. Polyethylene glycol diacrylate (PEGDA), was synthesized by acyl chloride esterification of PEG with acryloyl chloride. The inner wall of polyethylene glycol diacrylate was modified by PEGDA. The microscopic morphology was observed by electron microscope. The proliferation of rat nerve Schwann cell RSC96 in polyethylene glycol modified poly (lactic acid) nerve conduit (PEG-PLA) pretreatment medium was detected by CCK-8 experiment. Thirty SD rats were randomly divided into autogenous nerve graft(ANG) group, poly (ethylene glycol) modified poly (lactic acid) nerve conduit (PEG-PLA) group, and poly (lactic acid) nerve conduit (PLA) group , with 10 rats in each group. The rat model of sciatic nerve 10 mm defect was repaired with autogenous nerve graft, PEG-PLA conduit, and PLA conduit, respectively. Gross observation was performed after operation, nerve regeneration speed was evaluated at 2 weeks after operation, gait analysis was performed at 4, 8 and 12 weeks after operation, sciatic nerve function index was measured. At 12 weeks after operation, samples were taken for histological evaluation of regenerated nerve, wet weight recovery rate of gastrocnemius muscle and histological examination. Results Scanning electron microscope showed that the inner wall of the PEG-PLA nerve conduit was loose and well arranged, and the proliferation of RSC96 cells was not inhibited after the nerve conduit was co-cultured with RSC96 cells, suggesting that the biocompatibility of the nerve conduit was good. At 2 weeks after operation, the results of immunofluorescence staining showed that the axon regeneration in the PEG-PLA group was better than that in the PLA group and lower than that in the ANG group. At 12 weeks after operation, the gait analysis, gastrocnemius muscle wet weight recovery rate and Masson staining in PEG-PLA group were superior to those in PLA nerve catheter group, which was closer to the repair outcome of autogenous nerve transplantation. Conclusion Poly (ethylene glycol) modified polylactic acid nerve conduit has good histocompatibility and can improve the effect of nerve regeneration. -
Key words:
- peripheral nerve injury /
- nerve regeneration /
- nerve conduit /
- polylactic acid /
- polyethylene glycol
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图 1 PEG-PLA和PLA神经导管电镜 A：PEG-PLA和PLA神经导管外壁；B：PEG-PLA神经导管内壁；C：PLA神经导管内壁
Figure 1. Scanning electron microscope of PEG-PLA nerve conduit A: PEG-PLA and PLA nerve conduit outer wall; B: PEG-PLA nerve conduit inner wall; C: PLA nerve conduit inner wall
图 2 术后12周各组大鼠3D步态足印
Figure 2. At 12 weeks after operation, the 3D gait footprints of rats in each group were observed
图 3 术后4周、8周、12周各组坐骨神经功能指数(aP＜0.05)
Figure 3. Sciatic nerve function index of each group at 4, 8 and 12 weeks after surgery (aP＜0.05)
图 4 术后12周大鼠腓肠肌大体观
Figure 4. Gross view of gastrocnemius muscle in rats at 12 weeks after surgery
图 5 腓肠肌湿重恢复率(左)和腓肠肌肌纤维横截面积(右)(aP＜0.05；bP＜0.01)
Figure 5. Gastrocnemius muscle wet weight ratio (left) and mean cross-sectional area of gastrocnemius muscle fiber (right) (aP＜0.05; bP＜0.01)
图 6 术后12周大鼠腓肠肌肌肉横截面Masson染色
Figure 6. Masson staining of cross section of gastrocnemius muscle in rats at 12 weeks after operation
图 7 术后2周各组移植段再生神经的免疫荧光染色
Figure 7. Results of immunofluorescence staining of regenerated nerve in each group at 2 weeks after surgery
图 8 术后12周，各组移植段再生神经中段的免疫荧光染色结果。免疫荧光染色可见NF200阳性的再生神经纤维被标记为红色荧光，CD31阳性的血管内皮细胞被标记为绿色荧光
Figure 8. Immunofluorescence staining results of the middle segment of regenerated nerve in each group at 12 weeks after operation. Immunofluorescence staining showed that NF200-positive regenerated nerve fibers were marked with red fluorescence and CD31-positive vascular endothelial cells were labeled with green fluorescence
表 1 神经导管材料对RSC96细胞增殖的影响(n=5)
Table 1. Effect of nerve conduit on the proliferation of RSC96 cells in the three groups (n=5)
Time
pointOD value F P PLA PEG-PLA Control Day 1 0.413±0.180 0.413±0.007 0.432±0.014 3.291 0.073 Day 3 1.324±0.151 1.266±0.117 1.409±0.059 1.957 0.184 Day 5 2.111±0.217 2.089±0.188 2.294±0.183 1.638 0.970 
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