硫酸软骨素酶ABC-PLGA缓释微球处理去细胞同种异体神经移植修复大鼠坐骨神经

Repairing sciatic nerve in rats by acellular allogeneic nerve transplantation treated with chondroitinase ABC-PLGA microspheres

目的探讨硫酸软骨素酶ABC(chondroitinase ABC,ChABC)-聚乳酸-聚乙醇酸共聚物缓释微球处理改良化学法去细胞同种异体神经移植修复大鼠坐骨神经的可行性。方法用复乳法制备硫酸软骨素酶ABC-聚乳酸-聚乙醇酸共聚物缓释微球。取成年雄性SD大鼠40只,随机分为4组(n=10):硫酸软骨素酶ABC-聚乳酸-聚乙醇酸共聚物缓释微球处理改良化学法去细胞同种异体神经移植组(A组)、自体神经移植组(B组)、改良化学法去细胞同种异体神经浸泡ChABC神经移植对照组(C组)、改良化学法去细胞同种异体神经移植09%氯化钠注射液对照组(D组)。取A、C、D 3组动物人工切取右侧坐骨神经10 mm,用改良化学法制备移植神经段:A、C两组浸泡在PBS液中,D组浸泡在含2 U/ml ChABC的PBS液(pH7.4)中。A组动物取C组制备神经行同种异体神经移植,外膜无张力缝合,并在移植神经段距近心端缝合处2 mm、4 mm、6 mm、8 mm处分别注入0.2μl制备好的硫酸软骨素酶ABC-PLGA缓释微球。B组在右侧离断10 mm坐骨神经倒置后行外膜缝合。C组取D组制备神经行神经移植。D组取A组制备神经行神经移植后,按照A组的位置注入等量等渗0.9%氯化钠注射液。术后4、8、12周进行大体标本观察,术后12周行电生理检测、HE染色、镀银染色及电镜组织学检测、图像分析对比。结果制备所得硫酸软骨素酶ABC微球表面光滑,球体大小各异且均匀,无粘连及成簇等现象,Weibull方程曲线显示硫酸软骨素酶ABC微球体外释药稳定。采用硫酸软骨素酶ABC-聚乳酸-聚乙醇酸共聚物缓释微球处理改良化学法去细胞同种异体神经移植能够促进大鼠神经缺损处的轴突再生,提高神经修复的速度和质量,再生神经直径较粗,粘连较轻,电生理检测和组织学观察显示各项指标均优于两对照组,且较两对照组修复效果更接近于自体神经移植。结论硫酸软骨素酶ABC-聚乳酸-聚乙醇酸共聚物缓释微球处理改良化学法去细胞同种异体神经移植可以修复大鼠神经损伤。

Objective To investigate the feasibility of repairing sciatic nerve in rats by acellular allogeneic nerve transplantation treated with chondroitinase ABC-PLGA microspheres. Methods Chondroitinase ABC-PLGA microspheres were prepared by double emulsion methods. Forty adult male SD rats were randomly selected and divided into four groups （n=10）： freeze-thrawed combined optimized acellular nerve （FTOAN） group treated with chondroitinase ABC-PLGA microspheres （Group A）, autologous nerve transplantation group （Group B）, FTOAN soaking chondroitinase ABC group （Group C）, FTOAN treated with normal saline group （Group D）. Ten millimeters of nerve from the right sciatic nerve of animals in three groups （Group A, C, D） was artificially cut and transplanted with FTOAN. Group A and C were soaked in PBS, while group D were soaked in PBS solution containing 2 U/ml ChABC （pH 7.4）. Preparation line nerve of group C was taken by animals in group A and allograft nerve grafts were taken in both two groups, the outer membrane was sutured without tension, and preparated chondroitinase ABC-PLGA microspheres accounting for 0.2 ~ul was injected in the transplanted neural segments from the seams of proximal part of 2 mm, 4 mm, 6 ram, 8 mm respectively. Outer membrane suture was taken by group B on the right side of 10 mm broken sciatic nerve after inversion. Preparation line nerve of group D was taken by animals in group C and nerve-grafting was taken in both two groups. Group D made allograft nerve grafts with preparation line nerve of group A, and equal amount of isotonic saline was injected according to the position of group A. Gross specimen was observed 4,8,12 weeks after operation. Electrophysiological detection, HE staining, silver staining, electron microscope histological detection and image analysis were compared 12 weeks after operation. Results The preparated chondroitinase ABC microspheres had smooth surface and sphere uniform sizes, and no adhesion and clusters were found. Weibull equation curve showed the stability of in vitro release of chondroitinase ABC-PLGA microspheres. The axonal regeneration of neural defect in rats could be promoted by freeze-thrawed combined optimized acellular nerve group treated with chondroitinase ABC-PLGA microspheres, the quality and speed of nerve repairing were also improved. The regeneration nerve was thicker in diameter and lighter in adhesion. Electrophysiological detection and histology observation showed that all indicators were better than the two compared groups, and also the repair effect was much closer to autologous nerve grafts. Conclusion Freeze-thrawed combined optimized acellular nerve group treated with chondroitinase ABC-PLGA microspheres can repair the nerve injury in rats.