电纺丝壳聚糖/聚乳酸神经导管修复大鼠周围神经缺损的实验研究

EFFECT EVALUATION OF ELECTROSPUN CHITOSAN/POLYLACTIC ACID NERVE CONDUITS FOR REPAIR OF PERIPHERAL NERVE DEFECT IN RATS

目的研究利用电纺丝壳聚糖/聚乳酸(chitosan/polylactic acid,ch/PLA)神经导管修复大鼠坐骨神经缺损的方法和效果。方法采用电纺丝方法制备ch/PLA神经导管,通过扫描电镜、生物力学测定、表面润湿性测定和体外生物相容性检测,并与纯PLA比较,测试ch/PLA神经导管的性能。选取健康SD大鼠54只,随机分成3组,每组18只,制作右侧坐骨神经10 mm缺损模型,分别利用ch/PLA神经导管(A组)、自体神经(B组)移植修复,并与切除旷置(C组)进行对照。术后4、8、12周,通过大体观察、坐骨神经功能指数(sciatic functional index,SFI)、神经电生理、肌肉湿重恢复率、肌细胞横截面积检测和组织学、免疫组织化学染色、透射电镜观察,对大鼠神经再生进行评价。结果随着chitosan的加入,神经导管的均一性、物理性能、亲水性及体外生物相容性均得到了明显改善。术后4周A组再生神经已通过神经缺损。术后A、B组SFI不断改善,两组恢复速度相似,差异无统计学意义(P>0.05)。术后8、12周,A、B组可引出神经肌电图表现,且神经传导速度与动作电位波幅均不断恢复(P<0.05)。术后12周,A、B组肌肉湿重恢复率与肌细胞截面积优于C组(P<0.05),但A、B组间差异无统计学意义(P>0.05)。生长相关蛋白43、神经纤维丝蛋白160亚单位、S-100蛋白免疫组织化学染色示,A、B组轴突与髓鞘恢复情况相似。术后12周A、B组再生神经纤维直径相似(P>0.05),而A组再生神经髓鞘厚度优于B组,差异有统计学意义(P<0.05)。结论电纺丝ch/PLA神经导管具有促进大鼠周围神经再生的作用,有可能成为治疗周围神经缺损的新方法。

Objective To investigate the effect of electrospun chitosan/polylactic acid（ch/PLA） nerve conduit for repairing peripheral nerve defect in rats.Methods Nerve conducts loaded with ch/PLA was made by the way of electrospun.The mechanical property,hydrophility,biocompatibility were tested,and the scanning electron microscope was used to observe the ultrastructure.The same experiments were also performed on pure PLA nerve conducts as a comparison.Then,54 Sprague Dawley rats were divided into 3 groups randomly,18 rats in each group.Firstly,the 10 mm defects in the right sciatic nerves were made in the rats and were respectively repaired with ch/PLA（group A）,autografts（group B）,and no implant（group C）.At 4,8,and 12 weeks after operation,general observations,sciatic functional index（SFI）,electrophysiological evaluation,wet weight of gastrocnemius and soleus muscles,histological examination,immunohistological analysis,and transmission electron microscopy were performed to evaluate the effects.Results Compared with pure PLA nerve conducts,the addition of chitosan could improve the mechanical property,hydrophility,biocompatibility,and ultrastructure of the nerve conducts.At 4 weeks postoperatively,the regenerated nerve bridged the nerve defect in group A.The SFI improved gradually in both group A and group B,showing no significant difference（P〉0.05）.Compound muscle action potentials and nerve conduction velocity could be detected in both group A and group B at 8 and 12 weeks after operation,and significant improvements were shown in both groups（P〈0.05）.The wet weight and myocyte cross section of gastrocnemius and soleus muscles showed no significant difference between group A and group B（P〉0.05）,but there was significant difference when compared with group C（P〈0.05） at 12 weeks postoperatively.Immunohistological analysis revealed that S-100 positive Schwann cells migrated in both group A and group B,and axon also regenerated by immunohistological staining for growth associated protein 43 and neurofilaments 160.Transmission electron microscopy showed no significant difference in the diameter of nerve fiber between group A and group B（P〉0.05）,but the thickness of myelin sheath in group A was significantly larger than that in group B（P〈0.05）.Conclusion The electrospun ch/PLA nerve conduits can effectively promote the peripheral nerve regeneration,and may promise an alternative to nerve autograft for repairing peripheral nerve defect.