温敏性壳聚糖神经导管的绿色制备方案及性能研究

A green route for the fabrication of thermo-sensitive chitosan nerve conduits and their property evaluation

目的探寻一种温敏性壳聚糖神经导管的绿色制备方案,并分析其提高神经导管力学强度、减缓降解速率的效果。方法利用壳聚糖的离子特异性效应,将注模成型的壳聚糖浸泡于NaCl溶液中0、4、12、24、36、48、72 h进行离子相变,再经漂洗、冻干、60Co灭菌后得到神经导管。取于NaCl溶液中浸泡0~72 h获得的神经导管,行大体观察、外径测量以及力学强度测试,并根据测量结果选择最佳相变时间点样品行微观结构观察、体外酶降解性能测试及细胞相容性。取20只雄性SD大鼠制备左侧坐骨神经15 mm缺损模型后,分别采用自体神经(对照组,n=10)和最佳相变时间点神经导管(实验组,n=10)桥接缺损。术后8周测量复合肌肉动作电位(compound muscle action potential,CMAP),大体及甲苯胺蓝染色观察再生神经,HE染色观察腓肠肌。结果随离子相变时间延长,神经导管颜色逐渐加深,外径逐渐减小,至12 h时无改变;神经导管拉伸强度逐渐增大,48 h时与12~36 h时最大拉力比较差异有统计学意义(P<0.05),与72 h时比较差异无统计学意义(P>0.05),故后续实验采用离子相变48 h的神经导管进行观察。扫描电镜显示神经导管为均匀的多孔结构,体外降解速率较未经离子相变导管明显降低;大鼠雪旺细胞能在神经导管内层黏附并增殖。体内实验显示,术后8周实验组、对照组CMAP分别为(3.5±0.9)、(4.3±1.1)m/V,均显著低于健侧CMAP(45.6±5.6)m/V(P<0.05);实验组及对照组间比较,差异无统计学意义(P>0.05);实验组神经导管可桥接神经缺损,再生神经纤维及腓肠肌组织形态与对照组比较无明显差异。结论利用壳聚糖离子特异性效应设计的神经导管绿色制备方案未添加任何毒性试剂,步骤简便,有利于壳聚糖神经导管的产品转化,制备的神经导管可修复大鼠周围神经缺损。

Objective To explore a green route for the fabrication of thermo-sensitive chitosan nerve conduits,improve the mechanical properties and decrease the degradation rate of the chitosan nerve conduits.Methods Taking advantage of the ionic specific effect of the thermo-sensitive chitosan,the strengthened chitosan nerve conduits were obtained by immersing the gel-casted conduits in salt solution for ion-induced phase transition,and rinsing,lyophilization,and 60Co sterilization afterwards.The nerve conduits after immersing in NaCl solutions for 0,4,12,24,36,48,and 72 hours were obtained and characterized the general observation,diameters and mechanical properties.According to the above results,the optimal sample was chosen and characterized the microstructure,degradation properties,and cytocompatibility.The left sciatic nerve defect 15 mm in length was made in 20 male Sprague Dawley rats.The autologous nerves(control group,n=10)and the nerve conduits(experimental group,n=10)were used to repair the defects.At 8 weeks after operation,the compound muscle action potential(CMAP)was measured.The regenerated nerves were investigated by gross observation and toluidine blue staining.The gastrocnemius muscle was observed by HE staining.Results With the increased ionic phase transition time,the color of the conduit was gradually deepened and the diameter was gradually decreased,which showed no difference during 12 hours.The tensile strength of the nerve conduit was increased gradually.The ultimate tensile strength showed significant difference between the 48 hours and 12,24,and 36 hours groups(P<0.05),and no significant difference between the 48 hours and 72 hours groups(P>0.05).As a result,the nerve conduit after ion-induced phase transition for 48 hours was chosen for further study.The scanning electron microscope(SEM)images showed that the nerve conduit had a uniform porous structure.The degradation rate of the the nerve conduit after ion-induced phase transition for 48 hours was significantly decreased as compared with that of the conduit without ion-induced phase transition.The nerve conduit could support the attachment and proliferation of rat Schwann cells on the inner surface.The animal experiments showed that at 8 weeks after operation,the CMAPs of the experimental and control groups were(3.5±0.9)and(4.3±1.1)m/V,respectively,which showed no significant difference between the two groups(P<0.05),and were significantly lower than that of the contralateral site[(45.6±5.6 m/V),P>0.05].The nerve conduit of the experimental group could repair the nerve defect.There was no significant difference between the experimental and control groups in terms of the histomorphology of the regenerated nerve fibers and the gastrocnemius muscle.Conclusion The green route for the fabrication of thermo-sensitive chitosan nerve conduits is free of any toxic reagents,and has simple steps,which is beneficial to the industrial transformation of the chitosan nerve conduit products.The prepared chitosan nerve conduit can be applied to rat peripheral nerve defect repair and nerve tissue engineering.