Evaluation Of Electrospun Chitosan/ Polylactic Acid Nerve Conduits Used For Peripheral Nerve Regeneration: An Experimental Study In Rats

Objective: In this study, a new chitosan/ polylactic acid nerve conduit was produced by the method of electrospun. After the physical properties and biocompatibility was tested through in vitro experiment, the nerve conduct was implanted into Sprague-Dawley rats to repair a sciatic nerve defect. The effect of repairment was examined at different time after operation.Methods: The physical properties and biocompatibility was tested through in vitro experiment. Scanning electron microscope was used to observe its fiber formation. Fifty-four adult male Sprague–Dawley(SD) rats weighing 200-250 g were randomly divided into four nerve grafting groups, with eighteen rats in each group. Firstly, 10 mm defect in the right sciatic nerves were made in the rats and were respectively repaired with electrospun chitosan/ polylactic acid conduits graft(group A),autografts(group B) and non-graft(group C). At 4, 8 and 12 weeks after the operations, sciatic function index(SFI) and electromyogram of each rat was tested. Then, the right sciatic nerves were taken out,and the comparative evaluation was made on the repairing effects by electrophysiological evaluation,wet weight of gastrocnemius and soleus muscles,histological examination,immunohistological analysis and electron microscopy observation.Results: In vitro experiments demonstrated that the tensile force, compressive stress, and hydrophilisy of the nerve conduct was enhanced as the addition of chitosan. At the meantime, the fiber structure became more disciplinary. No rat was found significant signs of systemic or regional inflammation and serious surgical complications after implantation either in experimental group or in control groups. Four rats developed ulcerofsole in their right foot in the electrospun chitosan/PLA group at three weeks postoperatively. Three rats developed ulcerofsole in their right foot in the autograft group at three weeks postoperatively. Seven rats developed ulcerofsole in their right foot in the non-graft group at three weeks postoperatively. At 4 weeks postoperative, the electrospun chitosan/PLA tube showed degradation without collapse. The regenerated nerve have bridged the sciatic nerve defect. At 8 weeks and 12 weeks postoperatively, the diameter of the regenerated nerve increased gradually. At 12 weeks after surgery, the appearance of the regenerated nerve manifested similar to that of the autografts. The nerve guidance tube was covered by connective tissue. The sciatic function index(SFI) was measured at 4, 8 and 12 weeks postoperatively. At 4 weeks after surgery, there was no statistically significant differences between electrospun chitosan/ polylactic acid conduits graft group,autografts group and non-graft group(p>0.05). At 8 weeks and 12 weeks after operation, walking track analysis showed a significantly lower score in group C graft than in group A and group B(p<0.05), whereas there was no significant difference between group A and group B(p>0.05). Compound muscle action potentials(CMAP) and nerve conduction velocities(NCV) were detected at 4, 8 and 12 weeks after surgery on the hindlimbs of the operated side. At 4 weeks postoperatively, no reaction was detected among all groups. At 8 weeks postoperatively, group A and group B had a similar NCV(p>0.05), while no reaction was detected in group C, still. But group B had a larger CMAP than group A(p>0.05). No significant diference on NCV could be observed between group A and group B at 12 weeks postoperatively(p>0.05), but group A had a larger CMAP than group B(p<0.05). At 4, 8 and 12 weeks after surgery, both group A and group B had a bigger gastrocnemius and soleus muscles wet weight recovery rate than group C(p<0.05), but there was no significant difference between group A and group B(p>0.05). At 4 weeks after surgery, all the three groups had a similar myocyte cross section(p>0.05). At 8 weeks and 12 weeks after surgery, both group A and group B had a bigger myocyte cross section than group C(p<0.05), but no significant diference could be observed between group A and group B(p>0.05). Histologically, newly regenerated nerve fibers could be observed in both group A and group B at 4 weeks postoperatively, but fewer and more disorder in group A. At 8 weeks after surgery, nerve tracts could be found in group A, which were obviously myelinated. In group B they ranged more closely and neatly. Newly regenerated nerve fibers increased farther more at 12 weeks postoperatively, and the nerve looks thicker and maturer. Immunohistochemically, the average optical density(AOD) of GAP-43 in group A was significantly greater than in group B at 4 and 12 weeks after surgery(p<0.05), but similar in this two groups at 8 weeks postoperatively(p>0.05). The AOD of NF-160 was obviously greater in group A than in group B at 4 weeks after surgery(p<0.05), but similar in this two groups at 8 and 12 weeks postoperatively. There was also no significant diference on the AOD of S-100 between group A and group B at 4, 8 and 12 weeks postoperatively(p>0.05). This two groups had similar nerve fiber diameters at 12 weeks after operation(p>0.05), but the thickness of the myelin sheath in group A was significantly greater.Conclusion: The addition of chitosan improved the physical properties and biocompatibility of the nerve conduct. The electrospun chitosan/ polylactic acid nerve conduct could promote the regeneration of peripheral nerve as well as autograft, thus proved its possibility in the treatment of peripheral nerve defect.