Development And Evaluation Of Chitosan/ Poly (Vinyl Alcohol) Nerve Guidance Conduits Used For Peripheral Nerve Regeneration

Part one Evaluation of chitosan/ poly (vinyl alcohol) nerve guidance conduits used for peripheral nerve regeneration: an experimental study in ratsPurpose: In the present study, poly(vinyl alcohol) (PVA) was added to chitosan to form a composite material, with improved mechanical properties, for use as nerve conduits by the method of repeated freezing-thawing.We examined the effects of chitosan/polyvinyl alcohol nerve conduit in repairing peripheral nerve defect at different time.Methods: Eighty-four adult male Sprague–Dawley (SD) rats weighing 180-220 g were randomly divided into four nerve grafting groups, with eleven rats in each group. Chitosan/poly (vinyl alcohol) nerve guide conduit group, poly(lactic-co-carprolactone) tubes, silicon tubes and autografts group respectively, (groups A, B and C; control group D). A segment of left sciatic nerve was resected and removed, leaving a 15-mm long defect following retraction of the nerve ends at all rats. The defects were bridged by chitosan/poly (vinyl alcohol) nerve guide conduit, poly(lactic-co-carprolactone) tubes, silicon tubes and autografts group respectively. Four-week, 8-week and 12-week postoperative,a series of examinations was performed, including Sciatic Functional Index (SFI), electrophysiological methods, Weight of the gastrocnemius and soleus muscles, and histological examination. 12-week postoperative, True Blue retrograde tracing was examined.Results: In experimental and control groups, all rats showed no noticeable signs of systemic or regional inflammation and serious surgical complications after implantation and the locomotor function of their injured limbs gradually recovered. Three rats developed mild decubital in their left heel in the chitosan/PVA group, 4 rats in the P(LLA-CL) group, 6 in the silicon group, and 2 in autograft group because of the sciatic nerve injury at two weeks post-surgery. At 4 weeks postoperative, the chitosan/PVA tube showed degradation. The regenerated nerve have bridged the sciatic nerve defect, but maintained its initial shape without lumen collapse. At 8 weeks postoperative, diameter of the regenerated nerve became thick. At 12 weeks postoperative, the appearance of the regenerated nerve similar to that of the autografts. Adhesions between the tube and the peripheral tissue were insignificant. The surface of the tube was replaced with well-vascularized connective tissue. The sciatic function index (SFI) was measured 4, 8 and 12 weeks post-operation. At 4 weeks, there were no statistically significant differences among chitosan/PVA grafts, P(LLA-CL) grafts,silicon grafts and autografts. At 8 weeks and 12 weeks, walking track analysis showed a significantly lower score in the P(LLA-CL) graft and silicon graft group than in the chitosan/PVA graft (p<0.05).There was no significant difference between chitosan/PVA graft and autograft groups. Four weeks, Eight weeks and Twelve weeks post-surgery, compound muscle action potentials (CMAP) were detected at hindlimbs of the operated side for each rat in all groups, and the peak amplitude (AMP) and nerve conduction velocities (NCV) were measured. At 4 weeks, CMAP could not be detected among all groups. At 8 weeks, the highest NCV was observed in the autograft group. There was no significant difference between chitosan/PVA graft and the P(LLA-CL) graft(p>0.05). At twelve weeks, NCV showed a significantly lower value in the P(LLA-CL) graft and silicon graft group than in the chitosan/PVA graft (p<0.05). There was no significant difference between chitosan/PVA graft and the autograft graft(p>0.05). At 8 weeks, the highest AMP was observed in the autograft group(p<0.05). The peak amplitude value of chitosan/PVA grafts superior to P(LLA-CL) grafts (p<0.05). At twelve weeks, NCV showed a significantly lower value in the P(LLA-CL) graft and silicon graft group than in the chitosan/PVA graft (p<0.05). The Wet Weight of Gastrocnemius and Soleus Muscles were measured 4, 8 and 12 weeks post-operation. The lowest value was observed in the silicon group(p<0.05). There was no significant difference between chitosan/PVA grafts ,PLLA-CL grafts and autografts(p>0.05).At twelve weeks, the nerve fiber density showed a significantly lower value in the P(LLA-CL) graft and silicon graft group than in the chitosan/PVA graft (p<0.05).There was no significant difference between chitosan/PVA graft and autograft groups(p > 0.05).The diameter of the regenerated nerve fibers showed a significantly lower value in the P(LLA-CL) graft and silicon graft group than in the chitosan/PVA graft (p<0.05).There was no significant difference between chitosan/PVA graft and autograft groups(p>0.05).The thick of myelin sheath showed significantly higher value in autograft group than other group(p<0.05). There was no significant difference between chitosan/PVA grafts and PLLA-CL group(p>0.05). At twelve weeks, True Blue -labeled neuron cell bodies were found within both anterior horn of gray matter in the spinal cord and dorsal root ganglions (DRGs) ipsilateral to the operated side of the tested rats on illumination with ultra-violet light 1 week after the injection of True Blue. The number of labeled neurons of True Blue fluorescence at anterior horn of gray matter in the spinal cord showed a significantly lower value in the P(LLA-CL) graft and silicon graft group than in the chitosan/PVA graft (p<0.05).There was no significant difference between chitosan/PVA graft and autograft groups(p>0.05).Conclusion: The chitosan/PVA blend may be useful for functional peripheral nerve regeneration, and may be potential possibility of using the newly developed nerve grafts as a promising alternative to nerve autograft. Part two Evaluation of chitosan/ poly (vinyl alcohol) nerve guidance conduits used for peripheral nerve regeneration: an experimental study in MacaquesPurpose: Chitosan/poly (vinyl alcohol) complex is a new kind of material used to repair the peripheral nerve defect. In this content, we examined the effects of chitosan/polyvinyl alcohol nerve conduit in repairing peripheral nerve defect.Methods: Twelve adult Macaques weighing 3.26-5.35 KG were randomly divided into three nerve grafting groups, with four Macaques in each group, Chitosan/poly (vinyl alcohol) nerve guide conduit group, negative group and autografts group respectively, (groups A, and B; control group C). A segment of left radial nerve was resected and removed, leaving a 20-mm long defect following retraction of the nerve ends at all Macaques. In the chitosan/poly (vinyl alcohol) nerve guide conduit group the nerve defect was bridged with the chitosan/poly (vinyl alcohol) nerve guide conduit; in the autograft group the removed nerve segment was reversed 180o and re-implanted across the nerve defect. As to the non-grafted group, the nerve defect was left unbridged. Eight-months postoperative,a series of examinations was performed, including electrophysiological methods and histological examination.Results: A monkey of non-grafted group died of the anaesthetic excessive. Other Macaques showed no noticeable signs of systemic or regional inflammation and serious surgical complications after implantation and the locomotor function of their injured limbs gradually recovered. At 8 months post-surgery, compound muscle action potentials (CMAP) were detected at forelimbs of the operated side and contralateral unoperated side for each Macaque in all groups, and the peak amplitude (AMP) and nerve conduction velocities (NCV) were measured. CMAP could not be detected at negative group. AMP showed a significantly higher value in the contralateral unoperated normal radial nerve than in the chitosan/PVA graft and the autograft group(p<0.05). There was no significant difference between chitosan/PVA graft and the autograft graft(p>0.05). NCV showed a significantly higher value in the contralateral unoperated normal radial nerve than in the chitosan/PVA graft and the autograft group(p<0.05). The nerve conduction velocities value of autografts superior to chitosan/PVA grafts (p<0.05). At 8 months post-surgery, the nerve fiber density was measured. The nerve fiber density showed a significantly higher value in the contralateral unoperated normal radius nerve than in the chitosan/PVA graft and the autograft group(p < 0.05). There was no significant difference between chitosan/PVA graft and the autograft graft(p>0.05). But the diameter and the thick of myelin sheath of the regenerated nerve fibers showed a significantly higher value in the contralateral unoperated side than in the chitosan/PVA graft and the autograft group.Conclusion: The chitosan/PVA blend may be useful for functional peripheral nerve regeneration, and may be potential possibility of using the newly developed nerve grafts as a promising alternative to nerve autograft.