Enhancement Of Nerve Regeneration Speed And Peripheral Nerve Defect Length Repaired With Chemically Extracted Acellular Nerve Allograft

Background:The treatment of peripheral nerve injury is a tough problem clinically. The standard clinical approach for bridging nerve defects is an autologous nerve graft, or autograft.Disadvantages of the autograft include limitation of the source of autologous nerve and loss of function at the donor site.Therefore,many researchs are focused on developing proper biomaterials as an alternate to autograft.Chemically extracted acellular nerve allograft(CEAN) which removes immunogenicity such as Schwann cells and myelin while restoring the nerve structures such as basal lamina tubes and extracellular matrix,is a promising material alternative to autograft.In nerve grafting,muscles may show irreversible atrophy and lose function before axonal regeneration into the distal targets.For CEAN,the speed of recovery of nerves regeneration must be increased and muscle atrophy decreased.In addition,the results of repiring the peripheral nerve defect with CEAN became worse while the length of peripheral nerve defect increasing.It becomes an important research subject that how to increase the length of repairing peripheral nerve defect.Objective:1.Electrophoretic analysis of rat CEAN protein is to be performed to know about the protein of CEAN.Dorsal root ganglia of chicken are cultured onto the surface of CEAN slice in order to observe the effect of CEAN structure on the fiber regeneration. 2.Sustained of nerve growth factor is to be deliveried for CEAN,used to repair injured nerves,to evaluate effect of the method on the speed of nerve regeneration, muscular atrophy and nerve tissue regeneration.3.The protein of Chondroitin Sulfate Proteoglycan(CSPG) in CEAN is to be degraded by Chondroitin enzyme ABC,and the peripheral nerve large defects is to be repaired with these CEAN in order to evaluate the effect of degrading Chondroitin sulfate proteoglycan of CEAN on the length of repairing nerve defects. Methods:ⅠThe protein and structure characteristics of CEAN The CEAN were perpared according to Sondell method and electrophoresed to observed whether the electrophoresis strip of 28-30kDa(Myelin Protein) exited or not.Dorsal root ganglia of chicken was cultured onto the surface of CEAN slice and dyed with nerve fiber fluorescence to observe the orientation of nerve fiber growth on the surface of CEAN slice.ⅡExperiment on the effect of controlled nerve growth factor on peripheral nerve defect repaired by CEANThe microspheres of NGF were prepared drug microsphere technology and fixed with the fibrin gels to make the complicated controlled release NGF.The Wister rats were randomly divided into four groups:Autograft group(A group),acellular nerve allograft+the double controlled release NGF(B group),acellular nerve allograft(C group) and acellular nerve allograft+fibrin glue(D group).The nerve regeneration length was measured 2 weeks after operation.The effects of peripheral nerve regeneration were evaluated by neural electrophysiology,the recovery rate of triceps surae muscular tension and weight and histological assessment 4 monthes after operation.ⅢEffect of CSPG degraded by Chondroitin enzyme ABC in CEAN on repairing peripheral nerve defectCEAN of which chondroitin sulfate proteoglycan was degraded by chondroitin enzyme ABC was immunohistochemistrily dyed with anti-CSPG in order to observe the remain of chondroitin sulfate proteoglycan and the other elements in CEAN.The Wister rats which left sciatic nerve was cut for 20 mm defect were randomly divided into two groups:CEAN degraded by chondroitin enzyme ABC(A group),CEAN(B group).The result of nerve regeneration was evaluated 3 monthes after operation.Results:ⅠElectrophoretic analysis of rat CEAN protein showed that the electrophoresis strip of 28-30kDa was totally disappeared.The large number of nerve fibers growed from DRG along the basement membrane of CEAN,while as the little nerve fibers grew outsite CEAN.ⅡThe NGF was detected from the complicated controlled release drug of NGF at least 60 days and the bioactivity of released NGF stimulated the neuritis growth of chick dorsal root ganglion.At second week after nerve repair,the datum showed that the length of nerve regeneration in autografting was larger than the other groups (P＜0.05),NGF-treated acellular grafting better than acellular grafting alone and acellular grafting with fibrin glue(P＜0.05),and there were no difference between acellular grafting alone and acellular grafting with fibrin glue.Four months after nerve repair,nerve regeneration was assessed functionally and histomorphometrically. The percentage of the triceps surae muscular tension in autografting were better than the other groups(P＜0.05).There were no significant differences between the other three groups(P＜0.05).The percentage of the triceps surae weight in autografting were better than the other groups(P＜0.05),and that in NGF-treated acellular grafting better than the other two control group(P＜0.05).There were no significant differences between acellular grafting alone and acellular grafting with fibrin glue (P＜0.05).Based on the histological observation,the image analysis indicated that axonal diameter,axon number and myelin thickness in NGF-treated acellular grafting were better than those in acellular grafting alone and acellular grafting with fibrin glue (P＜0.05).There were no significant differences between NGF-treated acellular grafting and autografting.ⅢThe slice of CEAN degraded by chondroitin enzamy ABC was immunohistochemistrily dyed:the CSPG could be degraded by chondroitin enzamy ABC and the laminin could be perserved.Animal experiment evaluated by functional and histological accessment 12 weeks after operation showed that there was autotomy in both groups.There was only one of 6 rats that had autotomy foot in A group but 6 of 8 rats in B group.Assessment of autotomy was carried out using the scale described by Wall,there were significant difference between both groups(P＜0.05). Five of six rats had positive pain reaction test in A group while as 1 of 8 rats did in B group.There were significant difference between them according to Chi-square test of four-fold table was performed(P＜0.05).The percentage of the triceps surae muscular tension and triceps surae weight in A group were better than the B group(P＜0.05). Nerve fibers structure dyed by HE staining were better in A group than in B group.Conclusions:1 There was no myelin protein remained in the CEAN,while the basement membrane structure of CEAN can induce the nerve fiber growing.2 NGF bioactivity can be protected by NGF complicated controlled release drug. The speed of peripheral nerve regeneration can be improved repaired by CEAN with NGF complicated controlled release drug.3 CSPG can be removed from the CEAN degraded by chondroitin enzyme ABC. Chondroitinase treatment increases the effective length of acellular nerve grafts.