Brachial Plexus Defect Bridging By Extracellular Matrix-modified Tissue Engineered Nerve Graft In Rat

Background:Brachial plexus injuries(BPIs),especially brachial plexus defects,are difficult to cure and result in serious dysfunction.Therefore,it remains an urgent challenge in medicine.Currently,brachial plexus defects can be treated using autogenous nerve grafts(autografts),nerve transfer,tendon transfer,and free functioning muscle transfer.Although autografts are viewed as the gold standard for peripheral nerve injury treatment,their applications are limited by drawbacks such as donor-site morbidity,high risk of surgical complications,limitations of grafting materials,and diameter and/or fascicle mismatch.With the development of tissue engineering,tissue engineered nerve graft(TENG)has emerged as a promising alternative to autografts.Although TENG has been extensively studied in lower extremities,their use in upper extremities remains underexplored,especially for the brachial plexus.Therefore,we developed a TENG modified with extracellular matrix(ECM)and expand its application in upper trunk defects of brachial plexus in rats,which aims to provide a theoretical basis for TENG' s clinical transformation.Objective:To conduct a TENG modified with extracellular matrix,which was used to bridge an 8 mm gap in the upper trunk of brachial plexus in rats,and examine the efficacy of the TENG in repairing brachial plexus defect.Methods:1.SKP-SCs were co-cultured with chitosan neural conduits or silk fibers and subjected to decellularization treatment.Ten bundles of ECM coated silk fibers(five fibers per bundle)were placed into an ECM coated chitosan neural conduit in parallel to obtain the modified TENG.2.To characterize the modified TENG with immunostaining and scanning electron microscopy(SEM)imaging analysis.3.The modified TENG was applied to bridge an 8 mm gap in the upper trunk of brachial plexus.As control,we also established another three groups:the scaffold group,in which a chitosan/silk fiber neural conduit without ECM coated was used to bridge the nerve defect;the defect group,in which the nerve stumps were embedded into adjacent muscles without bridging;the autograft group,in which an autograft was used to bridge the nerve defect.4.Two weeks after surgery,the nerve grafts from the scaffold,autograft and TENG groups were excised and immunostained with anti NF-200 to evaluate the length and the area of the regenerated nerve fibers.5.Behavioral tests(Terzis grooming test,foot fault test and rotarod test)were carried out every two weeks after surgery to evaluate the functional recovery of upper extremities.6.Eight weeks after surgery,to assess functions of the regenerated nerves,electrophysiological evaluations were performed to analyze compound muscle action potential(CMAP)at the proximal end of the grafted sites and nerve conduction velocity(NCV).7.Seven weeks after surgery,FITC-CTB was injected into multiple locations of the affected biceps brachii.Seven days later,the C5 and C6 spinal cord segments as well as the corresponding dorsal root ganglions were excised and immunostained to investigate whether the regenerated nerves integrated.8.Eight weeks after surgery,the distal ends of regenerated nerves were evaluated with immunofluorescence staining and transmission electron microscopy(TEM)scanning to analyze the numbers of regenerated nerve fibers and the degree of myelination.9.Eight weeks after surgery,wet weight ratio of the target muscles was measured and the biceps brachii muscles were stained to analyze the percentage of fully reinnervated NMJs as well as the fragment number of the ACh Rs.Results:1.ECM was abundantly deposited on the surface of the chitosan conduit and silk fibers,suggesting the modified TENG was constructed successfully.2.Two weeks after surgery,the regeneration lengths in the autograft group and the TENG group were comparable and significantly exceeded that in the scaffold group.In accordance with the results of regeneration length evaluation,compared with that of the scaffold group,the areas of NF-200-positive regenerating axons in the autograft and TENG groups were significantly higher.There was no difference between the autograft and TENG groups.3.Two weeks after surgery,the results of behavioral tests were comparable among the groups,suggesting successful operation.Eight weeks after surgery,the results of behavioral tests in autograft group and TENG group were significantly better than that of scaffold group,with no difference observed between autograft and TENG groups.4.Compared with that in the scaffold group,the CMAP amplitudes at the proximal end in autograft and TENG groups were significantly higher.Moreover,compared with that in the scaffold group,the NCVs in autograft and TENG groups were significantly higher.5.The percentages of FITC-CTB-labeled sensory neurons in the DRG in autograft and TENG groups were significantly higher than that in the scaffold group.Similarly,the percentages of FITC-CTB-labeled motor neurons in the anterior horns of the spinal cords in autograft and TENG groups were significantly higher than that in the scaffold group.As expected,no differences in these two parameters were observed between the autograft and TENG groups.In total,the regenerated nerves in the TENG group had already reached the targeted muscles.6.Immunohistochemical staining of distal regenerated nerves showed that compared with that in the scaffold group,the numbers of distal regenerated nerve fibers in the autograft and TENG groups were significantly higher.More importantly,compared with the scaffold group,the autograft group and TENG group exhibited obviously higher myelination percentages.TEM analysis indicated that compared with that in the scaffold group,results of all the three parameters(the diameters of myelinated axons,the numbers of myelin layers and the thicknesses of the myelin sheath)in the autograft and TENG groups were significantly better.No differences were observed between the autograft and TENG groups.In summary,these results suggest that the TENG and the autograft effectively promoted the regeneration and remyelination of the defective upper brachial plexus.7.The wet weight ratio related to contralateral muscles in the scaffold group was significantly lower than those in autograft and TENG groups.For the immunohistochemical staining,compared with that in the scaffold group,the percentages of fully reinnervated NMJs in autograft and TENG groups were significantly higher.Similarly,the numbers of fragments per ACh R in the autograft and TENG groups were significantly lower than that in the scaffold group.There were no differences in these parameters between the autograft and TENG groups.Conclusions:1.In this study,we constructed a modified TENG by creatively recruited the silk fibers in the chitosan conduit which increased the surface area of the deposited ECM.2.We established an upper brachial plexus defect model and applied the modified TENG to the bridge the brachial plexus defect successfully.3.Treatment with the developed TENG allows the effective bridging of brachial plexus defects in the upper extremities,comparable with that of autograft.