Repair Peripheral Nerve Defects With Naturally Nerve-derived Extracellular Matrix Scaffold And Mesenchymal Stem Cells

With the development of modern society, the number of nerve injuried patients is increasing as result of accidents, war, earthquakes, and cancer surgery. Cases of peripheral nerve injuries is increased 60 to 90 million each year, which is about 30 million to 45 million cases need nerve transplant. If the patients aren't treated properly, it will result in permanent disability and lead to serious economic and social burden. Current "gold standard" treatment of peripheral nerve injury is the autograft, but it is restricted by the lack of donor nerve, loss of donor nerve function and the neuronal structure and size does not match needed. Therefore, the development of an effective substitute for autologous nerve graft is focus at home and abroad.The development of tissue engineering provides a new method to repair peripheral nerve defects. Tissue engineering nerve is consists of the scaffold material, seed cells and growth factors, and it should have the composition and spatial structure of normal nerve. Natural Neural Extracellular Matrix is not only provide a support structure and attachment site for seed cell, but also contain a lot of biological information which can influence cell adhesion, migration, proliferation, differentiation and regulation cell gene expression. Natural Neural Extracellular Matrix shows superiority in guiding and promoting nerve regeneration. However, the recently commercial nerve regeneration conduits are use ofⅠ,Ⅲcollagen, which are only using part of the extracellular matrix. The hot topic is to keep the main component of ECM and construct ideal tissue engineering nerve. Objective:This study is constructed better tissue engineering nerve and evaluated the effect of repair peripheral nerve defects. The tissue engineering nerve is consist of the composition and structure of natural neural ECM scaffold and promoting nerve growth, low immunogenicity of the seed cells.Method:1. Nerve extracellular matrix was prepared by freeze-thaw, mechanical grinding and centrifugation. Then its morphology and composition was evaluated by SEM and immunofluorescence staining. Schwann cells or dorsal root ganglia were used to evaluation of nerve extracellular matrix biocompatibility.2. Orientation of ECM scaffold was prepared by oriented crystallization technique. Then mechanical properties of ECM scaffold were evaluated by biomechanical tester. Nerve axons were measured to evaluation of short-term results by repair of peripheral nerve defect.3. Rat bone marrow mesenchymal stem cells, rat adipose stem cells and human umbilical cord Wharton mesenchymal stem cells were induced into Schwann-like cells in vitro and proved phenotype by immunofluorescence staining. The survival of BMSCs in fibrin glue and neurotrophic factors was detected by Dead/live staining and ELISA. The effect of different number of BMSCs in fibrin glue was quantitative evaluated by axonal growth length of dorsal root ganglion, Schwann cell migration and axon area index.4. To improve the effect of CEANA to repair of peripheral nerve defects, Schwann-like cells were injected in acellular nerve or change adding seed cells styles, and through functional assessment and histological evaluation of its the effect of peripheral nerve defect.Results:1. Nerve extracellular matrix showed 30～130 nm diameter nano-filaments. Type I collagen, laminin, fibronectin were positive proved by immunofluorescence staining. Rat dorsal root ganglia and Schwann cells were growing well on the extracellular matrix and maintain normal cell phenotype. 2. The orientation of ECM scaffold was showed orientation of the porous honeycomb structure in longitudinal section by SEM. Rupture strength, ultimate stress, strain and fracture power of oriented ECM scaffolds and acellular nerve was significant difference to normal nerve (P<0.05). There was no difference in elastic modulus between normal nerve and acellular nerve, but elastic modulus of oriented ECM scaffolds was lower than acellular nerve's (P<0.05). The length of axons in oriented ECM scaffolds group was less than acellular nerve group (P<0.05).3. The morphology of Schwann-like cells were similar to Schwann cells and p75 and GFAP immunofluorescence staining was positive. The length of axonal growth, Schwann cell migration and axon area index in BMSCs co-culture with DRG group were significantly higher than he control group (P<0.05).4. Adding dADSC, dMSC and the SCs into the acellular nerve, it was increased contraction of triceps muscle, recovery rate of muscle, the number of sciatic nerve myelinated fibers and myelin thickness. It has similar results in functional assessment and histological evaluation to added MSCs inside or outside of acellular nerve.Conclusion:1. Prepared the nerve extracellular matrix was maintained natural extracellular matrix composition and it has satisfactory cell compatibility.2. Orientation of ECM scaffold was prepared by oriented crystallization technique, but its mechanical properties should be improved in future. The tensile strength of acellular nerve meets the requirements of the nerve mechanical properties. Orientation of ECM scaffold was proved have satisfactory biocompatibility, but it has less effect to compare with acellular neve in peripheral nerve injury. The acellular nerve has composition and structure bionic of normal nerve and it can guide nerve regeneration. 3. Rat BMSCs, ADSC and human umbilical cord Wharton mesenchymal stem cells can be induced to Schwann-like cells and it has similar phenotypic of Schwann cells. BMSCs in fibrin glue can survival well and the secretion of factors wasn't affected. The difference number of BMSCs in fibrin glue has a dose-effect relationship with the growth of DRG.4. The Schwann-like cells injected into acellular nerve can mprove peripheral nerve repair results and it has similar effect to injected Schwann cells. Compared to directly injected BMSCs into acellular nerve, supply BMSCs around acellular nerve have the similar effect, which was not restricted by the number of seed cells and maintain the internal structure of acellular nerve.