The Mechanism Of Sciatic Nerve Regeneration

In clinical practice, direct end-to-end suturing techniques are suggested for a short nerve injury. When large gaps remain between the ends of injured peripheral nerves, nerve autografting has been the first choice for repairing nerve gaps. However, this approach has inevitable disadvantages, such limited supply of available nerve autografts and certain donor site mobidity. Xenografts and allografts have been evaluated as alternatives to autografts but have poor successful rate and problems of immune rejection. A wide range of materials has been developed for use as a synthetic nerve guidance channel. Of these materials , nondegradable materials such as silicone rubber have been widely used because of their inert and mechnical properties. However, upon completion of regeneration these material no longer serve any purpose and may become detrimental due to mechanical impingement or infection. Contrarily, biodegradable materials potentially avoid these problems and make secondary surgery unnecessary. Biodegradable material such as polyglycolic acid (PGA)and polylactic acid (PLA) are used for tubulization to bridge both ends of amputated peripheral nerve, Nonetheless ,upon degradation by hydrolysis, PLA and PGA produce lactic acid and glycolic acid respectively. These degradation by -products would lead to a lowering of local pH and could bedetrimental to the surrounding cells and tissue. Nerve guidance channels have also been fabricated out of collagen with favorable results in nerve repair. But collagen is rather expensive and difficult to handle during suturing because of mechanical weakness.We previously developed a biodegradable biocompatible artificial tendon with Human hair keratin(HHK) which were specially treated .The experiment shows that HHK is a biodegradable material with excellent biocompatibility, plasticity and adhesiveness. It suggested that HHK is an excellent biomaterial with great potential for clinical operation. Some researchers also apply a nerve guidance channel made of HHK to restore the sciatic nerve with long gap, it rurnes out that it can guide the nerve regeneration, and improve their function of the limbs ,and there are a lot of schwann cells adhere to the HKK, that means HHK has good cellcompatibility. but the mechanism of sciatic nerve regeneration with HHK is still not clear. In this study, we observe the morphological changes by means of HE, Immunochistochemistry and Electric microscope in order to clearify the mechanism of sciatic nerve regeneration induced by HHK.The aims of grafted tubes are to create surfaces to preferentially attach migrated Schwann cells and growth cones, directing outgrowing axons from the amputated nerve ends. While there isn't anything within the tube, so it is not easy for schwann cells to adhere to the HHK nerve tube, the HHK nerve tube is very loose so that it can't accumulate all kinds of factor which could promote nerve regeration and prevent in-growth of fibroblasts into the nerve gap. When we design a nerve guidance channel from tissue engineering approach, besides conduit material, incorporation of neurotrophic factors and cells supporting axon regeneration should also be considered, nerve regeneration has been shown to be enhanced across gaps utilizing guidance channels filled with neutrophic factors such as nerve growth factor(NGF),brain derived neurotrophic factor(BDNF).Besides NGFs, several studies have shown enhanced regeneration when Schwann cells were seeded within nerve guidance channels. Therefore, we design a HHK nerve guidance tube which filled with cocultured Schwann cells which were tansfect by NGF and BDNF and HHK to evalue its function.Chapter 1 The morphological mechanism of damaged rat sciatic nerve regeneration induced by human hair keratin in vivoObjective To observe morphological changes of regenerated sciatic nerve induced by HHK in order to illuminate the mechanism of sciatic nerve regeneration and to evaluate the effect of HHK in repairing injured peripheral nerve. Methods Twenty-eight normal SD rats were used in this study, which were divided into 3 groups including a control group (n=4) and 2 experiment groups(n=24). The sciatic nerves of rats in the 2 experiments groups were transected to create a 10-mm gap, then insert either HHK tissue engineering artificial nerve(group n, n=12) or HHK+collagen membrane tissue engineering artificial nerve (group ni,n=12) between the gap ,and routinely sutured the epineurium of the nerve stumps with tissue engineering nerve using 9-0 nylon.Anatomic and histological examinations were performed at different time points after surgery. Results Noteworthy improvement in healing could be seen from Anatomic histological results of group II and group III, in which HHK conduits were partially degraded and embedded in white tissue, crisp and fragile. 2 days to 2 weeks after HHK implantation,the schwann cells dedifferentiated, and 3 weeks after HHK implantation, there are large amount of macrophages and megakaryocytes surrounded the HHK, and a large amount of regenerated schwann cells and blood vessles were observed between the HHKs which was partially degraded and absorbed. 6 weeks after HHK implantation, there are plenty of inmature Schwann cells in the nerve close to the surgery site,andsome HHKs were degraded completely. 9 weeks after the HHK implantation, HHKdegraded apparently ,much more nerve fibrils in the myelino 12 weeks after theoperation, almost all HHKs completely degraded, the severed sciatic nerves wereperfectly repaired and Schwann cell proliferation and myelinization around HHK,there were nerve fibrils in the myelin.Chapter II Construction of HHK tissueengineering nerve in vitroSection I Cloning of p-NGF and BDNF and construction of their eukaryoticexpress vectorObjective: In order to improve SC's activity of secreting P-NGF and BDNF, extractethe rat brain total RNA from rat brain tissue, clone P-NGF and BDNF and constructtheir eukaryotic express vector.Methods and result: P-NGF and BDNF was obtained from rat brain tissue byreverse transcription polymerase chain reaction (RT-PCR). Their eukaryoticexpression vector were successfully constructed by directional cloning the targetgene into pEGFP-C2 vector.Section II Gene modifying SCs and construction of tissueengineering nerve invitroObjective: Construction of in vitro tissueengineering nerve with absobtive anddegraded HHK and p-NGF and BDNF gene modified rat SCs.Methods: New borned rat's sciatic nerve was digested with two enzymes to getSCs, and the SCs was transfected with p-NGF and BDNF through liposome, thenscreen the positive cells with G418, and coculture the HHK and the positive SCs todetect their compatibility.Result: The SCs were obtained from new borned rats' peripheral nerves. The SCspurity was up to 96% examined by S-100 protein staining. Successfully transfectedP-NGF and BDNF into SCs. The gene modified SCs grew well on the surface ofHHK. Put the cocultured HHK and SCs into the HHK conduit to construct the tissueengineering nerve in vitro.Conclusion 1. As an ideal material for nerve injury repair, HHK can guide sciatic nerve elongation across a 10mm gap.We discovered that an ideal inside environment was formed along the HHK whether with or without a collegen membrane. 2. High differentiated schwann cells dedifferentiated into young one safter damage. Apocytosis plays key role during the dedifferentiation.Damaged axon was enclosed and relieved, and growing buds which has a lot of pseudo-ographs were formed on the top of the healthy axon. The pseudographsengrapp edone or more schwann cells,and only one pseudograph can developeinto a complete axon after competition. Nerve fiber barrier membrane was derived from the capillary meenchymal stem cells and the outboard blood vessel barrie -r membrane.In a word, the regeneration pattern of the schwann cells ,the axon and the nerve membrane is necessary for self-organization., and the schwann cells, the axon and the nerve membrane correspond with each other i n phase. 3. |i-NGF and BDNF was obtained from rat brain tissue, Their euka-ryotic expression vector pEGFP-C2-P-NGF/pEGFP-C2-BDNF were successfully constructed. 4. The SCs were obtained from new borned rats' peripheral nerves. Successfully transfected 0-NGF and BDNF into SCs. The genemodified SCs grew well on the surface of HHK. Put the cocultured HHK and SCs into the HHK conduit to construct the tissueengineering nerve in vitro.