Experimental Study On Repairing Sciatic Nerve Gap Of Rat With Tissue Engineering Artificial Nerve Graft

PREFACEPeripheral nerve defects caused by trauma are encountered commonly in clinic. If you want to recover function of the nerve you must restore their continuity. We can utilize microsurgical technique to connecte the nerve stump directly by separate nerve, change the path of nerve or flex articulation ect for nerve defects of <3cm.But the long gaps ( > 3cm) must be connected by graft. At present,autologous nerve transplantation has been considered the best method to repairing the long nerve defect. Because of the limited availability and donor-site morbidity of autograft,the mismatch of nerve cable dimensions between the donor graft and the recipent nerve ect, autologous nerve transplantation cannot be applied in clinic extensively. So many researchers concentrated on searching for a new graft to replace these autografts. Allograft represent a reconstructive alterative ,but are limited because of great rejection and the need for long-term immunosuppression. A study has shown that allografts freezed by—196℃decrease their antigon and immune response,but it cannot meet clinical needs. Other non-nerve tissues such as arteries,veins,muscle ect ,were used as alternatives to suture repair of nerve to successfully bridge nerve gaps in animal experiment. But there is a large gap to use in clinic. With the development of material science and engineering technique,artificial synthetic materials such as silicone tube ,PGA,PLA ect, have been used for repairing peripheral nerve defects,but the functional recovery is not satisfactory.With the emergement of tissue engineering in 1980s,there is a new project in using tissue engineering approaches to bridge peripheral nerve defects. Tissue engineering is formally defined as the application of the principles and methods of engineering and the life science toward the fundamental mammalian tissues and the development of biological subsitutes that restore,maintain,or improve tissue function. The principle of tissue engineering is :a small number of cells can be harvested from the patient using a biopsy ,and then the harvested cells are cultured in vitro in order to get the appropriate number in the laboratory. These cells can then be grown within a biocompatible ,absorbable natural or synthetic scaffold and implant into the defective site. The scaffold degrade gradually,the cells secrete various matrix materials to create an actual living tissue ultimately. The basic principles of tissue engineering of the peripheral nerve is: the cells,which can promote peripheral nerve regeneration, are seeded into natual or synthetic scaffold to bridge peripheral nerve long gaps. A tissue engineering scaffold should provide a necessary mechanical support as well as a physical structure for the transplanted cells to attach,grow, and maintain differentiaed functions. It has shown that Schwann cells display a very imporment role in peripheral nerve regeneration by proliferating and forming bands of Bungner,and releasing neurotrophic factors and directing the proximal axon to distal stump. Although Schwann cells are very effective in inducing nerve regeneration,their clinical use is limited because it is diffcult to obtain a sufficient number of cells to satisfy the requirement of tissue engineering. In the cultivation of Schwann cells used in autotransplantation, another peripheral nerve have to be sacrificed and that frequently leads to multiple surgical procedures and the loss of function at the donor site.Bone Mesenchymal Stem cell(BMSc),which are also know as stromal stem cells,are believed to be multipotent cells that can replicate as undifferentiated cells. Recent experimental studies suggested that cultured BMSc can differentiate into neural cells and glial cells in vitro and vivo, and promote axonal regeneration in peripheral nerve defect. In addition, autologous BMSc are easy to isolate and culture to obtain a sufficient number of cells.Therefore,BMSc overcomed the difficulties encountered with the use of autologous SCs for transplantation,it can be considered to be an alternative to SCs.There are some general requirements for a favourable peripheral nerve tissue enginerring graft: first,the graft should be biocompatible.Second,the graft material should be flexible and strong. Third, it should be permeable. In addition,the intrinsic structure of graft should not only provide a great surface for the attachment of implanted cells,but also imitate the function of bungner bands in which Schwann cell columns were formed to guide nerve regeneration. We have developed a chemical method to treat allograft, acellular nerve allograft was made successfully. This nerve scaffold has an structure of natural three-diamensional space. It may provid a more suitable environment for the transplanted cells to attach ,grow,and maintain differtiated functions. In our experiment,cultured BMSc were microinjected into the scaffold to repair a 15mm defect in the rat sciatic nerve. To investigate the effectiveness of this graft promote functional recovery,and provid experimental support for clinic work and the further study of peripheral nerve tissue engineering.MATERIALS AND METHODSAcellular nerve allograft were made by chemical extraction. The BMSc were isolated and cultured ;It was subcultured for three times and marked by Brdu. BMSc,marked by Brdu,were microinjected into the accelular nerve allograft and cultured for 7 days. The connection of graft and cells were observed by scanning electron microscope. A 15mm defect in the rat sciatic nerve was bridged by accelular nerve allografts filled with BMSc , accelular allograft filled with culture medium solution and autologous nerve respectively. 12 weeks after operation,the effectiveness of functional recovery were assessed by tibial former muscle wet weight comparison ,electrophysiological evaluation, transmission electron microscope of gastrocnemius,immunohistochemical staining of the s-100 protein of foot skin,HRP ect.RESULTS1. The period of making accelular nerve allograft by identified methods is 11 days;The Schwann cells,myelin sheath,axon were disappeared in normal nerve,the basal membrance consuit of SCs only were remained.Laminin,which could promote axon regeneraion and adhere to cells, was observed in basal memberance tubes by immunohistochemical staining.2. Bone Mesenchymal Stem cell performed the long spindle-shaped morphology after subcultured for three times. The scanning electron microscope observation manifested that BMSc show an ellipsoidal shape and attach to tube tightly.3. The tipitoes of experimental rat can separate and support ground 12 weeks after operation.The immunohistochemical staining of the s-100 protein of foot skin is possitive.The HRP was tested in spine and ganglion. Analysis of tibial former muscle wet weight comparison , electrophysiological evaluation,myelin sheath tickness,the diameter of nerve fiber showed no statistically significant difference between experimental group and autograft.CONCLUSION1. The accelular nerve graft eliminated the cells,and remained Schwann cells basal memberance tubes. The scaffold,which three diamensional spsce structure , can be used as tissue engineering graft.2. The Laminin was remained during making accelular nerve graft,which can guide the axon regeneration.3. The affinith of accelular nerve graft to bone marrow stromal cells is good.4. The artificial nerve graft construced by BMSc and accelular nerve graft are able to promote peripheral nerve regeneration and functional recovery.5. The sensory recovery can be tested by immunohistochemical staining of the s-100 protein of foot skin.