Experimental Study About The Injured Rat Sciatic Nerve Restored By Tissue-engineered Peripheral Nerve

ChapterⅠThe preparation of Scaffold of acellular tissue-engineerednerve derived from rats and study of its morphologyObjective: To develop a chemical method to remove the cells andmyelin in peripheral nerve of rats, so as to obtain a acellular scaffold forfabricating tissue-engineered nerve.Methods:10 SD rats,20 sciatic nerves were divided into twogroups,10 nerves in every group. Normal nerve as control group, acellularnerve by chemical disposal as test group. The characters of the structurewere observed under light and electronic microscope, and the componentswere analyzed by immunohistochemistry.Results: Acellular nerve were milk white columned and translucent,it's tractility was slightly increased than before disposal. After disposal,schwann cells, myelin sheath and axon of nerve fibers were eliminated,but the basement membrane tube of schwann cells and three-dimensionalsupports of extracellular matrix of epineurium and perineurium werereserved.Conclusion: The acellular nerve that disposed by chemical methodhas accord with the demand in structure and component, more experimentis need to validate whether it be a effective nerve graft. ChapterⅡThe culture of adipose-derived stem cells and induce it todifferentiate into schwann-like cellsObjective: To master the method for the isolation, culture andamplification of rat adipose derived stem cells(ADSCs) in vitro andexplore their biological characteristics and inducing condition forschwann-like cell differentiation of ADSCs.Methods: The inguinal fat pads of inbreds train of rat were excisedwith sterile technique, followed by digestion with collagenaseⅠ, thenthe cells were cultrued in Dulbecco modified Eagle medium with 10%new-born calf serum. The morphology of ADSCs was observed withinserted microscope constantly. Passage cells were examined using flowcytometry about CD antigens: 29, 34, 44. Growth curve of passage 1, 4and 10 cells using MTT. Passage 4 cells were induced to schwann-likecells. The proliferation and morphology before and after induction ofADSCs was observed with inserted microscope constantly.Immunocytochemistry of S-100 and GFAP, the ratio of positively stainedcells and staining grey scale measurements was counted.Results: Primary cells began to be adherence after culture for 3 daysand were fibroblast-shaped. Cells confluenced more than 90% after 8days and present colony whirlpool. Passage cells began to be adherencewithin 2 or 4 hours after sub-cultrue. After temporal stage of latencypassage cells proliferated rapidly and confluenced within 3 days. Latencyphase of cells prolonged slightly and proliferation became slowly throughpassage 10. Flow cytometry showed that 95% and 96% of cells expressed CD44 and CD29 respectively, while only 5% of cells expressed CD34.The Growth curve showed that cell proliferation ability of passage 1 and4 was more powerful than passage 10. The morphology of a part of cellswere convert to schwann-like cell and the Immunocytochemistry ofS-100 and GFAP were positively stained after induction.The ratio ofpositively stained cells and staining grey scale measurements of 4d afterinduction were superior to that of 2d and 8d.Conclusion: 1.ADSCs that characteristics were similar tomesenchymal stem cells (MSCs) could be isolated and culturedeffectively from adipose of rat. 2. ADSCs could be induced intoschwann-like cell in vitro.ChapterⅢStudy on using the differentiated adipose derived stemcells of rat into a tissue-engineered peripheral nerveObjective: Make of a tissue-engineered peripheral nerve byacellular nerve graft and differentiated ADSDs. Study of thebiocompatibility between the graft and cells and the effect of the tissue-engineered peripheral nerve on the sciatic nerve gap, so that provideproof for clinical research.Method: The adipose derived stem cells of inbreds train of rat wereinduced to differentiate into schwann-like cells and the cells were injectedinto the acellular nerve allograff.The proliferation or adhersion of these cells on the graft were observed by inverted microscope or ScanningElectronic Microscope. Activity of cells were detected using MTT. 10mmnerve gap of inbreds train of rats in two group were bridged by tissue-engineered peripheral nerve or autogenous nerve, the effect wereappraised by naked eye, recovery rate of sciatic function index (SFI%),nerve-electrophysiological (NEP), histology, Transmission ElectronMicroscopy and quantitative analysis of recovery rate of myelinated fiberpopulations,diameter of myelinated fiber and thickness of myelin sheat.Result: Schwann-like cells disperse in the hole of nerve graft at oneweek under Scanning Electronic Microscope, the processes of thesatellite-like cells adhere to the wall of graft.There was no statisticaldifference of activity of cells in both group(P＞0.05) using MTT. At 12weeks,the result of appearance, histology and TEM were similar, therewere no statistical difference in SFI%, NEP and quantitative analysis ofrecovery rate of myelinated fiber populations, diameter of myelinatedfiber and thickness of myelin sheat.Conclusion: 1. Schwann-like cells were compatible with acellularnerve graft without any rejection. 2. The tissue-engineered peripheralnerve can restore the gap of sciatic nerve of rat effectively and there wasno marked difference of nerve regeneration when compared withautogenous nerve.