| Objective: To plant fetus rabbit Schwann cells that were cultured and purified in vitro into the extracted nerve graft of homogeneity and made it into bridging complex. Then transplanted the bridging complex into the rabbit defective sciatic nerve. The experiment was designed to investigate the effect of acellular nerve allografts on the functional recovery and reconstruction of the nerve-muscle structure of the sciatic nerve defect in Rabbit. Accordingly, it could confirm that the bridging complex not only offered favourable supporting effects but also induced and promoted the regeneration of neuraxises and medullary sheathes. This method would provide experimental basis for the repairing of defect of peripheral nerve.Methods: 1.Preparation of the Schwann cell cultures: Executed the adopted pregnancy New Zealand white rabbit and taken out the fetus rabbits of 28 days conceptus age. Under operation microscope, both sides of sciatic nerves were achieved and the external connective tissue of the nerve segments was stripped, and then the segments were scissored up to shreds. We applied enzyme digestion (trypsin / collagenase) to culture Scs. In order to remove fibroblast, the double 30min differential adhesion was applied. We even added NGF to FBS to promote SCs growing. Observed SCs under the microscope and made them go down to posterity. Collected and counted the good growth cells of the second filial generation. Accredited them with S-100.2. Preparation of Extracted Nerve Grafts (eNG): Two sides of the adult New Zealand white rabbit sciatic nerves were used to prepare the eNG. Before the extraction, sheared the adipose tissue and part of the epineurium with the operating microscope. Divided them into 3cm one section. Then they were dipped in 4oC distilled water immediately, Soaked for 6hs. After that, immerged them into 3% Triton X-100 solution. 12hs later, the sciatic nerves would be put into 4oC distilled water again for another 6hs. Then they would be treated by 3% Triton X-100 for the second 12hs. The process would not stop until the sciatic nerves were treated by 3% Triton X-100 totally for 96hs. The eNG were examined with paraffin section stained in Hematoxylin-Eosin .The other piece of nerve was observed with scanning electron microscope. 3. The experimental study of recellularized nerve grafts on the reconstruction of the nerve-muscle structure and functional recovery of the sciatic nerve gap:Cell suspension containing about 108/ml of SCs were micro-injected in the extracted nerve graft by a stainless needle, glass-cylinder 100ul microsyringe. The recellularized nerve grafts were composed of Schwann cell and eNG.And then transplanted it into the rabbit defective sciatic nerve quickly(experiment group).The control group did not transplant Schwann cells. Observed the ulcers on the feet of the rabbit 4,8 and 16 weeks after operation. Observed the recovery of nerve and the structure of muscle. Before the operation of the rabbits at 4th, 8th, 16th week, the electrophysiological studies were carried out .After the operation, detected the wet weight of tibialis anterior and the morphornetric studies were carried out .The electrophysiological indices included the conducted velocity of regenerated nerve and the time of incubation period. The EMG light morphometric indices included the diameter , the myelin sheath thickness of regenerating nerve fibers , the degree of the connective tissue, the structure of tibialis anterior and motor end plate. The transmission electron morphometric indices included the myelin sheath thickness of regenerating nerve fibers, the structure of the mitochondrion , the microtubule , the microfilament and schwann cells. Then analyzed them in statistics.Results: 1.SCs culture: Under operation microscope , observed and counted SCs that was gone down to posterity. The density of SCs achieved 1×108/ml .Most of cells were spindle, owed 2~3 dendrites. They had orbicular-ovate nucleus which could be seen obviously. They grew end to end and side by side just like the whirlpool or the palisade. Occasionally, we could see a few of fibroblasts that bigger and lighter than SCs. They were multi-tentacle flat and irregular, having 2~3 obvious chromatospherites. 2. eNG : The extracted nerves were ivory white without gloss. They were soft but ductile and shorter than fresh nerve. After 96hs treated by 3% Triton X-100,cells, axons and myelin sheaths were removed and only an acellular network structure left. In scanning electron microscope, the basal laminin tubes composed by collagen fibers were remained in their former position, with their former morphous and structural feature. 3. The experimental study of populating Schwann Cells into the extracted nerve graft of homogeneity in repairing of rabbit sciatic nerve: 4,8 and 16 weeks after operation, the rejection was not found in the operating field. The experimental group was better than the control group in healing of ulcers,the number of nerve fibres,the ultramicrostructure of medullary sheaths ,the regeneration nerve , the structure of the motor end plate and muscle. 4 weeks after operation, the NCV and the wet weight of tibialis anterior were not different between the two groups. But 8 and 16 weeks after operation, the experimental group was better than the control group.Conclusions: 1.We applied enzyme digestion (trypsin / collagenase) and the double 30min differential adhesion to culture Scs. The density of SCs was enough. We even added NGF to FBS to promote SCs growing. 2.Schwann cells,axons and myelin sheaths could be successfully removed after treated by Triton X-100 for 96hs. The basal tubes and collagen fibers structure remained intact. It could confirm that the bridging complex just only offered favourable supporting effects but also induced and promoted the regeneration of neuraxises and medullary sheathes.3. The acellular nerve allografts transplanted with SCs could significantly promote the reconstruction of the nerve-muscle structure and functional recovery of injured sciatic nerve.
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