The Experimental Study Of Populating Schwann Cells Into The Extracted Nerve Graft Of Homogeneity In Repairing Of Rabbit Sciatic Nerve

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. Observed the regeneration and the functional recovery of rabbit sciatic nerves. 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 populating Schwann Cells into the extracted nerve graft of homogeneity in repairing of rabbit sciatic nerve: Cell suspension containing about 10~8/ml of SCs were micro-injected in the extracted nerve graft by a stainless needle, glass-cylinder 100ul microsyringe. 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 12 weeks after operation. Detected the regeneration of neuraxises and medullary sheathes of rabbit sciatic nerves by EMG light microscope and electron microscope and so on .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 1×10~8/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 chromatosph- erites. 2. eNG : The extracted nerves were ivory white without gloss. They were soft but ductile and shorter than fresh nerve. After 96h 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 12 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 and the regene- ration nerve. 4 weeks after operation, the NCV were not different between the two groups. But 8 and 12 weeks after operation, the experimental group was better than the control group.Conclusions: 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. 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. The acellular nerve allografts transplanted with SCs could significantly promote the regeneration of injured sciatic nerve.