| Objective: To plant fetus rabbit Schwann cells that were cultured and purified in vitro into the chemical extracted acellular 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 the condition of nerve regeneration and the change of immunological cells around allograftst. 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, its immunogenicity is extraordinary low. 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.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 chromatospherites. 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, to shear the adipose tissue and part of the epineurium with the operating by microscope. To divid 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. 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: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. The bridging complex 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 infiltrating of immunological cells in the muscle tissue around allografts of the rabbit after 1,4 and 8 weeks, further, counted the amount of immunologic cell in every high power field. Observed the ulcers on the feet of the rabbit 4,8 and 16 weeks after operation.To observe sciatic nerve regeneration and myelination by light microscope. To observe the information of nerve regeneration and myelination and the ultrastructural change of the mitochondrion , the microtubule , the microfilament and Schwann cells by electron microscope. Making use of the analytical system on picture to treat the number of regenerated nerve fiber and the axon diameter and the thickness of myelin sheath of medullated nerve fibers. To detected the wet weight of the triceps surae. Then analyzed them in statistics.Results: The experimental study of populating Schwann Cells into the extracted nerve graft of homogeneity in repairing of rabbit sciatic nerve: After operation, the immunological rejection was not found in the operating field. There are lots of lympholeukocytes and macrophages in the muscle tissue around the transplanted sciatic nerve after 1 week, There are obviously more lympholeukocytes and macrophages in the experimental group than the control group. They decrease after 4 weeks in two groups, they decrease more seriously in the experimental group.Obviously decrease after 8 weeks. There is significant difference between the experimental group and the control group after 1 week, but, there isn't after 4 weeks and 8 weeks.The experimental group was better than the control group in healing of ulcers,the number of regenerated nerve fiber and the axon diameter and the thickness of myelin sheath of medullated nerve fibers. After 4 weeks, the wet weight of the triceps surae were not different between the two groups. But after 8 and 16 weeks, 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. Moreover, Its immunogenicity is extraordinary low. The acellular nerve allografts transplanted with SCs could significantly promote nerve regeneration and functional recovery of injured sciatic nerve. |
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