An Experimental Study On Repair Of Peripheral Nerve Defects With Chitosan-collagen Film And Activated Schwann Cell

Introduction:The effective repair of the peripheral nerve gap following injury continues to be a considerable clinical challenge. Peripheral nerve autograft is nearly clinically sole selection of repairing the gap. That has inevitable disadvantages, such as limited supply of available nerve graft material and permanent loss of the donor nerve function. An attractive to autografts seems to be artificial nerve with tissue engineering. The aim to this study was to develop a novel effective substitute composed of activated Schwann cells and chitosan-collagen film to repair peripheral nerve gap. This study includes five major parts.Part one Modified Schwann cell culture from adult ratsObjective: To introduce a modified technique of explants culture to obtain highly purified Schwann cells.Methods: 20 adult SD rats were randomly divided into 2 groups, group A for modified explants culture and group B for tranditional explants culture. The sciatic nerve was dissected bilaterally through a dorsal incision and collected in DMEM. The epineurium was gently stripped and nerve fascicles were subsequently cut in 0.5mm~3 segments. Group A: nerve segments were digested in 0.03% collagenase for 20min before translation to Petri dish. Grou B: nerve segments were transplanted to Petri dish directly. The individual nerve explants were transplanted to a new dish every 5 days, totally 3times. The cell number were counted separately and compared. Schwann cell purity was determined by immunostainning the cells for S-100.Results Cell population in group A was several folds to that in group B. Accordingly Schwann cell purity in group A was also higher than group B. In modified culture, the cell obtained from the first time explants culture were mainly fibrocytes. It contributed to remove fibrocytes from explants. Schwanncell purity reached 85% in the second time explants culture by modified methods. And Schwann cell purity was 95% after third replantation with modified method, while explants were disappeared and fully used.Conclusion: Modified explants culture is a quick way to obtain highly purified Schwann cells.Part two Activated Schwann cell culture and growth ruleObjective: To culture activated SC from predegenerated nerve by addition of activated liquid and explore the growing rule of the activated SC in culture.Methods: 10 male SD rats, weight 200g～250g. Activated SC culture:The right sciatic nerve of SD rats were transected for predegeneration. 1 week later, the distal segment of the transected right sciatic nerve was harvest. The epineurium was gently stripped and each nerve was weighted. Nerve segments were digested in 0.03% collagenase and active liquid (0.1ml/ml) for 20 min according to modified explant culture. Normal SC culture: The untreated left sciatic served as control. The epineurium was gently stripped and each nerve was weighted. Nerve segments were digested in 0.03% collagenase without of active liquid for 20 min according to modified explant culture. Cell number per mg was compared. 1×10~5 cells of second generation were planted on 35 petri dish. The numbers of activated SCs and normal SCs were counted at various growing point (2d,4d,6d,8d,10d,12d,14d), then the growing curve was drawn. Doubling time was also calculated. At the 8 day, protein was extracted after cell number counting. CDK1 was measured by western blot. Schwann cell purity was determined by immunostainning the cells for S-100 at 1 and 14 days.Results: Acivated SC was 7. 5×10~4/mg from pregenerated nerve after modified explant culture. Normal SC was 1.5×10~4/mg from normal nerve. After subcultivation, activated SC amounts were more than normal SC in various point except first 2 days. The doubling period of activated SC was 5 days, while normal SC was 7days. Activated SC proliferates faster than normal SC. The CDK1 expression was also higher in activated SC than in normal SC. At 14 days onculture, purity of activated SC was 95% and normal SC was 92%.Conclusion: A large mount of activated SC can be obtained from predegenerated nerve. Activated SC proliferate faster than normal SC. it may be partly explained by CDK1 over-expression in activated SC.Part three Gene expression of BDNF, GAP43, c-Jun and p21 in activated Schwann cellObjective: To compare activated Schwann cell and normal Schwann cell in BDNF, GAP43, c-Jun and p21 gene expression.Methods: 10 male SD rats, weight 200g～250g. The right sciatic nerves of SD rats were transected for predegeneration. 1 week later, the distal segment of the transected right sciatic nerve was harvest. The untreated left sciatic nerve was harvested as control. Activated schwann cell were cultured from predegenerated nerve and normal Schwann cells were harvest from contralateral norm sciatci nerve. rt-PCR was employed for gene enlargement. mRNA was distilled from activated Schwann cells and Schwann cells respectively. Then the mRNA was reverse transcript to cDNA with SuperScriptTM, and cDNA worked as template for PCR enlargement. The product of PCR was separated with 1% agarose gel electrophoresis for 40min～50min. PCR products of GAP43,BDNF, C-JUN,P21 was measured and then compared between the experiment group and control group.Results:GAP43, C-JUN,P21 mRNA of activate Schwann cells are much more than that of the normal Schwann cells. BDNF mRNA was manifest in activated Schwann cells, however it was not showed in normal schwann cells.Conclusion1. BDNF may act as a marker for activated schwann cell.2. GAP43,BDNF,C-JUN,P21 gene expression is up regulated in Activated Schwann in contrast to normal Schwann cell. It may partly elucidate why activated Schwann cells proliferate quickly and promote nerve regeneration.Part four Growth rule of activated Schwann cells on Chitosan-collagen filmObjective: To explore the growth rule of activated SC cultured on the surface of Chitosan-collagen film.Methods: Adult SD rat sciatic nerves were transected and predegenerated for 7 days. Activated SCs were harvested by modified piece replant way. 1×10~5 cells were planted on the surface of Chitosan-collagen film. 1×10~5 cells were planted on petri dish severed as control. Activated SCs were observed through the phase contrast microscope. The numbers of activated Schwann cells were counted at various growing point (2d,4d,6d,8d,10d,12d,14d) , then the growing curve of activated Schwann cells was drawn. Doubling time was also calculated. Scanning electron microscope was employed to observe cell growth on chitosan-collagen film at 3d,7d,10d.Results: Activated SCs grow well on the surface of the Chitosan-collagen film under observation of phase contrast microscope and scanning electron microscope. Cell amount reached were up to 10×10~5 on Chitosan- collagen film and 7×10~5 petri dish separately after 2 weeks compared to the primary 1× 10~5. The doubling period of activated SCs was 4 days on chitosan-collagen film and 5 days on Petri dish. Activated proliferated faster on chitosan-collagen film than on Petri dish.Conclusions: Chitosan-collagen film posses an ability to promote activated SCs proliferation. The fine affinity made it possible to transplant activated SCs by chitosan-collagen film.Part five An experimental study on repair of peripheral nerve defects with Chitosan-collagen film and activated Schwann cellObjective: To study the efficiency of artificial nerve made of Chitosan-collagen and activated Schwann cells on promoting nerveregeneration.Methods: Activiated Schwann cells and normal Schwann cells from left sciaticnerve were cultured on Chitosan-collagen film. Then the films were sutured intoconduits to bridge 10mm defect of right sciatic nerve. Autograft and complainChitosan-collagen film were served as control. General observation, amplitudeof active potential (AMP) and nerve conduction velocity (NCV) were measuredat the end of 4, 8, 12 weeks. Evaluation included triceps surae measurementand histological examination was made at the end of 12 weeks. The myelinatednerve fibers density, axon area percentage and thickness of mylin sheath weremeasured by computerized image analysis.Results: There was no differenc between the group of (Chitosan-collagen withactivated Schwann cells) and the group of autograft in electromyogrphicexamination, triceps surae measurement and axon regeneration. Whereas thegroup of activated Schwann cells got better results than the normal Schwanncells. The group of Chitosan-collagen film without Schwann cell showed a poorresult of axon regeneration.Conclusion: Chitosan-collagen and activated Schwann cells can effectivelypromote nerve regeneration. It provides a novel method for repairing theperipheral nerve defects. Additionally, activated Schwann cell is better thannormal Schwann cells in encouraging nerve regeneration.