| The powerful kinetic energy of various firearms in modern war can easily lead to the defect of peripheral nerves.Peripheral nerve injuries are also common in traffic accidents,tumor resection,inflammatory diseases,congenital malformations and so on.It is difficult to repair the damaged peripheral nerve,which brings heavy burden to patients and society.The peripheral nerve defect can be treated by neurorrhaphy,the prognosis is usually better as the defect is less than 0.5cm.When the nerve defect is more than 4 cm,autotransplantation is used.However,autotransplantation is limited in source and difficult in matching,and the repair effect is not satisfactory.Although the allogeneic nerve can replace the autogenous nerve to repair the nerve defect,the repair effect of the allogneic nerve is far worse than that of autogenous nerve.In recent years,the rapid development of tissue engineering nerve（TEN）technology brings new hope for the repair of nerve defects.First,it can be used as a substitute of autogenous nerve and allogeneic nerve.Second,it can repair longer nerve defects.Third,it can be"customized"according to the length and diameter of nerve defects.Although a small number of tissue engineering nerves have been used in clinic,there are still some problems such as slow nerve regeneration,poor recovery of nerve structure and function.In previous studies,scholars improved the TEN technology from the following aspects.First,the synthesis,screening of scaffold materials and the production of neural conduit.PLGA is recognized as the most ideal scaffold materials at present.Second,the selection of more suitable seed cells.ESCs,NSCs,BMSCs,ADSCs,DPSCs,SCs,OECs were choosed to be seed cells in the past.Epidermal Neural Crest Stem Cells（EPI-NCSCs）are easy to obtain,have strong ability of self-renewal,multi-directional differentiation,secreting neurotrophic factors,and have low risk of tumorigenesis.It is a more suitable new seed cell.Third,better extracellular matrix should be selected to strengthen the induction and promotion of nerve regeneration in the process of repair.At the same time,it has also been found that inflammatory immune response has a very important effect on the repair of nerve defects,which has become a hot topic in recent years.It will be helpful to explore the mechanism to break through the existing dilemma of nerve defects repair.Inflammatory immune response plays an important role in the process of tissue wound repair,and it also has a key impact on the local pathological microenvironment in the process of nerve defect repair.Inflammatory immune response is a complex network composed of immunocompetent cells and inflammatory cytokines.In recent years,people have gradually realized that macrophages are the key immunocompetent cells in this network.Macrophages can polarize to M1 type and produce neurotoxicity,and also polarize to M2 type which is conducive to neural damage repair in the process of neural defect repair,At present,people recognize that there are two main types of inflammatory factors.One is pro-inflammatory factors,such as IL-1β,IL-6,TNF-αand IFN-γ,which can aggravate inflammatory response.The other is anti-inflammatory factors,such as IL-4,IL-10,IL-13 and TGF-β,which can inhibit inflammatory response.On the basis of the method used by our research group to prepare artificial nerves,this research made a new type of TEN that was composed of the EPI-NCSCs which is a neural stem cell obtained from the automatic hair follicles,PLGA stent and ECM matrix.We observed the repair effect of the new TEN in long segment sciatic nerve（SN）defect,and studied the mechanism of its action from the regulation of inflammation microenviroment.We bridged the sciatic nerve defect,then observed the polarization state of the macrophages,the expression of inflammatory factors,the repairing effect of the structure and function of the defect sciatic nerve using different modern biological research techniques within 9weeks after bridging.The key scientific question to be solved is whether tissue engineering nerve can regulate macrophage polarization in local nerve tissue.Can new TEN regulate the expression of local inflammatory factors?Is the regulated inflammation microenvironment more conducive to the transplantation and repair of peripheral nerve defects?Materials and methods1.EPI-NCSCs culture,tisuuse engineering nerve production and sciatic nerve bridging（1）Cell culture:EPI-NCSCs were dissected out from whisker follicles GFP transgenic SD rats.The cells were cultured in DMEM/F12/FBS/bFGF medium for primary culture.The slides covered with cells were washed with PBS solution for 5 min x 3times.Cultured EPI-NCSCs were identified by rabbit anti SOX10 and mouse anti Nestin primary antibodies,visualized with Alexa Flour 594 goat anti-rabbit IgG and Cy5 goat anti-mouse IgG secondary antibodies.The cell purity was 99%as determined by the ratio of Nestin-SOX10double positive cells and GFP-cells in the same view field.（2）PLA/PGA was mixed at 85/15 ratio and dissolved in trichloromethane to prepare 5%solution.The solution was evaporated in glass Petri dish to form PLGA film with 25 um thickness,then the film was rolled into PLGA catheter,sterilized by gamma ray irradiation,and infiltrated with DMEM/F12 before using.（3）SD rats were anesthetized routinely.The right sciatic nerve was exposed,10 mm sciatic nerve was excised,and the stump retracted to form a 15 mm defect.PLGA catheter was inserted into the broken end of the nerve,25 mL EPI-NCSCs/ECM was injected,8-0suture was used to suture the broken end,the muscle and skin were sutured layer by layer.2.Regulation of macrophage polarization（1）Immunofluorescence identification:SD rats were anesthetized,the sciatic nerve was exposed and removed about 20 mm long.20 micron frozen sections were cut.The sciatic nerve was permeabilized with Triton X-100 for 15 minutes,then blocked with Goat serum for 1 hour.The sections were incubated with rabbit anti-iNOS and mouse anti-CD68（M1macrophage）,rabbit anti-arginase-1 and mouse anti-CD206（M2 macrophage）at 4℃overnight,followed by incubation with TRITC-IgG/FITC-IgG for 2 h.Cells nuclear were stained with Hoechst 3334 for 30 minutes.（2）Fibroblasts and SCs immunofluorescence:SD rats were anesthetized and the sciatic nerve was removed,20μm frozen sections were prepared and incubated with Vimentin primary antibody（fibroblasts）and S-100 primary antibody（SCs）.The secondary antibody was TRITC-IgG/FITC-IgG and cells nuclear were stained with Hoechst33342.3.Inflammatory cytokine regulation（1）Western-blot:The sciatic nerve was homogenized,the total protein was extracted and the protein concentration was quantified.Then,the proteins were subjected to SDS-PAGE gels,transferred to PVDF membrane and then blocked.The membranes were incubated overnight at 4℃with IL-4/IL-6/IL-13/TNF-α/actin primary antibody,then incubated for 2h at 37℃with goat anti-rabbit or mouse IgG horseradish peroxidase conjugated.Proteins were visualized by using the ECL procedure and the intensity of each band was expressed relative to that of actin.（2）Immunohistochemical staining:SD rats were anesthetized.The segments of sciatic nerve were removed as above and post-fixed.The segments were embedded in paraffin.Five micrometer longitudinal sections were cut,then treated with 3%H2O2 and blocked with goat serum for 1 h.The sections were incubated overnight with rabbit anti-IL-4,rabbit anti-IL-6,mouse anti-IL-13,rabbit anti-TNF-α,followed by incubation with secondary biotinylated goat anti-rabbit/mouseIgG antibody,then stained by DAB,finally counter stained with hematoxylin.（3）Immunofluorescence staining:SD rats were anesthetized,perfused and fixed.20μm frozen sections were prepared and placed in 0.3%TritonX-100 for 15 min at room temperature,then blocked with goat serum at room temperature for 1h,and sections were incubated with anti-IL-4/IL-6/IL-13/TNF-αat 4℃overnight,followed by incubation with TRITC-IgG/FITC-IgG for 2 h.The cell nuclei was stained with Hoechst33342.4.Repair of sciatic nerve defect（1）Histological observation:At 9 weeks after operation,rats were anesthetized,these gments were fixed and embedded in paraffin.4 micrometer sections were cut stained with HE,and observed by light microscopy.（2）Sports end plate inspection:At 9 weeks after operation,the gastrocnemius muscle of rats were dissected,30μm frozen sections were cut,stained with AChE,observed under light microscope,and analyzed by Image ProPlus image software.（3）Sensory function assessment:At 9 weeks after operation,rats’hind feet were immersed in hot water at 50℃.The retraction time of hind feet was recorded.The pain and temperature were expressed by LFRT.（4）Motor function evaluation:At 9 weeks after operation,the hind footprint of rats was recorded,TS,ITS and PL were measured,and SFI was calculated according to the formula.（5）Neurophysiological examination:The rats were anesthetized at 9 weeks after operation,the experimental and normal sciatic nerves were exposed and stimulated with double hook electrodes（polar space was 2 mm）,and the bipolar needle electrodes were recorded.The stimulation interval was 0.25 ms,the stimulation intensity was 10 mA,and the stimulation frequency was 1 Hz.Each rat was repeatedly recorded 5 times and the results were analyzed using PowerLab software.（6）Gastrocnemius wet weight recovery test:At 9 weeks after operation,the gastrocnemius muscle of the experimental side and the normal side were cut off and weighed by electronic balance immediately.The results was showed as:the weight of gastrocnemius muscle of the experimental side/theweight of gastrocnemius muscle of the normal side×100%.5.Statistical analysisAll values were expressed as mean±standard deviation.Two-sample t-test was used to determine the difference between the groups.SPSS（version 22.0）statistical software was used for data analysis.P values less than 0.05 was considered statistically significant.Result1.EPI-NCSCs were obtained from the follicles of GFP rats.The purity of EPI-NCSCs was 99.23±2.1%identified by SOX10 and Nestin double labels.After transplantation to the defected 10 mm sciatic nerve,EPI-NCSCs survived for at least 9 weeks in the host tissue.The surface of the PLGA vessel wall was a network structure with many micro-holes about 6μm in diameter,which provided a structural basis for EPI-NCSCs adhesion and growth,and also provided a structural guarantee for seed cells to obtain environmental nutrition.2.One week after the sciatic nerve defect bridged with TEN,M1 macrophages had a lower proportion in EPI-NCSCs group compared to control group.The proportion of M2macrophages in EPI-NCSCs group was significant higher than that of control group.At 3weeks after bridging,the number and activity of SCs in favor of nerve regeneration were significantly increased,while the number and activity of fibroblasts hindering nerve regeneration were significantly decreased.The seed cells EPI-NCSCs play a key role in these beneficial effects among the components of TEN.3.Western-blot,immunofluorescence and immunohistochemical methods confirmed that the expression of anti-inflammatory factors IL-4 and IL-13 in the host nerve tissue increased gradually,while pro-inflammatory factors IL-6 and TNF-a decreased gradually at1,3,7,14 and 21 days after the sciatic nerve defect was bridged with TEN.The increase of anti-inflammatory factors and the decrease of proin-flammatory factors provide a more suitable inflammatory immune microenvironment for nerve fiber regeneration after sciatic nerve injury.4.At 9 weeks after operation,morphological and histological observation showed that artificial nerve bridging restored the continuity of defective sciatic nerve.Compared with the DMEM group,the diameter of nerve was thicker,the structure of nerve was more compact,the composition of nerve fibers was more,the number of motor endplates was more and the volume was larger.LFRT measurements showed better recovery of sensor y function.SFI evaluation showed that the recovery level of motor function was significantly improved.Meanwhile,the recovery rate of muscle wet weight was significantly better than that of the control group.Neuroelectrophysiological examination finally confirmed that the latency was shortened and the potential amplitude was increased in EPI-NCSCs group,which indicated that nerve conduction velocity was faster and the response to stimulation was stronger.ConclusionEPI-NCSCs in the new TEN could survive in host tissues at least 9 weeks after transplantation.On the one hand,they promoted the polarization of macrophages in host tissues to M2 type and activate SCs which are beneficial to the regeneration of deity fibers;on the other hand,they reduced the number of M1 macrophages and inhibited the proliferation of fibroblasts which are not conducive to the regeneration of nerve fibers.The polarization of macrophages regulated by transplantation coincided with the expression of anti-inflammatory factors（IL-4,IL-13）and pro-inflammatory factors（IL-6,TNF-α）in host tissues.These results indicated that TEN could be able to regulate and provide more suitable inflammation microenvironment for the repair of defected sciatic nerve. |
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