The Effects Of Epidermal Neural Crest Stem Cells And Olfactory Ensheathing Cells Co-transplanted On Local Inflammation Microenvironment In The Defected Sciatic Nerve Of Rats

It has been a difficult neurosurgery problem for repairing the structure and function of the defected sciatic nerve for a long time.Usually caused by traffic accidents,mining accidents and gun injury,the site of injury is mostly broken or defective.So surgical anastomosis is difficult and often need nerve transplantation repair.However,the repair effect of peripheral nerve defected is not satisfactory.It is not only because of the highly differentiated neural tissue,peripheral nerve fiber regeneration slowly and difficultly to form synaptic connections with target tissue,inflammatory/immune response after peripheral nerve injury may also has an important influence.The inflammatory/ immune regulation of peripheral nerve mainly includes the secretion of inflammatory factors and the infiltration of inflammatory cells.Therefore,it is critical to understand and maintain the dynamic balance of inflammatory factor secretion and activation of inflammatory cells.Tissue engineering makes cells transplantation for the treatment of nerve injury has become a hot research topic in recent years.Epidermal neural crest stem cells(EPI-NCSCs)can secrete many neurotrophic factors,angiogenesis factors and candifferentiate into neurons,astrocytes and oligodendrocyte cells.Olfactory ensheathing cells(OECs)can secrete various neurotrophic factors(NT-3/4/5,BDNF,GDNF,CNTF,NGF),the expression of inflammatory proteins(S-100,Nexin,glial derived connexin)to promote angiogenesis and myelin regeneration,and stimulate the guidance of regenerated axon.At present,these two kinds of cells have good application prospects in tissue engineering,which were used in the repair of spinal cord and sciatic nerve injury.Polylactic acid polyglycolic acid copolymer(PLGA)has a good biodegradability,is widely used in the research of the artificial nerve conduit.Extracellular matrix(ECM)contains laminin and collagen to promote cell adhesion and growth.Therefore,the PLGA conduit placed the ECM,which is conducive to the survival and growth of the seed cells in the conduit.In order to investigate the inflammatory/immune role of EPI-NCSCs and OECs co-transplanted in the repair of peripheral nerve defect in rats.The primary culture of EPI-NCSCs,OECs and EPI-NCSCs+OECs as seed cells,mixed the equal amount of ECM,filled with a PLGA conduit,the control group with equal Dulbecco's Modified Eagl's medium(DMEM)instead of cells,used to repair the right sciatic nerve with a 10 mm defect.Compared with the DMEM groups,the Co-trans group showed significantly increased the expression level of IL-4 at 1-3 weeks,and the expression of IL-4 in the Co-trans group was significantly higher than those in the single cells transplantation group at 1,2 and 3 weeks;the Co-trans group showed significantly decreased the expression level of IL-6 at 3 days and 1 week,and the relative expression of IL-6 in the Co-trans group were significantly lower than those of single cells transplantation group at 1 day and 1 week;the Co-trans group showed significantly increased the expression level of IL-13 at 1 day,1 week and 3 weeks,and the relative expression of IL-13 in the Co-trans group was significantly higher than EPI-NCSCs group at 3 weeks;the Co-trans group showed significantly decreased the expression level of TNF-? from 1 day to 3weeks,and the relative expression of TNF-? in the Co-trans group was significantly lower than OECs group at 3 weeks by western blot detection.Immunohistochemistry and immunofluorescence staining showed that the expression of anti-inflammatory cytokines(IL-4 and IL-13)was increased,but decreased for the expression of pro-inflammatory cytokines(IL-6 and TNF-?)compared to the DMEM group,which was consistent with increase of M2 macrophages and decrease of M1 macrophages at 1week after transplantation.Likewise,the activated fibroblasts was significantly decreased for in their number at 3 weeks.In vivo,transplanted green fluorescence cellswere round and spindle morphology,which survived with wide distribution in the nerve stump and the regenerated nerves at least for 9 weeks after bridging.The recovery of the structure and function of the nerve in the Co-trans group was significant superior to the other three groups by HE staining under light microscope and by examination of right lateral foot retraction time,sciatic function index(SFI),recovery rate of gastrocnemius wet weight and electrophysiology at 9 weeks.These results indicated that EPI-NCSCs and OECs co-transplanted could regulate and provide more suitable inflammation and regeneration microenvironment for repair the structure and function of the defected sciatic nerve.