Repair Of The Rat Sciatic Nerve Defect With Novel Silk Fibroin Sustained-release Carrier Nerve Conduit

Peripheral nerve injury is a common clinical disease.For the repair of nerve rupture,suture broken nerve end-to-end can be performed.For the repair of nerve defect with long distance,it is necessary to bridge the nerve graft between the end of the broken end.The use of autologous nerve transplantation is still the "gold standard" for nerve defect repair in clinically.However there are many limitations of autologous nerve transplantation such as the shortage of donor nerve,causing donor site morbidity and so on.At present,nerve conduits made of natural or synthetic materials have become the effective substitutes to autologous nerve transplantation for peripheral nerve defect repair,but their repair effects still need to be improved.Studies have shown that longitudinally oriented microchannel structure set inside the conduit using silk fibroin to simulate normal nerve fibers distribution is conducive to guiding axon regeneration.Silk fibroin has the similar structural characteristics with autologous nerve.In order to further improve the repair effect of nerve conduit,on the basis of constructing silk fibroin microchannels,exogenous addition of fibronectin and N-cadherin promotes the proliferation and migration of Schwann cells,so that reconstruction microenvironment is beneficial to nerve regeneration.Because protein drugs generally have short half-life and are easy to lose biological activity,the protein drug sustained-release carrier is constructed by compounding calcium alginate on the surface of silk fibroin,which can maintain the activity of drugs and prolong their action time.In this study,three types of novel silk fibroin sustained-release carrier nerve conduits were constructed,namely fibronectin silk fibroin sustained-release carrier nerve conduit(FN/SF NC),N-cadherin silk fibroin sustained-release carrier nerve conduit(N-Cad/SF NC),complex protein silk fibroin sustained-releas carrier nerve conduit(FN&N-Cad/SF NC).In order to investigate the repair effect of the novel silk fibroin sustained-release carrier nerve conduit,we used autograft and pure silk fibroin nerve conduit(SF NC)as control groups to bridge the 10 mm defect gap of the sciatic nerve in rats.The specific research contents and results of this article are summarized as follows:(1)Characterization of silk fibroin sustained-release carrier in vitro.Enzyme-linked immunosorbent assay(ELISA)results showed that the silk fibroin sustained-release carrier can release fibronectin and N-cadherin for 10 days.The release curves of fibronectin and N-cadherin were about the same.A small amount of the drug released from the 1st day to the 2nd day.The drug released rapidly from the 2nd day to the 7th day.The accumulative release of drug increased linearly and continuously.The release rate of the drug significantly slowed down from the 7th day to the 10 th day.The accumulative release of the durg kept stable basically from the 10 th day to the 12 th day.The final accumulative release of fibronectin was 0.95 ?g and N-cadherin was 0.92 ?g.Schwann cells were co-cultured with silk fibroin sustained-releas carrier.The results of CCK-8 showed that FN/SF,N-Cad/SF,FN&N-Cad/SF all had good biocompatibility without affecting cell proliferation activity.After 3 days,Schwann cells showed significant proliferation behavior in the FN/SF group and the FN&N-Cad/SF group compared with the control group.(2)Evaluation the repair effect of silk fibroin sustained-release carrier nerve conduit.After nerve transplantation,the sensory function and motor function of rats were tested.The results showed that at 12 weeks after surgery,the withdrawal time of FN&N-Cad/SF NC group was not significantly different from that of autograft group,but significantly lower than SF NC group,FN/SF NC group,N-Cad/SF NC group.The value of SFI in autograft group was not significantly different from FN/SF NC group,N-Cad/SF NC group,FN&N-Cad/SF NC group,but significantly higher than SF NC group.Gastrocnemius wet weight measurement and Masson staining showed that at 12 weeks after surgery.The gastrocnemius wet weight rate of FN&N-Cad/SF NC groupwas not significantly different from the autograft group,but it was significantly higher than SF NC group,FN/SF NC group,N-Cad/SF NC group.The collagen positive area percentage in the autograft group was significantly lower than that in four nerve conduit groups.However,the collagen positive area percentage in the FN/SF NC group,N-Cad/SF NC group,and FN&N-Cad/SF NC group was significantly lower than that in the SF NC group.Histological staining and immunofluorescence staining showed that FN/SF NC group,N-Cad/SF NC group,FN&N-Cad/SF NC group can significantly promote Schwann cell migration and proliferation compared with SF NC group in the first 4weeks after surgery.At 12 weeks,the number of neonatal axons was significantly increased in the FN&N-Cad/SF NC group compared with the SF NC group,and the difference was not significant compared with the FN/SF NC group and the N-Cad/SF NC group.(3)Preliminary inquiry on the repair mechanism of silk fibroin sustained-release carrier nerve conduit.At 14 th day after surgery,FN/SF NC group,N-Cad/SF NC group,FN&N-Cad/SF NC group could up-regulate the expression of Schwann cell proliferation-related genes(Cyclin D1,PCNA),migration-related genes(NCAM,Itgb1)and nerve growth factor(NGF).Among them,FN&N-Cad/SF NC group is more effective.From the 21 st day to the 30 th day,the relative expression level of each gene in every group returned to normal.In summary,the three types of novel silk fibroin sustained-release carrier nerve conduits prepared in this study have good drug loading and biocompatibility.Compared with SF NC group,the repair effect is improved.Among them,FN&N-Cad/SF NC group is the best,and there is no significant difference compared with autograft group.The FN/SF NC group and N-Cad/SF NC group are next.This means that the exogenous addition of fibronectin and N-cadherin can effectively promote the proliferation and migration of Schwann cells and the release of nerve growth factor,thereby the repair effect of nerve conduits is improved.