The Research Of Regenerated Electrospun Silk Fibroin Nanofibers As The Role For Facial Nerve Repair

Objective: To research whether regenerated electrospun silk fibroin nanofibers(SF)as never conduits could improve nerve regeneration microenvironment and induce thefacial never regeneration of SD rats or not. Its feasibility as a kind of novel graft forassisting the regeneration process of defects in the facial never was explored in order toprovide experimental evidence for clinical use.Methods:Firstly, fabrication of silk fibroin nanofibrous scaffolds:the nanofibershad a smooth surface with solid voids among the fibers, interconnecting a porousnetwork, constituting a fibriform three-dimensional structure and that the averagediameter of the fibers was about305±84nm; Secondly, preparation of SF nanofibergrafts: the SF nanofiber tubes had a bilayered structure with aligned nanofibers in theinner layer, and random nanofibers in the outer layer.The inner diameter is1.5mm and0.2mm thickness.Last, both were immersed in75%ethanol,dried in vacuum at roomtemperature,sterilized by radiation prior to use.1. In vitro:Schwanns cell (SCs) culture:the rat dorsal root ganglia (DRG) of neonatal SD rats with1–2d old,stripped in Hank'sbalanced salt solution, at the density of1.0×105cells/well seeded on the pre-wetted SFand poly-L-lysine(PLL)-coated cover slips in35mm Petri dishes respectively aftertrypsinized. Schwanns cell adhesion, growth and immigration were observed underinverted light microscopy, fluorescence microscope and scanning electronmicrograph(SEM). Schwanns cell proliferation viability examination by MTT and cellprotein expression examination(ELISA) about neurotrophic factors (CNTF,BDNF,NGF)were analysed.2. In vivo: Set up the animal surgery models: the facial nerve transectedleaving a5-mm long defect was bridged with SF graft or autograft in SD rats.Three months after implantation, morphological and functional evaluation that includedelectrophysiology, histology, Fluorogold retrograde tracing and transmission electronmicrographs were carried through in terms of nerve repair.Results:1. From SEM, light and fluorescence microscopy we observed longer cellprotrusions and network formation of SCs on the random electrospun SF. SCs formed amore effective and complex interconnecting network through the longer neurites. Incontrast, in the absence of the topographical guide of the scaffold, SCs cultured on PLLadopted an unorganized, disordered morphology with a random orientation. A greaternumber of cells on SF extended tightly along the silk scaffolds forming longer cell chainsthan on PLL which is the basis of regenerating axons across a nerve gap graft to a distalaxon. On the other hand,according to the results of MTT and ELISA, this material did notexert any significant cytotoxic effects on their phenotype and had no any negativeinfluence on expression of neurotrophic factors at protein level.2. In vivo:The effects ofnerve regeneration were observed by a range of morphological and functional assessments.There were no distinct regional inflammation response and scar formation in the rats in theSF graft group over a3-month period after implantation, similar changes were observed inthe autograft group. At3months after implantation, the SF graft had disappeared due todegradation, and the original5-mm long nerve defect was replaced with a tissue that had anerve-like appearance between both stumps. CMAPs amplitude has decreased significantlywhen compared with the normal CMAPs amplitude value recorded at the contralateralunoperated side, there was no significant difference between the SF graft and autograftgroups. The FG retrograde tracings were found in the facial nucleus ipsilateral to theoperated side of the rats in the SF graft and autograft groups. Moreover, statistical analysisshowed that there was no significant difference in the morphometric data, including theaverage thickness of regenerated myelin sheaths, the mean diameter of myelinated fibers,the number of retrograde-labeled motoneurons and the CMAPs amplitudes between thesilk fibroin graft and autograft groups(P>0.05).Conclusions: Regenerated electrospun silk fibroin nanofibers was beneficial to the adherence, proliferation and migration of SCs. The distribution of nanofibrous scaffoldscould serve as a suitable substrate for cell spreading,neurites extending, which offered thenerve regeneration channels under the topographical guide of the scaffold.The SF tubecould induce the nerve stump across the facial never gap defect, and regenerated nervefibers in SF group succeeded in reconnection. Hence regenerated electrospun silk fibroinnanofibers could replace nerve autografts to facilitate nerve repair following facial nerveinjury and maybe become a promising alternative in repairing peripheral neural structureand function. It was of potential value in clinical applications.