Preparation Of New Nerve Guidance Conduit For Peripheral Nerve Regeneration

Peripheral nerve tissue has complex structure and function,and peripheral nerve defects has the disadvantages of high disability rate,poor prognosis and complex treatment,which is the most serious problem in clinical.Traditional autologous transplantation and allogeneic are still the common treatment for peripheral nerve defects.However,there some limitations of traditional treatments to limit their application.The development of nerve tissue engineering provides a new direction for peripheral nerve defects.Currently,the research in the field of nerve tissue engineering focuses on scaffold materials,scaffold structure,nerve cells,nerve growth factors,electrical stimulation sginal and so on.In this study,three kinds of new nerve guidance conduit(NGC)were prepared from the three factors of tissue engineering: scaffold,cell and growth factor.First of all,the polypyrrole(Ppy)polymer,which has good biocompatibility and conductivity,it was coated on electrospun poly(L-lactic acid-co-e-caprolactone)/silk fibroin(PLCL/SF)nanofibers to obtain Ppycoated NGC.It was achieving the innovation in sacffod materials.And it was combined with electrical stimulation for in vitro study.Then,in order to imitate the structure of endoneurium for scaffold structure innovation,the nanofiber sponges were used as filler to prepare nanofiber sponges-containig(NS-containing)NGC.Finally,the polydopamine nanospheres(PDA NPs)were used to load the nerve growth factor(NGF)to prepare composite hydrogel fiber-containig NGC,which has good biocompatibility and NGF delibery carrier ability.It realized the innovation of growth factor and scaffold structure.Spectifically,the major contents are presented as follows:(1)Ppy-coated nanofiber membranes were obtained by coating Ppy on PLCL/SF(75/25,w/w)nanofiber membarnes using in situ polymerization and electrospinning technique,with pyrrole as monomers.Three kinds of Ppy-coated nanofiber membranes(PLCL/SF-Ppy-1,PLCL/SF-Ppy-2 and PLCL/SF-Ppy-3)were prepared by in situ polymerization with three different concentrations of pyrrole monomers.Scanning electron microscopy(SEM),atomic force microscopy(AFM)and Fourier transform infrared spectroscopy(FTIR)confirmed that Ppy was successfully coated on the surface of PLCL/SF nanofibers.Thermal stability,degradation stability,electrical properties,mechanical properties and hydrophilicity properties of three Ppy-coated nanofiber membranes were characterized.The results show that the Ppy-coated membranes have good thermal stability,degradation stability,and good conductive properties.It also proved that the Ppy coating improves the modulus and hydrophilicity of PLCL/SF nanofiber membrane,and this effect was increased with the increase of pyrrole concentration.Combined with electrical stimulation,Schwann(SC)cells and PC12 cells were cultured on three kinds of Ppy-caoted nanofiber membrane,it was found that the Ppy coating can significantly promote SC cell proliferation and PC12 cell differentiation,and this promotion effect was also increased with the increase of pyrrole concentration.Therefore,the above results indicate that PLCL/SF-Ppy-3 nanofiber membrane has better potential for nerve tissue engineering application.Furthermore,the Ppy-caoted NGC was fabricated and used to bridge 10 mm length rat sciatic nerve defect in vivo.Histological staining,immunohistochemistry,immunofluorescence and transmission electron microscopy(TEM)results show that polypyrrole coating can significantly promote nerve cells proliferation,axons and myelin regeneration.The analysis of the sciatic nerve function index(SEI)showed that Ppy-caoted NGC could promote the recovery of nerve function,and close to that of autologous transplantation group.Therefore,Ppycoated NGC have good potential in the application of nerve tissue engineering.(2)In this section,PLCL/SF(20/80,w/w)nanofibers were prepared by electrospinning at first,and then the nanofibers were immersed into tert-butanol solution to disperse using high speed homogenization technology,eventually the nanofiber sponges were obtained by freeze-drying.SEM results show that the nanofiber sponge was composed of nanofibers.The porosity of nanofiber sponges is 88.81 + 1.13% and the pore size distribution is from 0 to 300 μm.The cyclic compression test showed that the nanofiber sponge had good anti compression elasticity.The nanofiber sponges were used as filler to prepare NS-containing NGC,in vitro study showed that SC cell could infiltrate 1.5-2.0 mm into NS-containing NGC after cultering for 7 days.It was indicated that the NScaotinign NGC has a large pore structure which could promote the growth and adhesion of SC cells.After transplantation surgery,SFI value,Masson staining and triceps surae muscle(TSM)wet weight ratio stastical results showed that the nerve function recovery in the three groups was: autologous group > NS-containing NGC group > Hollow NGC group.Similarly,histological staining,immunofluorescence,immunohistochemistry and TEM results showed the same results.It was found that the NS-caontaining NGC could provide more space for SC cells adhesion and proliferation,which promote axons,myelin regeneration,and nerve regeneration.Therefore,the scaffold can promote the regeneration of nerve and the recovery of nerve function,and it has potential application in nerve tissue engineering.(3)Polydopamine nanospheres(PDA NPs)were prepared by oxidative polymerization using dopamine as monomer.Response surface methodology was used to optimize the reaction conditions for the preparation of PDA NPs.The optimum reaction conditions were as follows: dopamine concentration was 10 mg/mL,pH value was 9,reaction time was 9 h.The particle size of PDA NPs was 324.2 + 13.9 nm by SEM and laser particle size gauge characterization,and the particle size distribution was uniform(PDI value was 0.231).FTIR and Raman spectroscopy(RS)results showed that PDA NPs was successfully synthesized.PDA NPs were set as carrier,carbodiimide/Nhydroxysuccinimide(EDC/NHS)used to grafted NGF on the surface of PDA NPs,to obtain NGF@PDA NPs.The results of SEM and laser particle size analyzer showed that the grafting of NGF did not affect the particle size and particle size distribution of PDA NPs.The results of FTIR and RS confirmed that NGF was successfully grafted on the surface of PDA NPs.In addition,the in vitro release experiment and MTT results showed that the NGF on the surface of NGF@PDA NPs could be released slowly,and its biological activity could be maintained to promote the proliferation of SC cells.Combined with three-dimensional(3D)printing technology,NGF@PDA NPs was mixed with gelatin / hyaluronic acid hydrogel,and then printed the composite hydrogel fiber.Then,the composite hydrogel fiber was filled into the hollow PLCL/SF(75/25,w/w)nanofiber NGC,to obtained the composite hydrogel fiber-containing NGC.In vitro degradation experiments showed that the hydrogel fibers had good biodegradability.In vitro MTT experiment showed that the composite hydrogel fiber-containing NGC with sustained release of NGF could not only have good biocompatibility,but also showed the ability to promote the proliferation of SC cells.therefore,the obtained results demonstrated that the fabricated of sustained release NGF which mediated with PDA NPs composite hydrogel fiber-containing NGC is a promising biomaterial for peripheral nerve defect.In conclusion,this study prepared three kinds of new NGC by combining the current research hotspots,and the application of these NGCs in peripheral nerve defects was investigated both in vivo and in vitro.This study provides theoretical guidance for the application of nerve tissue engineering in peripheral nerve defects.