| Repair of nerve tissue defects and injuries is a challenge in clinical treatment today.The use of nerve tissue engineering scaffold to guide nerve regeneration is an effective alternative to autologous transplantation in this field.Due to the promoting effect of electrical stimulation(ES)on nerve regeneration,electroactive nerve tissue engineering scaffolds exhibit a good potential for nerve repair.The excellent properties,such as biacompability and electroconductive property of regenerated silk fibroin(RSF)and poly(3,4-ethylenedioxythiophene)(PEDOT)make RSF/PEDOT materials to be good candidates for electroactive nerve tissue engineering scaffolds with outstanding properties and unique advantages.However,few research was reported in this field until now.In this study,RSF and PEDOT and its derivative materials were respectively selected as the matrix material and conductive functional materials to fabricate conductive biomaterials for nerve repair.A modified chemically oxidative polymerization and deposition method and a novel method of macromolecular embedding were proposed.As a result,new types of conductive RSF/PEDOT film with good electroconductive property,transparency,and adhension property between functional body and matrix were fabricated.The effects and mechanisms of various process conditions on the prepared conductive films were studied.And the application potential of the conductive films in nerve tissue engineering was demonstrated by in vitro culture of rat pheochromocytoma cells(PC12cells)on the films.On this basis,a conductive RSF/poly(hydroxymethyl-3,4-ethylenedioxythiophene)(PEDOT-OH)scaffold with microfluidic channels was prepared.A culture method combined dynamic perfusion and ES was carried out to test the feasibility of conductive RSF/PEDOT-OH microfluidic scaffold for PC12 culture in vitro.This study provided a reference for researches and applications of RSF/PEDOT electroactive nerve tissue engineering scaffolds.At the beginning,hydroxymethyl-3,4-ethylenedioxythiophene(EDOT-OH)had been polymerized and deposited on the surface of RSF film as ammonium persulfate(APS)was used as oxidant.In order to improve the efficiency of this reaction,sodium dodecyl sulfate(SDS)was adopted as surfactant to construct a well-organized and stable poly(hydroxymethyl-3,4-ethylenedioxythiophene(PEDOT-OH)coating on RSF film surface and a conductive RSF/PEDOT-OH film was fabricated.The effects of dosages of surfactant and oxidant,initial p H value and monomer concentration on the structure and properties of the RSF/PEDOT-OH film were studied.Under the optimal condition,the RSF/PEDOT-OH film exhibited a sheet resistance(Rs)of 3.28×105?/sq corresponding conductivity of 6.1×10-3 S/cm.And favorable electrochemical stability,structural stablility of conductive coating and hydrophilicity of the produced film were demonstrated.In addition,PC12 cells cultured on the RSF/PEDOT-OH film had a good behavior and performed better cell viability than that on pure RSF film,which indicated the biocompatibility of the RSF/PEDOT-OH film.In order to further improve the electroconductive property of RSF/PEDOT-OH film and make it transparent,Fe Cl3 was used to replace APS as the oxidant.Because of the complex between SDS and Fe Cl3,the electroconductive property of RSF/PEDOT-OH film was improved.To overcome the trade-off between transparency and electroconductive property of the PEDOT-OH coating,a composite oxidants recipe of Fe Cl3 and APS was developed.Due to Fe3+regeneration through APS and the electrostatic interaction from oppositely charged doping ions in PEDOT-OH,structural defects and excessive deposition of the conductive coating were both avoided.As a result,a well-organized conductive nanoscale coating formed and a transparent conductive RSF/PEDOT-OH film was produced.Whereas,the coating was hardly achieved in traditional single oxidant system.Under the optimal condition,the produced film performed a Rs of 5.12×104?/sq corresponding to conductivity of 8.9×10-2 S/cm,and a transmittance above 70%at maximum in the visible range.Favorable electrochemical stability,adhesion property between PEDOT-OH coating and RSF film and hydrophility of the film were demonstrated.Furthermore,the transparent conductive RSF/PEDOT-OH film provided a supportive environment for adhesion,growth,and differentiation of PC12 cells.The feasibility of real-time observation and ES of PC12 cells on the RSF/PEDOT-OH film were demonstrated.In order to prepare a modified RSF film with superior electroconductive property and transparency than the RSF/PEDOT-OH film.An one-step facile PEDOT:PSS modification approach for RSF film based on comformation transformation and embedding of macromolecules was developed and a transparent conductive RSF/PEDOT:PSS film was prepared.During the immersion treatment of RSF fillm in the PEDOT:PSS aqueous dispersion/ethanol mixture system,PEDOT:PSS was successfully embedded into the shallow surface of RSF film,and a tightly conjunct conductive layer on the film surface was formed based on the conformation transformation of RSF.The prepared films had the best comprehensive performance with 70 vol.%ethanol proportion in formula,which presented a sheet resistance of 3.833×103?/sq corresponding to conductivity of1.003 S/cm,and a transmittance over 80%at maximum in the visible range.The outstanding properties allow the further modification by electropolymerization and deposition on the film surface and applications such as cell photostimulation.Favorable electrochemical stability and adhesion property between PEDOT:PSS coating and RSF film were demonstrated.In addition,the real-time observation of PC12 cells and an effective ES-cell response based on the transparent conductive layer were demonstrated.In order to fabricate conductive 3D RSF scaffold,a RSF microfluidic scaffold was fabricated through soft lithography,molding,and encapsulating process.The widths of the five sections of the RSF microfluidic channel were 1750?m,885?m,720?m,630?m and 520?m,respectively,and the heights were all 250?m.Microchannels in the scaffold were modified through the chemically oxidative polymeriazation and deposition of PEDOT-OH,while the composite oxidants recipe was adoped in presence of SDS.A conductive RSF/PEDOT-OH microfluidic scaffold was successfully achieved,with resistance of 2.35×105?between two ends in wet state.Compared to the pure RSF microfluidic scaffold,the RSF/PEDOT-OH scaffold provided a better support to the adhesion of PC12 cells.With pre-differentiation treatment of the nerve growth factor(NGF)and dynamic microfluidic perfusion culture in the RSF/PEDOT-OH microfluidic scaffold,an obvious differentiation phenomenon of PC12 cells was observed.The real-time observation of PC12 cells and an effective ES-cell response were demonstrated in the transparent conductive microfluidic scaffold.With stimulation of 100 m V DC signal for two days(2.5 h per day),the statistic average number of axons in PC12 cells increased.In this study,4.57×10-3 Pa was evaluated as the critical shear stress to allow a good growth of PC12 cells,which reflected the adhesion force between PC12cells and the RSF/PEDOT-OH scaffold.In summary,based on the application potential of SF/PEDOT materials in the field of nerve tissue engineering,the RSF/PEDOT-OH,RSF/PEDOT:PSS films and RSF/PEDOT-OH microfluidic scaffold prepared in this study are expected to achieve further applications in nerve regeration and bioelectronics.Meanwhile,it may provide a reference for the development of novel biomaterials in biomedical applications. |
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