Structure Design And Properties Of Biomimetic Composite Materials Of Silk Fibroin/graphene

Repair of nerve defects and injuries is a major problem in clinical treatment.The implanted catheters or scaffolds for repairing nerve tissue require not only desirable biocompatibility,but also good electrical and mechanical properties.Regenerated silk fibroin(RSF)mats fabricated by conventional electrospinning method can induce the growth and regeneration of nerve cell,but have slight effect on the differentiation of neuron due to poor electrical properties.In order to prepare RSF tissue engineering scaffolds with good mechanical and electrical properties,graphene materials were selected as the nano modified compound additives.In this thesis,the structure of concentrated RSF/graphene oxide(GO)aqueous solution and the interaction between RSF and GO were firstly investigated.Secondly,RSF/GO hybrid fibers fabricated by dry spinning method were used as the research model to explore the fiber-reinforced mechanism of GO and the confined crystallization of RSF micro-region.Then,RSF/GO,RSF/reduced graphene oxide(RGO),RSF/graphene(Gr)and RSF/Gr-X embedded composite scaffolds were prepared by electrospinning and dipping methods.The effect of different types of graphene materials on the structure and mechanical properties of composite scaffolds was analyzed.In addition,the ability of these composite scaffolds to induce nerve cell growth and differentiation was studied,which laid the foundation for expanding the application of RSF/GO fibers on the recovery of injured nerve tissues.The results from the structure of the concentrated RSF/GO mixed aqueous solution indicated that the existence of GO increased the viscosity of RSF solution and delayed the solution/gel transformation of RSF solution under shear,which was not beneficial for the formation of ordered liquid crystal structure in the RSF solution.The results of Synchrotron Radiation Small Angle X-ray Scattering(SR-SAXS)showed that the mean-square radius of gyration(R_g)of RSF/GO mixed solution and the interface thickness parameters between RSF and GO both increased under shear.On the basis of the preparation of RSF/GO spinning dope with good spinnability,the RSF/GO hybrid fibers were obtained through a custom-built capillary dry-spinning device which was used to biomimic the spinning method of silkworms.The condensed structures of RSF/GO hybrid fibers were studied,and the relationship between the crystalline structure,orientation structure,phase interface,GO content and mechanical properties were analyzed.It was found that GO resulted in the confined crystallization of RSF.With the increase of GO content,?-sheet content,crystallinity and crystallite size of the hybrid fibers decreased,while fraction of mesophase increased.In addition,an appropriate amount of GO promoted the orientation of crystalline region and mesophase of fibers.Compared with RSF fibers,the interface thickness parameters and the scattering object misorientation of the RSF/GO hybrid fibers were both larger,while their correlation length was smaller.Furthermore,the mesophase of RSF/GO fibers transferred to crystalline region during the stretching process.When the strain of the hybrid fibers exceeded the critical value,the crystalline region and amorphous region gradually changed to mesophase.Within elastic deformation zone,no obvious changes in the orientation of the crystalline region and mesophase were observed.However,when the fiber entered plastic deformation zone,the orientation of the crystalline region firstly increased,then the orientation of the mesophase gradually increased with the increase of strain.When the mass ratio of GO/RSF was 1/1000,the breaking strength of the RSF/GO hybrid fibers was about(435.5±71.6)MPa,which was about a 23%improvement in comparison to degummed silk and a 72%improvement for neat RSF fiber.Based on the single RSF/GO fiber with excellent mechanical properties,RSF/GO blend scaffolds were fabricated by electrospinning method.Furthermore,in order to fabricate silk fibroin tissue engineering scaffolds with excellent mechanical performance and conductivity,RSF/RGO scaffolds were obtained via reduction of GO.It was found that with the increase of RGO content,the diameter of the RSF/RGO fibers increased firstly and then decreased,while the?-sheet conformation fraction,crystallinity and crystallite size gradually decreased.Hydrothermal treatment increased the disorder of RGO in fibers.Moreover,the composite fiber mats had the best mechanical properties when the mass ratio of RGO/RSF was 1/100.The lowest surface resistance of the RSF/RGO fibers was 1.1×10~8?/sq.When the mass ratio of RGO/RSF was less than 1/100,RSF/RGO scaffolds had no obvious effect on the growth and proliferation of Schwann cells(SCs).With further increase of RGO content,the toxicity of the composite scaffolds increased,which had obvious inhibitory effect on cell proliferation.However,the RSF/RGO fiber mats benefit the adhesion and spreading of SCs.It was found that Gr had the greatest effect on the morphology,?-sheet content,crystallinity and crystallite size of the composite fibers than GO and RGO.RGO and Gr increased the electrical properties of composite scaffolds,but no significant difference of the electrical properties was found.In addition,Gr and GO strengthen and toughen composite scaffolds,respectively.RSF/GO,RSF/RGO and RSF/Gr scaffolds promoted the proliferation of SCs,which grew and spread well on the RSF/RGO scaffolds.All of the above composite scaffolds had certain antibacterial properties to staphylococcus aureus,and the RSF/RGO scaffolds showed outstanding antibacterial properties.It was noted that the improvement of electrical properties of RSF/RGO and RSF/Gr scaffolds prepared by the solution blending method were limited.In order to further improve the electrical properties of composite scaffolds,a dipping method which immersed the as-spun RSF mats into the Gr ethanol solution was applied in this thesis on the basis of contraction of RSF fiber network during the post-treatment of ethanol.The RSF/Gr-X-embedded composite scaffolds with good embeddability of Gr and excellent conductive network were prepared.With the increase of Gr concentration and immersion time,the quality of Gr loading on the scaffolds increased,and the surface hydrophilicity and electrical resistance of composite scaffolds gradually decreased.The minimum surface resistance was about 0.3×10~3?/sq.The majority of Gr on the fiber surface was stripped down by ultrasonic and washing processing,while the Gr embedded into the pores remained in the fiber.The initial modulus of RSF/Gr-X-embedded composite scaffold increased significantly,the elongation at break reduced,while the breaking strength had limited change.Compared with RSF scaffolds,RSF/Gr-X-embedded scaffolds improved cell adhesion and spreading,but inhibited cell proliferation.To further study the ability of RSF/Gr-X-embedded scaffolds for nerve cells differentiation,the rat adrenal pheochromocytoma cells(PC12)were cultured on this scaffold with and without electrical stimulation(ES).It was noted that the adhesion and growth of PC12 cells cultured on the RSF/Gr-X-embedded scaffolds were better than those cultured on neat RSF scaffolds.When electrical stimulation was applied,the proportion of PC12 neurite-bearing cells and axonal length of PC12 cells increased with the increase of voltage and time.The proportion of PC12 neurite-bearing cells and average length were about66.9%and 8.9?m at 100 mV voltage and 6 h ES time,respectively.