| Silk fibroin(SF)protein hydrogel was studied and used in the restoration of soft tissue engineering,drug sustained release,genetic engineering and other fields because of its good biocompatibility,biodegradability,water retention and pore connectivity,it's the current research hotspot of biomaterials and regenerative medicine fields.As the soft tissue repair materials,SF hydrogel is commonly prepared through cross-linking,grafting,or mechanical shearing,however,the shortcomings were still existed such as long timescales,poor mechanical properties,introduction of organic solvent and the uncontrollable appearance during the preparation.These methods have made it difficult to meet the requirements of clinical application.Therefore,it is an urgent need to establish a new method to build a green and mild SF gel with good performance.In this study,we focused on the physical and chemical characteristics of SF protein macromolecules and used a green,mild and environmental electrostatic field assembly technology to build SF gel with good performance rapidly and effectively through regulating the pH value and the extra electric field,providing a new type of SF scaffolds for soft tissue repair engineering.On the basis of the deep understanding of the physical and chemical characteristics of SF protein macromolecules and the properties of the target material,we assumed that we could regulate the assembly behavior of SF macromolecular,reduce or offset the repulsive force between or inside the SF macromolecules,strengthen hydrophilic and hydrophobic interaction,mutual entanglement and looping state through the synergy of pH value and electrophoresis to obtain a stable secondary structure(beta sheet).Simultaneously,we gained a SF electron gel with good mechanical performance and stability by the changing the arrangement of SF macromolecules.And through the change of steady dc voltage,SF solution concentration,pH value,we found that the optimum conditions to get a silk fibroin electro gelation is that the steady dc voltage is 24 V,the solution concentration is 5 wt %,and the best pH value is 4.Electron gelation was evaluated by its water absorption and mechanical properties.Scanning electron microscopy(SEM)was used to observe the morphology of SF electron gelation.Fourier infrared spectrum(FTIR),X-ray diffraction(XRD)and Raman spectroscopy(Raman)were applied to investigate the secondary structure of SF electron gelation.The loading and releasing of model drug in vitro experiments were applied to explore drug sustained release properties of the SF electron gelation.Finally,the in vitro degradation experiments by protease XIV and rat renal tubular epithelial cell compatibility experiments were used to evaluate the biodegradation behavior and cell compatibility of SF electron gelation.The results showed that the SF electron gelation(SF,pH-e-gel)prepared by the synergy effect of the pH(~pI)adjustment and electrophoresis of low voltage electric field,had a superior mechanical properties when compared to that of the natural SF gel or SF pHgel obtained by controlling the pH.The compression strength and compression modulus are 7.91 MPa and 70.0 MPa respectively,more higher than that of SF gel with 0.25 MPa and 1.0 MPa respectively,and the water uptake ratio of SF pH-e-gel is 392%.SEM results indicated that SF pH-e-gel materials possess the uniform circular hole morphology with smaller size and thicker hole wall rather than the layer or flake pore morphology of classic silk hydrogel.The FTIR,XRD and Raman results demonstrated that SF pH-e-gel presented a low contention of beta-sheet structure,and the amount of alpha helix structure and silk I crystallization are higher when compared with that of SF gel or SF pH-gel.Again,with rhodamine B as the model drug,sustained drug release experiments results showed that the drug encapsulation efficiency reaches 87.5%,and the total drug release quantity percentage is about 60% within 120 hours when the initial drug concentration of SF solution is 10 ?g/ml.And also,in vitro cumulative degradation rate of SF pH-e-gel reached 80% within 28 days by the function of protease XIV which indicated that SF pH-e-gel has good biodegradable properties.Finally,the rat renal tubular epithelial cells were chosen to seed on the SF pH-e-gel,the morphology and vitality characterization of cells at different time points showed that the SF pH-e-gel can support the epithelial cell adhesion,migration,proliferation and differentiation,indicating that the SF pH-e-gel possessed good biocompatibility.In this study,we build a green,environmental and effective novel method for the preparation of SF electron gel with good mechanical properties,biocompatibility,slow-releasing potential and biodegradability,and clarify the mechanism of the formation of SF pH-e-gel.It mechanism is that: by regulating the pH of SF solution,the SF macromolecules chains approached due to the reduction or offset of the repulsive force between or inside macromolecules;and by the action of electrostatic field,the SF macromolecules were pulled,unbend and further intertwined to form a mass of ?-helix structure in the function of thermodynamics.At the same time,the approaching of SF macromolecules,thermal motion of the molecular chains and hydrophilic and hydrophobic interaction facilitate the formation stable beta sheet structures collectively,then together with the above ?-helix structure assembled into the insoluble and high crystalline content solid gel.The existence of silk I crystallization that mainly be formed of ?-helix structures contribute to the lower elastic modulus of SF electron gel,and the silk II crystallization that be formed of beta sheet structures facilitate to the improvement of strength and modulus of SF electron gel.The SF electron gel prepared through the method we have built shows significantly superior mechanical properties when compared to that of the traditional SF gel,and presented good biocompatibility and biodegradability,providing a new type of biomedical material for soft tissue repair engineering. |
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