Silk fibroin / chitosan / graphene oxide-based composite material as well as preparation method and application thereof

Abstract

The invention provides a silk fibroin / chitosan / graphene oxide-based composite material and a preparation method and application thereof, the composite material comprises a silk fibroin / chitosan / graphene oxide porous scaffold with radial orientation, and the porous scaffold is loaded with drug-containing hydrogel. By discussing the manufacturing method, the physical and biological characteristics and the potential in clinical application of the silk fibroin / chitosan / graphene oxide-based composite material, the important significance of the innovative scaffolds for improving the wound healing effect is clarified, and a direction is pointed out for future research in the field.

Description

Technical Field

[0001] The present invention relates to the field of biomedical materials, and particularly to a silk fibroin / chitosan / graphene oxide-based composite material, a preparation method thereof, and an application thereof. Background Art

[0002] The skin is the largest organ of the human body and plays a crucial role in protecting the underlying tissues from external threats and maintaining the stability of the internal environment. Skin injuries, such as wounds, burns, and ulcers, can severely affect the quality of life of patients and may lead to serious complications if not properly treated. Traditional methods for treating wounds usually involve topical application, suturing, or skin grafting; however, these methods may have some limitations, including the risk of infection, poor integration with surrounding tissues, and insufficient regeneration of the functional skin barrier. In recent years, the field of tissue engineering has gradually become a promising alternative for improving wound healing outcomes. Among various biomaterials, silk proteins and chitosan have received extensive attention due to their biocompatibility, biodegradability, and unique mechanical properties. Silk proteins are derived from silkworms and have excellent tensile strength, flexibility, and the ability to naturally support cell adhesion and proliferation. In addition, through different processing techniques, their structural properties can be customized to form scaffolds with ideal mechanical and biochemical characteristics. On the other hand, chitosan is a biopolymer obtained from chitin, and its structure and composition are similar to those of glycosaminoglycans present in the extracellular matrix (ECMs). It has inherent antibacterial properties and is known to promote wound healing by facilitating cell migration and proliferation. Its hydrophilic nature helps to maintain moisture, which is crucial for an effective healing process. Graphene oxide has powerful functionality. Graphene oxide is a derivative of graphene with many oxygen-containing functional groups on its surface, such as hydroxyl groups, epoxy groups, and carboxyl groups, which may endow it with better hydrophilicity and biocompatibility. It can cause aggregation reactions in platelets, thereby promoting thrombus formation. As a dressing, certain flexibility and strength may be required, and graphene oxide composites may have advantages in this regard, such as enhancing the mechanical properties of the material while maintaining breathability. Combining silk proteins, chitosan, and graphene oxide presents a promising strategy to create hybrid scaffolds that fully utilize the beneficial properties of these three materials. Herbal extracts have been widely used in wound treatment due to their natural properties, low toxicity, and effective biological properties. Puerarin (PUE), a flavonoid compound extracted from Pueraria lobata, has effective antioxidant and anti-inflammatory activities, can promote cell migration and angiogenesis, and can be used for wound repair. Liquid nitrogen directional freezing technology is an innovative material preparation method, widely used in the fields of tissue engineering and biomaterials, especially for preparing scaffolds with specific microstructures to promote cell growth and tissue regeneration. During the development of biomaterials, the microstructure of the scaffold plays a crucial role in cell adhesion, proliferation, and differentiation. By optimizing the physical and chemical properties of the scaffold, its biocompatibility and functionality can be significantly improved. The design of a radially oriented structure of the scaffold has been considered a key factor in tissue engineering, especially in skin repair. Radially oriented structure scaffolds can mimic the natural structure of the extracellular matrix and provide directional signals that affect cell behavior and tissue regeneration. By integrating silk fibroin, chitosan, and graphene oxide into a structured radially oriented scaffold, inducing cells to migrate from the wound periphery to the center, it is possible to create a microenvironment that better supports cell infiltration, angiogenesis, and overall tissue integration. Summary of the Invention

[0003] Technical problems to be solved: The purpose of the present invention is to provide a silk fibroin / chitosan / graphene oxide-based composite material, by exploring its manufacturing method, physical and biological properties, and potential in clinical applications, to clarify the significance of these innovative scaffolds in enhancing the wound healing effect, and to point the way for future research in this field.

[0004] Technical solution: A silk fibroin / chitosan / graphene oxide-based composite material, the composite material includes a silk fibroin / chitosan / graphene oxide porous scaffold with a radial orientation, and a drug-loaded hydrogel is loaded on the porous scaffold. The preparation method of the above-mentioned silk fibroin / chitosan / graphene oxide-based composite material includes the following steps: S1. Shorten silk fibers and add them to water, first perform physical shearing and then alkali hydrolysis, and then remove alkali and freeze-dry to obtain SF nanofibers; S2. Dissolve graphene oxide in water to obtain a GO dispersion; S3. Add SF nanofibers and water-soluble chitosan to the GO dispersion, form a uniform suspension by ultrasonic treatment, add the suspension to a mold and freeze-dry radially to obtain a porous scaffold, and crosslink the porous scaffold in ammonia water to obtain a radially oriented silk fibroin / chitosan / graphene oxide porous scaffold; S4. Inject the drug-loaded hydrogel into the porous scaffold to obtain a silk fibroin / chitosan / graphene oxide-based composite material. Preferably, the preparation method of the drug-loaded hydrogel includes the following steps: S11. Dissolve silk fibroin in a neutral salt solution, dialyze and adjust the concentration of the silk fibroin solution to obtain an RSF solution; S12. Add tannic acid to the RSF solution, stir and mix evenly, and let it stand to obtain the SF / TA mixed solution; S13. Add the wound repair agent to the SF / TA mixed solution to obtain the medicated hydrogel. Preferably, the concentration of tannic acid in the SF / TA mixed solution is 0.1 wt%; and / or, The concentration of silk fibroin in the SF / TA solution is 1 wt%; and / or, The volume concentration of the wound repair agent in the SF / TA mixed solution is 0.1 - 0.4 vt%. Preferably, in step S1, the rotation speed of the physical shearing is 25000 - 35000 r / min, and the time is 30 - 240 min; and / or, the OH in the alkali hydrolysis - has a concentration of 0.5 - 5 mol / L, and the hydrolysis time is 5 - 60 min; and / or, The temperature of the freeze-drying is -60 - 0 °C, and the freeze-drying time is 48 - 72 h; Preferably, in step S2, the concentration of the GO dispersion is 0.05 - 0.2 wt%. Preferably, in step S3, the mass-to-volume ratio of the SF nanofibers, water-soluble chitosan, and the GO dispersion is 0.1 g:0.4 g:15 mL; and / or, The temperature of the radial freeze-drying is -60 - 0 °C, and the freeze-drying time is 48 - 72 h; and / or, The cross-linking time is 12 h. The silk fibroin / chitosan / graphene oxide-based composite material prepared by the above preparation method is applied in wound repair. Beneficial effects: The silk fibroin / chitosan / graphene oxide-based composite material of the present invention has the following advantages: 1. The silk fibroin and chitosan (CS) used in the present invention are the necessary components in the extracellular matrix (ECM). The nanofibers can simulate the structure in the ECM, provide a microenvironment for cell growth, and are beneficial to cell proliferation and migration. Silk fibroin nanofibers and chitosan are both natural macromolecules with good biocompatibility and the necessary water retention in wound repair; by using the pH sensitivity of CS, ammonia vapor fumigation induces the deprotonation of amino groups in CS to provide stability for the scaffold; graphene oxide (GO) is a two-dimensional carbon nanomaterial with super affinity for water. The rich active groups of GO can improve the hydrophilicity and hemostatic ability of the SF / CS composite scaffold, and can also increase the porosity of the scaffold; the hemostatic sponge prepared from the composite of GO and natural polymer materials reduces the potential biosafety risk when free GO contacts tissues; 2. Inject the mixed solution of silk fibroin, chitosan, and graphene oxide into a copper tube and prepare it through the liquid nitrogen directional freezing technique. The radial temperature gradient generated by the round tube can be used to make ice crystals grow from the periphery to the center. Eventually, a radially porous material is formed after freeze-drying. The radially oriented structure plays a role in promoting wound healing by guiding the migration and proliferation of new tissues from the periphery to the center of the tissue defect site, accelerating the wound healing rate, and gradually reducing the wound defect from the periphery to the center. These porous structures provide a three-dimensional environment conducive to the in-growth of blood vessels and granulation tissue. Manufacturing a three-dimensional scaffold with aligned nanofibers and interconnected large channels similar to the extracellular matrix (ECM) can induce cells to migrate towards the wound center, thereby restoring cell function, promoting granulation tissue formation, and collagen deposition; 3. Puerarin is a natural isoflavone compound with strong antioxidant activity. It scavenges ROS free radicals and reduces oxidative damage to fibroblasts and endothelial cells. In addition, puerarin can promote the migration ability of HUVEC cells, and more importantly, it can promote angiogenesis. All of these are beneficial to wound healing; 4. In vitro cell experiments of the present invention demonstrate that the radially oriented scaffold can induce cells to migrate from the wound periphery to the center. The cell morphology of the SCG@PUE scaffold is significantly elongated along the scaffold direction. In vivo experiments confirm the presence of newly grown granulation tissue and re-epithelialization on the SCG@PUE scaffold, with high expression in the CD31 and α-SMA positive regions and good angiogenesis effect. The SCG@PUE scaffold has an obvious promoting effect on wound repair. Description of the Drawings Figure 1 It is a diagram of GO dispersion liquids with different concentrations and composite scaffolds prepared with GO at different concentrations. Among them, A is a physical diagram of GO dispersion liquids with different concentrations, and B is a composite scaffold prepared by mixing 2wt% SF, 4wt% CS, and GO at different concentrations; Figure 2 It is the performance characterization diagram of the SCG scaffold in Example 5 and Comparative Examples 1-2, 5. In the figure, A is the SEM image of GO, B is the AFM scanning image of GO; C is the FTIR of the SCG scaffold, and D is the porosity of the SCG scaffold; Figure 3 It is the hydrophilicity of the SCG scaffold in Example 5 and Comparative Examples 1-2, 8. In the figure, A is an image of the composite scaffold absorbing a drop of water at the two-second time point; B is the time for the composite scaffold to completely absorb a drop of water; Figure 4For the biocompatibility of the composite scaffolds in Example 5 and Comparative Examples 1-2, where A is the figure of NIH3T3 cells cultured on different scaffolds for 24, 48, and 72 h and stained with a live / dead cell double staining kit, and B is the figure of the absorbance at 450 nm measured by CCK8 for NIH3T3 cells cultured on different scaffolds for 24, 48, and 72 h; Figure 5 For the hemolytic activity figures of the composite scaffolds in Example 5 and Comparative Examples 1-2, 8, where A is the hemolytic activity figure and B is the hemolysis rate with different SCG scaffold dispersions; Figure 6 For the animal hemostasis figures of the composite scaffolds in Example 5 and Comparative Examples 1-2, 8, where A is the photo of the gauze, SF / CS, and SCG groups after in vivo hemostasis when there is a liver defect in rats, B is the clotting time in the rat liver volume defect injury, and C is the blood loss in the rat liver volume defect injury; Figure 7 For the stability characterization figure of puerarin-loaded SF hydrogel standing for different times; Figure 8 For the microscopic morphology figures of the ST@PUE hydrogel prepared in Examples 1-4; Figure 9 For the infrared and rheology of the ST@PUE hydrogel prepared in Examples 1-4; Figure 10 For the biocompatibility of the ST@PUE hydrogel prepared in Examples 1-4, where A is the figure of NIH3T3 cells cultured on different scaffolds for 24, 48, and 72 h and stained with a live / dead cell double staining kit, and B is the absorbance at 450 nm measured by CCK8 for NIH3T3 cells cultured on different scaffolds for 24, 48, and 72 h; Figure 11 For the puerarin drug release curves of the ST@PUE hydrogel prepared in Examples 2-3, where A is the puerarin drug release standard curve, B is the slow release figure of the hydrogel within 7 days, and C is the slow release figure of the hydrogel within 48 h; Figure 12 For the SCG@PUE scaffold prepared in Example 5; Figure 13 For the characterization of the biocompatibility of the composite scaffolds prepared in Examples 5-6 and Comparative Example 5, where A is the absorbance at 450 nm measured by CCK8 for NIH3T3 cells cultured on different scaffolds for 1, 3, and 7 days; B is the laser confocal scanning microscope image after rhodamine and Dapi staining for NIH3T3 cells cultured on the scaffold for 1, 3, and 7 days; Figure 14 For the SEM images of NIH3T3 cells cultured on the composite scaffolds prepared in Examples 5-6 and Comparative Example 5 for 1, 3, and 7 days; Figure 15 Cell migration diagrams of HUVEC cells on the composite scaffolds prepared in Examples 5-6 and Comparative Example 5 at 0, 6, and 12 h; Figure 16 Angiogenic lumen diagrams of the composite scaffolds prepared in Examples 5-6 and Comparative Example 5 at 4, 12, and 24 h; Figure 17 Shows the effects of the composite scaffolds prepared in Examples 5-6 and Comparative Example 5 on treating full-thickness skin defects. Among them, A are photos of the wounds treated with the composite scaffolds at 0, 7, and 14 days, B is the wound healing rate at 7 and 14 days, and C is a schematic diagram and comparison of the wound healing process in different groups. Blue represents 0 days, green represents 7 days, and red represents 14 days; Figure 18 Is the histological analysis of the composite scaffolds prepared in Examples 5-6 and Comparative Example 5 for treating wounds in vivo. Among them, A are H&E and Masson's trichrome staining sections of the regenerated tissues at 7 and 14 days after treatment with the composite scaffolds, B is the granulation tissue thickness in each group, C is re-epithelialization, and D is collagen deposition. The observation time is at 7 and 14 days; Figure 19 Is the histological analysis of the composite scaffolds prepared in Examples 5-6 and Comparative Example 5 for treating wounds in vivo. Among them, A are immunohistochemical staining diagrams of CD31 and α-SMA at 7 and 14 days, B is the quantitative result of the percentage of CD31-positive area in each group, and C is the quantitative result of the percentage of α-SMA-positive area in each group; Figure 20 Is the microscopic morphology diagram of the scaffold prepared in Comparative Example 6. Detailed implementation mode The present invention will be further described below in conjunction with the examples and the accompanying drawings of the specification. The following examples are explanations of the present invention and the present invention is not limited to the following examples: Example 1 The preparation method of the medicated hydrogel comprises the following steps: S11. Dissolve silk fibroin in a 9.3 M lithium bromide solution at 60 °C for 4 h. Subsequently, dialyze the silk fibroin solution against deionized water for 3 days to obtain a silk fibroin solution. Dilute the silk fibroin solution with deionized water to 1 wt% to obtain an RSF solution; S12. Add tannic acid to the RSF solution, with the concentration of TA being 0.1 wt%. Stir and mix evenly, and let it stand at 37 °C for 12 h to obtain an SF / TA mixed solution; S13. Add the wound repair agent - puerarin powder to the SF / TA mixed solution, with the volume concentration of puerarin powder in the SF / TA mixed solution being 0.1 vt% to obtain a medicated hydrogel, named ST@PUE1. Example 2 Example 2 is different from Example 1 in that the volume concentration of puerarin powder in the SF / TA mixed solution is 0.2 vt%, named ST@PUE2. Example 3 Example 3 is different from Example 1 in that the volume concentration of puerarin powder in the SF / TA mixed solution is 0.3 vt%, named ST@PUE3. Example 4 Example 4 is different from Example 1 in that the volume concentration of puerarin powder in the SF / TA mixed solution is 0.4 vt%, named ST@PUE4. Example 5 A method for preparing a silk fibroin / chitosan / graphene oxide-based composite material includes the following steps: S1. Short-cut silk fibroin fibers are added to water to obtain a 2% silk fibroin suspension. The silk fibroin suspension is first subjected to physical shearing at a rotational speed of 32000 r / min for 2 h. After shearing, alkali hydrolysis is carried out. The concentration of OH - in the alkali hydrolysis is 1 mol / L, and the hydrolysis time is 30 min. Then, the alkali is removed and freeze-dried to obtain SF nanofibers; S2. Graphene oxide is dissolved in water to obtain a GO dispersion with a concentration of 0.1 wt%; S3. The SF nanofibers and water-soluble chitosan are added to the GO dispersion. The mass-volume ratio of the SF nanofibers, water-soluble chitosan, and GO dispersion is 0.1 g:0.4 g:15 mL. A uniform suspension is formed by ultrasonic treatment. The suspension is added to a mold and radially freeze-dried at a temperature of -60 °C for 48 h to obtain a porous scaffold. The porous scaffold is placed in a closed environment containing NH3·H2O for 12 h until ammonia completely enters the sponge. Finally, the SF / CS scaffold is placed in an oven at 45 °C for 2 h to remove excess ammonia, and a radially oriented silk fibroin / chitosan / graphene oxide porous scaffold is obtained, named SCG0.1%; S4. The drug-loaded hydrogel prepared in Example 2 is injected into the voids of the porous scaffold to obtain a silk fibroin / chitosan / graphene oxide-based composite material, named SCG@PUE2. Example 6 Example 6 is different from Example 5 in that the drug-loaded hydrogel in step S4 is different. In this example, the drug-loaded hydrogel prepared in Example 3 is added to obtain a silk fibroin / chitosan / graphene oxide-based composite material, named SCG@PUE3. Comparative Example 1 The difference between Comparative Example 1 and Example 5 lies in that: the concentration of the GO dispersion in step S2 is different. In this example, the concentration of the GO dispersion is 0.05 wt%, and the radially oriented silk fibroin / chitosan / graphene oxide porous scaffold prepared in step S3 is named SCG0.05%. Comparative Example 2 The difference between Comparative Example 2 and Example 5 lies in that: the concentration of the GO dispersion in step S2 is different. In this example, the concentration of the GO dispersion is 0.2 wt%, and the radially oriented silk fibroin / chitosan / graphene oxide porous scaffold prepared in step S3 is named SCG0.2%. Comparative Example 3 The difference between Comparative Example 3 and Example 5 lies in that: the medicated hydrogel in step S4 is different. In this example, the medicated hydrogel prepared in Example 1 is added to obtain a silk fibroin / chitosan / graphene oxide-based composite material, named SCG@PUE1. Comparative Example 4 The difference between Comparative Example 4 and Example 5 lies in that: the medicated hydrogel in step S4 is different. In this example, the medicated hydrogel prepared in Example 4 is added to obtain a silk fibroin / chitosan / graphene oxide-based composite material, named SCG@PUE4. Comparative Example 5 A method for preparing a silk fibroin / chitosan / graphene oxide-based composite material includes the following steps: S1. Short-cut silk fibers are added to water to obtain a 2% silk suspension. The silk suspension is first subjected to physical shearing at a rotation speed of 32000 r / min for 2 h. After shearing, alkali hydrolysis is carried out. The concentration of OH - in alkali hydrolysis is 1 mol / L, and the hydrolysis time is 30 min. Then, the alkali is removed and freeze-dried to obtain SF nanofibers; S2. Graphene oxide is dissolved in water to obtain a GO dispersion with a concentration of 0.1 wt%; S3. SF nanofibers and water-soluble chitosan are added to the GO dispersion. The mass-volume ratio of SF nanofibers, water-soluble chitosan, and the GO dispersion is 0.1 g:0.4 g:15 mL. A uniform suspension is formed by ultrasonic treatment. The suspension is added to a mold and radially freeze-dried at a temperature of -60°C for 48 h to obtain a porous scaffold. The porous scaffold is placed in a closed environment containing NH3·H2O for 12 h until ammonia completely enters the sponge. Finally, the SF / CS scaffold is placed in an oven at 45°C for 2 h to remove excess ammonia, and a radially oriented silk fibroin / chitosan / graphene oxide porous scaffold SCG0.1% is obtained. Comparative Example 6 The difference between Comparative Example 6 and Example 5 is that the SF nanofibers in Example 5 are replaced with ordinary hydrolyzed silk fibroin; A method for preparing a silk fibroin / chitosan / graphene oxide-based composite material, comprising the following steps: S1. Dissolve silk fibroin in a 9.3 M lithium bromide solution at 60 °C for 4 h. Subsequently, dialyze the silk fibroin solution against deionized water for 3 days to obtain a silk fibroin solution, and dry it to obtain silk fibroin powder RSF powder; S2. Dissolve graphene oxide in water to obtain a GO dispersion with a concentration of 0.1 wt%; S3. Add RSF powder and water-soluble chitosan to the GO dispersion. The mass-volume ratio of RSF powder, water-soluble chitosan, and GO dispersion is 0.1 g: 0.4 g: 15 mL. Form a uniform suspension by ultrasonic treatment. Add the suspension to a mold and freeze-dry it radially. The temperature of radial freeze-drying is -60 °C, and the freeze-drying time is 48 h to obtain a porous scaffold. Place the porous scaffold in a closed environment containing NH3·H2O for 12 h until ammonia completely enters the sponge. Finally, place the SF / CS scaffold in an oven at 45 °C for 2 h to remove excess ammonia to obtain a radially oriented silk fibroin / chitosan / graphene oxide porous scaffold; S4. Inject the drug-loaded hydrogel prepared in Example 2 into the vacancies of the porous scaffold to obtain a silk fibroin / chitosan / graphene oxide-based composite material. In this comparative example, directly use silk fibroin solution as the scaffold. It can be seen from Figure 20 that the effect of forming the radial orientation structure is not as good as that of nanofibers, and the silk fibroin solution requires the use of a cross-linking agent, which may increase cytotoxicity; Comparative Example 7 The difference between Comparative Example 7 and Example 5 is that the freeze-drying method is different; A method for preparing a silk fibroin / chitosan / graphene oxide-based composite material, comprising the following steps: S1. Short-cut silk fibroin fibers and add them to water to obtain a 2% silk fibroin suspension. First, perform physical shearing on the silk fibroin suspension. The rotation speed of physical shearing is 32000 r / min, and the shearing time is 2 h. After shearing, perform alkali hydrolysis. The concentration of OH - in alkali hydrolysis is 1 mol / L, and the hydrolysis time is 30 min. Then remove alkali and freeze-dry to obtain SF nanofibers; S2. Dissolve graphene oxide in water to obtain a GO dispersion with a concentration of 0.1 wt%; S3. Add SF nanofibers and water-soluble chitosan to the GO dispersion. The mass-volume ratio of SF nanofibers, water-soluble chitosan, and GO dispersion is 0.1 g: 0.4 g: 15 mL. Form a homogeneous suspension by ultrasonic treatment. Freeze-dry the suspension at a temperature of -60 °C for 48 h to obtain a porous scaffold. Place the porous scaffold in a closed environment containing NH3·H2O for 12 h until ammonia completely enters the sponge. Finally, place the SF / CS scaffold in an oven at 45 °C for 2 h to remove excess ammonia, and obtain a radially oriented silk fibroin / chitosan / graphene oxide porous scaffold; S4. Inject the drug-loaded hydrogel prepared in Example 2 into the void of the porous scaffold to obtain a silk fibroin / chitosan / graphene oxide-based composite material. In this comparative example, the freeze-drying method is direct freeze-drying, resulting in a random porous structure, which is not conducive to the migration and proliferation of wound tissue defect sites from the periphery to the center growth direction. Comparative Example 8 The difference between Comparative Example 8 and Example 5 is that graphene is not added; A preparation method of a silk fibroin / chitosan / graphene oxide-based composite material, comprising the following steps: S1. Short-cut silk fibers are added to water to obtain a 2% silk suspension. The silk suspension is first subjected to physical shearing at a rotational speed of 32000 r / min for 2 h. After shearing, alkali hydrolysis is carried out. The concentration of OH - in the alkali hydrolysis is 1 mol / L, and the hydrolysis time is 30 min. Then, the alkali is removed and freeze-dried to obtain SF nanofibers; S2. Dissolve graphene oxide in water to obtain a GO dispersion with a concentration of 0.1 wt%; S3. Dissolve SF nanofibers and water-soluble chitosan in water. The mass-volume ratio of SF nanofibers, water-soluble chitosan, and water is 0.1 g: 0.4 g: 15 mL. Form a homogeneous suspension by ultrasonic treatment. Add the suspension to a mold and freeze-dry it radially. The temperature of radial freeze-drying is -60 °C, and the freeze-drying time is 48 h to obtain a porous scaffold. Place the porous scaffold in a closed environment containing NH3·H2O for 12 h until ammonia completely enters the sponge. Finally, place the SF / CS scaffold in an oven at 45 °C for 2 h to remove excess ammonia, and obtain a radially oriented silk fibroin / chitosan porous scaffold SF / CS; S4. Inject the drug-loaded hydrogel prepared in Example 2 into the void of the porous scaffold to obtain a silk fibroin / chitosan-based composite material. Performance test: Morphological characterization: The morphology of the samples was observed using a Regulus 8100 field emission scanning electron microscope (HITACHI, Japan) operating at 10 kV. The average diameter of TSn after alkali hydrolysis was evaluated using ImageJ software. In addition, the pore size of the disordered scaffold, the inter-pore wall spacing of the aligned scaffold, and the relationship between the pore size and the centers of the radial scaffolds within a central radius of 2 mm were measured and calculated using ImageJ software (n = 100). Atomic force microscopy (AFM) test: The dried GO was fixed on a silicon wafer and then attached to double-sided tape, and scanned on an atomic force microscope using long-strip tapping. The scanning frequency was 50 Hz, and the scanning range was 10 * 10 μm. Secondary structure test: The secondary structure of the TSn scaffold was studied using a NICOLET 5700is5 intelligent Fourier transform infrared spectrometer (Thermo Electron Corporation, USA). The Fourier transform infrared (FTIR) spectrum of the TSn scaffold was obtained with an average of 32 scans, a spectral resolution of 4 cm -1 and a wavenumber range of 400 cm -1 to 4000 cm -1 .. Porosity: The porosity of TSn scaffolds with different structures was measured using the liquid displacement method. The freeze-dried scaffold was immersed in a n-hexane solution with an initial volume of V0. After soaking for 20 minutes, the volume of the n-hexane solution was observed to be V1. Then the TSn scaffold was taken out of the solution, and the remaining volume of n-hexane was denoted as V2. Each group of samples was tested three times, and the average value was taken. The porosity (P) was calculated using formula (1). Hydrophilicity: Three samples were taken from each experimental group, and a dynamic water contact angle test was carried out using a water contact angle meter. The time for a sample to completely absorb a drop of water was measured, and the average value was recorded. Rheology test: The rheological behavior of the ST@PUE hydrogel was investigated using a DHR-2 rheometer, and a 20-mm parallel plate fixture was selected. The test temperature was 37 °C, the angular frequency was 6.28 rad / s, and the strain range was 0.1% - 100%. 300 μL of the freshly prepared hydrogel sample was taken for a dynamic strain sweep test to determine the equilibrium elastic and viscous behavior of the hydrogel. Antioxidant activity of ST@PUE hydrogel: The antioxidant capacities of SCG0.1% scaffolds and SCG@PUE scaffolds were investigated through ABTS radical scavenging experiments to demonstrate their ability to capture free radicals. A slight modification was made based on the method reported previously. Briefly, SCG0.1% scaffolds and SCG@PUE scaffolds were added to centrifuge tubes, followed by 1 mL of PBS, and then incubated in a water bath at 40 °C for 30 min. At room temperature, the solution was centrifuged at 10,000 rpm for 10 min, and the supernatant was taken. The operation was carried out according to the steps of the ABTS kit (the absorbance at 405 nm for preparing the ABTS working solution should be about 1.4). After thorough mixing, it was incubated in the dark at room temperature for 5 min, and the absorbance at 405 nm was measured. The percentage of ABTS scavenging effect was calculated according to formula (2). In the formula, Ablank is the absorbance of the blank group, Agel is the absorbance of the hydrogel group, and Acontrol is the absorbance of the control group. Drug sustained release: First, puerarin aqueous solutions with concentrations of 5, 10, 15, 20, 25, and 30 μg / mL were prepared. The absorbance at 250 nm was measured using a UV-visible spectrophotometer to establish the standard curve for PUE release, y = 0.07638x ± 0.06697, R 2 = 0.99764. 0.3 mL of ST@PUE hydrogel was placed into a dialysis bag with a molecular cut-off of 3500 and immersed in 30 mL of PBS. It was incubated with shaking at 37 °C. At different time intervals (0, 2, 4, 8, 12, 24, 48, 72, 96, 120, 144, 168 h), 3 mL of the supernatant was collected and replenished with 3 mL of fresh PBS. The absorbance at 250 nm was detected using a UV-visible spectrophotometer. The cumulative release rate was calculated according to the standard curve of PUE drug release. Formula (3) is as follows: Hemolysis test: According to GB / T 16886.4-202 / ISO 10993-4:2017 "Biological evaluation of medical devices - Part 4: Selection of tests for interactions with blood", after the test materials or medical devices directly or indirectly contact fresh anticoagulated blood and cause hemolysis reactions, the absorbance values of the blood supernatants in the sample test groups are measured. At the same time, the absorbance values of the negative test group and the positive test group are measured, and the hemolysis rate of the test samples is calculated to evaluate the hemolytic effect of the test samples. Take 2 mL of blood from the hearts of SD rats and add it to an anticoagulant tube containing 3.2% sodium citrate (1:9). Gently invert the anticoagulant tube up and down along the tube wall to fully mix the blood and the anticoagulant to prevent blood coagulation. Dilute the anticoagulated blood with 0.9% sodium chloride solution at a ratio of 5:4. Put 20 mg of the freeze-dried hydrogel sample into a centrifuge tube, add 10 mL of 0.9% sodium chloride solution, and incubate it in a 37 °C constant temperature water bath for 30 min. Then add 200 μL of the diluted blood and continue to incubate for 1 h. After the hydrogel fully reacts with the red blood cells, centrifuge at 2500 rpm for 5 min, take pictures and collect the photos. Take another 100 μL of the supernatant and add it to a 96-well plate, and use an enzyme-linked immunosorbent assay (ELISA) reader to measure the absorbance value at a wavelength of 545 nm. Set the negative control as 0.9% sodium chloride solution and the positive control as deionized water. There are 3 parallel samples in each group. Calculate the hemolysis rate of the sample according to formula (4): In the formula, A represents the absorbance of the experimental group, B represents the absorbance of the negative control group, and C represents the absorbance of the positive control group. Biocompatibility: Use NIH3T3 cells to evaluate the cytocompatibility of SCG scaffolds, ST@PUE, and SCG@PUE. All samples are sterilized under high temperature and high pressure conditions (125 °C, 200 kPa) for 30 minutes for cell culture. Cell viability and cytotoxicity staining: Prepare extracts by soaking SCG nanofiber scaffolds with different GO concentrations and ST@PUE gels with different drug loadings (0, 1, 2, 3, 4 mg / mL) at 0.2 g / mL in complete medium. Place 9 mm coverslips (n = 4 / group) in a 48-well plate, seed the digested cells at 1×104 cells per well, add 500 μL of fresh extract, and change the extract every two days. After 1, 2, and 3 days, aspirate the culture medium, gently wash the coverslips three times with PBS, and perform cell viability and cytotoxicity staining on the cells on the coverslips under light protection. The staining solution is prepared at a ratio of Calcein PI (1000X): buffer: CalceinAM (1000X) = 1:1000:1 (thaw the cell viability and cytotoxicity staining kit in advance). After the prepared staining solution is fully mixed, add 150 μL to each well, incubate at room temperature in the dark for 30 min, then aspirate the staining solution, add a small amount of PBS to keep the cells moist, and observe the cells with an inverted fluorescence microscope and collect images. Cell proliferation: All samples were placed in 24-well plates (Corning Inc., New York, USA) and soaked in Dulbecco's Modified Eagle Medium (DMEM) basal medium for 24 hours before cell culture, and then the basal medium was aspirated. NIH3T3 cells were seeded into the scaffolds at a density of 2×104 cells per well and incubated for 2 hours. Subsequently, 750 μL of complete growth medium was added to each well. NIH3T3 cells were cultured in DMEM medium (Gibco, China) supplemented with 10% fetal bovine serum and 1% streptomycin-penicillin. The cells were kept in an incubator with 5% CO2, 37 °C and 95% humidity, and the medium was changed every two days. Cell viability of NIH3T3 cells on the TSn scaffolds was measured using the Cell Counting Kit-8 (CCK-8) assay. After culturing NIH3T3 cells on the scaffolds for 1, 3, and 7 days, the scaffolds with adherent cells were carefully transferred to new 24-well plates. Then, a mixture of CCK-8 reagent and DMEM medium was prepared at a ratio of 1:10 (40 μL CCK-8 and 400 μL DMEM medium) under light protection. After culturing in a light-protected incubator at 37 °C and 5% CO2 for 2.5 h, 100 μL of the CCK-8 reaction mixture was transferred to a 96-well plate, and the absorbance (OD value) of each well at 450 nm was measured using a full-automatic microplate reader (DR-3518). The background absorbance was tested in the blank wells containing the CCK-8 reagent. Each sample and culture period were repeated four times. Cell fluorescence staining: The distribution and morphology of NIH3T3 cells on different structures were observed using a laser confocal microscope (Olympus, Japan). The liquid in the well plates after culturing for 1, 3, and 7 days was aspirated, and the cells were washed 3 times with phosphate-buffered saline (PBS) for 5 min each time. 500 μL / well of 4% (v / v) paraformaldehyde (PFA) solution (Biosharp, China) was added to the well plates to fix the cells for more than 12 h. After aspirating the PFA, the cells were washed 3 times with PBS for 5 min each time. 200 μL / well of 0.1% (v / v) Triton X-100 / PBS solution was added to the well plates to penetrate the cells for 10 min. After aspirating the Triton-100 / PBS solution, the cells were washed 3 times with PBS for 5 min each time. Under light protection, 200 μL / well of TRITC Phalloidin diluted with PBS (1:800) was added to each well plate to stain the cytoskeleton for 50 min. After aspirating the rhodamine dye solution, the cells were washed 3 times with PBS for 5 min each time. Finally, 200 μL / well of Dapi diluted with PBS (1:1000) was added to stain the cell nuclei for 15 min. After staining, the cells were washed 3 times with PBS for 5 min each time. Cell electron microscopy: The field emission scanning electron microscope was used to detect the surface adhesion and morphology of the NIH3T3 culture scaffolds. Each sample was washed with PBS to remove the culture medium, and then fixed with 4% paraformaldehyde for 12 h. The scaffolds containing cells were dehydrated with gradient ethanol (25%, 50%, 75%, 95% and 100%) for 5 min. After freeze-drying the dehydrated scaffolds, they were sputter-coated with gold, and the morphology of the cells on the samples was observed with a field emission scanning electron microscope. In vivo hemostasis test: The rats were randomly divided into 5 groups with 5 rats in each group. The rats were anesthetized and fixed on the operating board. After the abdomen was incised, the liver was exposed, and then the serous fluid around the liver was carefully removed to ensure the accuracy of blood loss assessment. The pre-weighed sample of the required size (diameter: 10 mm) was immediately applied to the liver wound (3×3 mm, depth: 2 mm) made with a circular punch. The observation results were recorded every 5 seconds until the bleeding completely stopped. Then the hemostatic material was weighed and the blood loss was calculated. After the experiment was completed, the rats were euthanized. Tube formation experiment of SCG@PUE scaffolds: Cell proliferation and migration: HUVEC cells were cultured in complete culture medium (89% high-glucose medium + 10% fetal bovine serum + 1% double antibody). After the HUVEC cells cultured to the 3rd to 5th passages were digested, they were resuspended and counted, and then evenly seeded in 6-well plates at a density of 30×10 4 cells / well. When the cells grew to about 80%, a linear midline was scratched at the bottom with a 200-μL sterile pipette. The bottom of the dish was gently rinsed with PBS to remove the residual cells, and 4 mL of the leaching solution of the composite scaffold was added to each well to culture the cells. The cell morphology was observed and pictures were collected with a fluorescence inverted microscope at 0, 6, and 12 h, and the cell-free blank areas at different time points were quantified with ImageJ. Angiogenesis assay: The 96-well plates were pre-cooled on ice, and 50 μL of Matrigel was added to evenly cover the bottom of the wells. Then the plates were placed in a 37°C CO2 incubator and incubated for 30 min to gel the Matrigel. When the HUVEC cells grew to 80%, the cells were digested and added to the 96-well plates, with 2×10 4 cells per well. 100 μL of the leaching solution was added to each well, and they were incubated for 4, 12, and 24 h respectively. At the designated time points, the plates were taken out and the cell morphology was observed and pictures were collected with a fluorescence inverted microscope. In vivo wound healing: To evaluate the effects of scaffolds with different structures on skin wound healing. Male SD rats (250±50 g) were selected for in vivo experiments and provided by the Experimental Animal Research Center of Soochow University. The animal experiments were approved by the Institutional Animal Care and Use Committee of Soochow University. All the composite scaffolds used in the animal experiments were sterilized by autoclaving for 30 min. A full-thickness skin defect model of rats was constructed to evaluate angiogenesis and tissue regeneration. Each rat was randomly assigned to a blank control group and three experimental groups (DS, AS, and RS groups). Anesthesia was induced by injecting anesthetic into the abdomen of the rats. After shaving the back hair manually and disinfecting the surface skin with iodophor, four circular wounds with a diameter of 8 mm were made on the back skin of the rats using the tissue punching method. The sterilized materials were filled into the wounds, and the materials were closely attached to the skin wounds of the rats with sterile gauze and breathable bandages. The rats implanted with the materials were placed under a warming lamp to prevent death due to hypothermia. At 0, 7, and 14 days after surgery, the wound images of the animals were taken with a camera (n = 5). The rats were sacrificed at 7 and 14 days after surgery, respectively, and samples were collected. The open wound area was measured using ImageJ software. The wound closure rate WoundHeal rate (WHR, %) was determined according to Equation (5). where S0 and St represent the initial wound area and the wound area after each time point, respectively. The tissues extracted from the wound area on the back of the rats were fixed in 10% formaldehyde solution for histological evaluation. After washing, the samples were dehydrated through a series of graded ethanol and embedded in paraffin. All samples were stained with hematoxylin and eosin (H&E) and Masson's trichrome to show the growth of new granulation tissue, the rate of epithelial reformation, and collagen deposition. In addition, immunohistochemical staining of CD31 and α-smooth muscle actin (α-SMA) was performed on the samples to evaluate angiogenesis in the wound area. Result analysis: Combined with Figure 1-2 , it can be seen from the SEM images that the prepared sponge has an interconnected porous structure. It can be seen that too high or too low concentration of the GO dispersion will affect the growth and migration of cells. As the GO content in the SCG composite scaffold increases, the pore wall spacing of the scaffold becomes smaller. When the concentration of the GO dispersion is 0.05%, the formation effect of the radial orientation structure in the central region of the scaffold is not good; when the concentration of the GO dispersion is 0.2%, it is difficult to form a radial structure of the scaffold, and the pore wall spacing is too small, which is not conducive to the growth and migration of cells. Therefore, the concentration of the GO dispersion will affect the porosity of the scaffold. The high porosity of the scaffold is beneficial to the exchange of nutrients and the clearance of metabolic wastes during tissue regeneration; the high-porosity sponge, thanks to its developed three-dimensional network structure and significantly increased specific surface area, can provide an ideal microenvironment for blood absorption and coagulation processes. This porous architecture can not only efficiently adsorb blood components but also promote the directional aggregation and interfacial adhesion of blood cells and coagulation-related proteins through physical enrichment, thus accelerating the initiation and progress of the coagulation cascade reaction; Combined with Figure 3, It can be seen from the figure that the hydrophilicity of the composite scaffold after adding GO is significantly improved compared with that of the SF / CS scaffold. With the increase in the concentration of the GO dispersion, the hydrophilicity also increases. Because the surface of GO contains a large number of hydrophilic groups. A wound dressing with good hydrophilicity can maintain a moist environment, which is beneficial to the movement of epithelial cells and fibroblasts, accelerating wound closure; by simulating the physiological moist environment, it provides ideal conditions for cell proliferation, collagen deposition and angiogenesis, absorbs excess exudate, and at the same time locks in moisture to prevent excessive drying or maceration; Combined with Figure 4 , It can be seen from the figure that the scaffold composite with GO still shows good biocompatibility. Live / dead staining shows the green color of live cells, and almost no red color of dead cells appears. With the increase of the culture time, the number of cells increases significantly, and the number of cells cultured in the gel of the SCG0.1% group is more and denser; Combined with Figure 5 , It can be seen from the figure that the hemolysis rate of the SF / CS and SCG composite scaffolds is less than 5%. The composite directly added with GO has better blood compatibility. TSF nanofibers have a high surface area ratio and high water absorption capacity, and can concentrate coagulation factors. By contacting the negatively charged platelet and red blood cell surfaces, chitosan can stop bleeding by absorbing moisture and converting it into an adhesion element that adheres to the damaged tissue. The two-dimensional sheet structure of GO can form a dense covering layer on the wound surface, mechanically blocking the outflow of blood. The high specific surface area enhances the contact efficiency with blood components, and the flexible structure can conform to the irregular surface of the wound to achieve a tight fit; Combined with Figure 6 , It can be seen from the figure that compared with the gauze, the blood loss of the SF / CS and SCG groups is lower. This result is confirmed by measuring the blood loss and bleeding time in different groups. The hemostasis time of the gauze group is 84.8 s, and the total blood loss is 1.58 g. The hemostasis times of the SF / CS and SCG0.05%, SCG0.1%, and SCG0.2% scaffolds are 31.7 s, 18.6 s, 14.6 s, and 14.9 s respectively, and the total blood losses are 0.23 g, 0.20 g, 0.17 g, and 0.17 g respectively. Compared with the gauze, the prepared scaffolds have better hemostasis effects, and the blood loss and hemostasis time decrease with the increase of GO; Combined with Figures 7-9 , It can be seen from Figure 7 that the SF solution will not gel without adding tannic acid (TA); after adding TA, the SF solution gels in about 12 h; the addition of puerarin will not affect the gel time of SF; Combined with Figure 8 , Figure 8 In the control experiment, the content of puerarin in the pure silk fibroin and the hydrogel containing the medicine in the composite scaffold is 0. It can be seen from the figure that the addition of tannic acid and puerarin will not affect the microstructure of the SF hydrogel; Figure 9The storage modulus G” of the ST@PUE hydrogel is greater than the loss modulus G’, indicating good structural stability and reaching the gel state; Combined with Figure 10 , it can be seen from the figure that the hydrogel after adding puerarin shows good biocompatibility. Live / dead staining shows the green of live cells, and almost no red of dead cells. As the culture time increases, the number of cells increases significantly, and the number of cells cultured in the gels of ST@PUE0, ST@PUE2, and ST@PUE3 groups is larger and denser, indicating that puerarin has little impact on biocompatibility. In further experiments, the hydrogels in Example 2 and Example 3 were selected and injected into the SCG scaffold; Combined with Figure 11 , it can be seen from the figure that the in vitro sustained-release behavior of ST@PUE2 and ST@PUE3 gels. The release rates reached 29.3% and 39.1% respectively at 8 h. After one day, the release rates of the two gels were 44.6% and 60.3% respectively. After that, the release rate of PUE tended to be balanced. At seven days, ST@PUE2 and ST@PUE3 gels reached 74.7% and 98.1% respectively, proving that ST@PUE3 gel can achieve high release rate and long-term sustained release, which is suitable for application in skin repair; Combined with Figure 12 , the effect of the radial orientation structure formation of the SCG@PUE scaffold prepared in Example 5 is very good. The wall spacing of the SCG@PUE scaffold shows a linear trend with the distance from the center. As it approaches the center, the wall spacing decreases. The SCG@PUE scaffold has obvious antioxidant ability, and the greater the drug loading, the more obvious the antioxidant effect; Combined with Figure 13 , it can be seen from the figure that there is no significant difference in cell viability among different groups of materials on the 1st and 3rd days of culture. After 7 days of culture, the biocompatibility of the SCG@PUE3 group composite scaffold is better than that of other groups, and significantly higher than that of the non-drug-loaded group scaffold. The incorporation of surface puerarin improves the biocompatibility of cells; Combined with Figures 14-16 , it can be seen from the figure that the cells on the SCG@PUE scaffold can grow and migrate along the lamellar direction of the material. PUE can enhance the migration ability of endothelial cells. The greater the drug loading, the stronger the migration ability, and it has an obvious effect on promoting angiogenesis; Combined with Figures 17-19, it can be seen from the figure that the wound healing rates of all material groups are faster than those of the control group, and the wound healing rate of the SCG@PUE3 scaffold group is the fastest, indicating that the SCG material promotes wound healing. The loading of puerarin accelerates the wound healing rate; the results of H&E stained sections show that the SCG@PUE3 scaffold group has the best effects on the growth of new granulation tissue and re-epithelialization; the results of Masson's trichrome stained sections show that the SCG@PUE3 scaffold group has the best collagen deposition. The SCG@PUE scaffold with a radial structure plays a positive role in stimulating the growth of granulation tissue, epidermal regeneration and collagen deposition; the angiogenesis amount in the SCG@PUE3 scaffold group is the highest, indicating that the composite material prepared by the present invention has a good effect on wound repair. The materials provided by the present invention have been introduced in detail above. Specific examples are used in this article to illustrate the structure and working principle of the present invention. The description of the above embodiments is only used to help understand the method and core idea of the present invention. The above is only the preferred embodiment of the present invention and is not used to limit the present invention. Moreover, the above embodiments are only examples given for clear illustration and are not limitations on the implementation manners. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A silk fibroin / chitosan / graphene oxide-based composite material, characterized in that: The composite material includes a silk fibroin / chitosan / graphene oxide porous scaffold with radial orientation, and a drug-loaded hydrogel is loaded on the porous scaffold.

2. The preparation method of the silk fibroin / chitosan / graphene oxide-based composite material according to claim 1, characterized in that, It includes the following steps: S1. Shorten silk fibroin fibers and add them to water. First, perform physical shearing and then alkali hydrolysis, and then remove alkali and freeze-dry to obtain SF nanofibers. S2. Dissolve graphene oxide in water to obtain a GO dispersion. S3. Add SF nanofibers and water-soluble chitosan to the GO dispersion, form a uniform suspension by ultrasonic treatment, add the suspension to a mold and freeze-dry radially to obtain a porous scaffold, and add the porous scaffold to ammonia water for cross-linking to obtain a silk fibroin / chitosan / graphene oxide porous scaffold with radial orientation. S4. Inject the drug-loaded hydrogel into the porous scaffold to obtain a silk fibroin / chitosan / graphene oxide-based composite material.

3. The preparation method of the silk fibroin / chitosan / graphene oxide-based composite material according to claim 1, characterized in that, The preparation method of the drug-loaded hydrogel includes the following steps: S11. Dissolve silk fibroin in a neutral salt solution, dialyze and adjust the concentration of the silk fibroin solution to obtain an RSF solution. S12. Add tannic acid to the RSF solution, stir and mix evenly, and let it stand to obtain an SF / TA mixed solution. S13. Add a wound repair agent to the SF / TA mixed solution to obtain a drug-loaded hydrogel.

4. The silk fibroin / chitosan / graphene oxide-based composite material according to claim 3, wherein: The concentration of tannic acid in the SF / TA mixed solution is 0.1 wt%; and / or, The concentration of silk fibroin in the SF / TA solution is 1 wt%; and / or, The volume concentration of the wound repair agent in the SF / TA mixed solution is 0.1 - 0.4 vt%.

5. The silk fibroin / chitosan / graphene oxide-based composite material according to claim 2, wherein: In step S1, the rotation speed of the physical shearing is 25,000 - 35,000 r / min, and the time is 30 - 240 min; and / or, The OH in the alkali hydrolysis - has a concentration of 0.5 to 5 mol / L, and the hydrolysis time is 5 to 60 minutes; and / or, The temperature of the freeze-drying is -60 - 0 °C, and the freeze-drying time is 48 - 72 h.

6. The silk fibroin / chitosan / graphene oxide-based composite material according to claim 2, wherein: In step S2, the concentration of the GO dispersion is 0.05 - 0.2 wt%.

7. The silk fibroin / chitosan / graphene oxide-based composite material according to claim 2, wherein: In step S3, the mass-volume ratio of the SF nanofibers, water-soluble chitosan and the GO dispersion is 0.1 g:0.4 g:15 mL; and / or, the temperature of the radial freeze-drying is -60 - 0 °C, and the freeze-drying time is 48 - 72 h; and / or, The cross-linking time is 12 h.

8. The application of the silk fibroin / chitosan / graphene oxide-based composite material prepared by the preparation method according to claims 2 - 7 in wound repair.

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