A photocured silk fibroin double-layer hydrogel and a preparation method thereof

Abstract

The application discloses a kind of photo-cured silk fibroin double-layer hydrogel and preparation method thereof, belong to medical dressing technical field.The double-layer hydrogel includes upper silk fibroin hydrogel, lower hyaluronic acid hydrogel.The application is covalently bridged by interface transition layer mussel myopic double mechanism through ultraviolet light excitation, realizes upper and lower covalent crosslinking, improves interlayer bonding strength.Two-step photo-curing process is used, and the whole process time is less than 70 seconds, and the preparation is simple, fast and easy to operate.The double-layer hydrogel can simulate the "epidermis-dermis" double-layer structure of skin, has good biocompatibility and moisturizing property, and effectively promotes wound repair.

Description

TECHNICAL FIELD

[0001] The application discloses a photocured silk fibroin double-layer hydrogel and a preparation method thereof, and belongs to the technical field of medical dressings. BACKGROUND

[0002] The repair of non-chronic wounds (such as small wounds, abrasions and cuts), chronic wounds (such as diabetic ulcers and pressure sores) and acute trauma (such as surgical incisions and burns) urgently needs a bionic dressing to simulate the "epidermis-dermis" double-layer structure of the skin: a dense and wear-resistant surface layer to resist external mechanical stress, and a porous and hydrophilic bottom layer to maintain a moist microenvironment. The combination of silk fibroin and hyaluronic acid is highly expected: silk fibroin provides high strength and oxygen permeability similar to the epidermis, and hyaluronic acid provides water retention (water content > 95%) and immune regulation functions similar to the dermis. However, existing double-layer dressings face technical bottlenecks:

[0003] 1. Weak interlayer bonding: The interface strength of composite dressings stacked physically or bonded by hydrogen bonds is generally low, far below the dynamic wound stress threshold, and is prone to delamination and shedding. Although chemical cross-linking agents (such as EDC / NHS) can improve the strength, the residual toxicity makes the L929 cell survival rate < 70%, and the acidic cross-linking environment (pH ≤ 5.5) destroys the wound healing microenvironment.

[0004] 2. Poor light curing compatibility of heterogeneous materials: The light reaction efficiency of tyrosine phenolic hydroxyl (-OH) in silk fibroin is < 10%, and direct UV irradiation causes molecular chain breakage, which cannot be cured by light alone with the groups of silk fibroin; the light curing of hyaluronic acid is limited: although methacrylated hyaluronic acid has a methacrylate photosensitive group, it can only be cross-linked within the homogeneous layer, and there is no covalent bonding site between silk fibroin. SUMMARY

[0005] In view of the deficiencies of the prior art, the application provides a photocured silk fibroin double-layer hydrogel and a preparation method thereof. Through specific ingredients and a preparation method, the interface cross-linking mechanism of mussel mucin is used to realize the covalent cross-linking of the upper silk fibroin hydrogel and the lower hyaluronic acid hydrogel, and the interlayer bonding strength is improved. The preparation method is simple, fast and easy to operate, does not require the use of complex instruments, and the prepared silk fibroin double-layer hydrogel can simulate the "epidermis-dermis" double-layer structure of the skin, realize the synergistic effect of "upper layer protection + lower layer healing", has good biocompatibility and moisturizing properties, effectively promotes wound repair, and has a wide application prospect.

[0006] One aspect of the object of the application is achieved by the following technical solutions:

[0007] A photocured silk fibroin double-layer hydrogel, characterized in that it comprises an upper silk fibroin hydrogel, a lower hyaluronic acid hydrogel, and an interface transition layer of mussel mucin.

[0008] Further, the upper layer silk fibroin hydrogel comprises 6-8 wt% of methacrylated silk fibroin and 0.1-0.3 wt% of photoinitiator.

[0009] Further, the lower layer hyaluronic acid hydrogel comprises 2-5 wt% of methacrylated hyaluronic acid and 0.05-0.2 wt% of photoinitiator.

[0010] Further, the interface transition layer comprises 1-3 wt% of mussel adhesive protein and has a pH value of 6.0-7.5.

[0011] Further, the photoinitiator is lithium phenyl-2,4,6-trimethylbenzoylphosphinate.

[0012] Another aspect of the object of the present application is achieved by the following technical solutions:

[0013] A preparation method of a photocured silk fibroin double-layer hydrogel, characterized by comprising the following steps:

[0014] (1) Lower layer pre-curing: inject a hyaluronic acid hydrogel prepolymer solution into the lower layer, irradiate with 365 nm ultraviolet light (10 mW / cm²) for 5-8 seconds to form a partially cross-linked gel base;

[0015] (2) Interface treatment: coat a mussel adhesive protein solution on the surface of the incompletely cured lower layer;

[0016] (3) Upper layer curing: cover a silk fibroin hydrogel prepolymer solution, irradiate with 365 nm ultraviolet light (10 mW / cm²) for 40-60 seconds to trigger synchronous covalent cross-linking of the upper and lower layers.

[0017] Further, the specific preparation method of the hyaluronic acid hydrogel prepolymer solution is as follows: dissolve hyaluronic acid in a 30 wt% N,N-dimethylformamide solution at a mass fraction of 1 wt%, mix well, then add 2-4 wt% methacrylic anhydride, maintain the solution pH at 8.5 with sodium hydroxide, react at 4°C for 24 hours, then precipitate with 3 times the mass of anhydrous ethanol, remove the supernatant after centrifugation, dissolve in water, dialyze with deionized water for 3 days, freeze-dry to obtain methacrylated hyaluronic acid, and finally dissolve the methacrylated hyaluronic acid at a mass fraction of 2-5 wt%, add 0.05-0.2 wt% of a photoinitiator, and dissolve to obtain the hyaluronic acid hydrogel prepolymer solution.

[0018] Further, the specific preparation method of the mussel adhesive protein solution is as follows: dissolve mussel adhesive protein in an aqueous solution at a mass fraction of 1-3 wt%, and adjust the pH to 6.0-7.5.

[0019] Further, the specific preparation method of the silk fibroin hydrogel prepolymer solution is as follows: after degumming and dissolving of silk, 0.05-0.15 wt% of methacrylic anhydride is added, and dialysis is performed with deionized water for 3 days, and methacrylated silk fibroin is obtained after freeze-drying; the methacrylated silk fibroin is dissolved according to a mass fraction of 6-8 wt%, and 0.1-0.3 wt% of a photoinitiator is added, and the silk fibroin hydrogel prepolymer solution is obtained after dissolving.

[0020] Compared with the prior art, the present application has the following advantages:

[0021] (1) The present application innovatively utilizes the covalent bridging of the dual mechanism of the mussel adhesive protein under the excitation of ultraviolet light: under the trigger of 365 nm ultraviolet light (10 mW / cm²), the catechol group of the mussel adhesive protein is oxidized into benzoquinone, the benzoquinone forms a quinone-amine bond (C=N) with the primary amino group of the lower layer of methacrylated hyaluronic acid, and simultaneously forms a quinone-phenolic ether bond (C-O-C) with the tyrosine phenolic hydroxyl group of the upper layer of methacrylated silk fibroin, and the interlayer bonding strength under the synergistic action of the double covalent bonds is better than the interlayer bonding strength under the action of traditional physical stacking / hydrogen bonds, thereby solving the problem of clinical delamination and shedding.

[0022] (2) The present application adopts a neutral pH (6.0-7.5) mussel adhesive protein solution and a low-toxicity photoinitiator, thereby avoiding the cytotoxicity of chemical cross-linking agents (EDC / NHS) and having good biocompatibility.

[0023] (3) The present application innovatively adopts a two-step light curing step-by-step process: lower layer pre-curing (5-8 seconds), silk fibroin interface coating, and upper layer synchronous curing (40-60 seconds), and the whole process takes <70 seconds, which is 10 times more efficient than the traditional multi-step chemical cross-linking (>10 minutes), and the preparation process is simple, fast and easy to operate, and only a portable 365 nm UV light source is needed. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 is a schematic diagram of the preparation method of the light-cured silk fibroin double-layer hydrogel provided by the present application;

[0025] Figure 2 is a picture of the light-cured silk fibroin double-layer hydrogel prepared in Examples 1-2 of the present application;

[0026] Figure 3 is a cell growth promoting diagram of the silk fibroin double-layer hydrogel prepared in Examples 1-2 and Comparative Examples 1-2 of the present application;

[0027] Figure 4 is an AO / EB staining diagram of the silk fibroin double-layer hydrogel prepared in Examples 1-2 and Comparative Examples 1-2 of the present application;

[0028] Figure 5 Figure 1 is a graph showing the in vitro degradation of the silk fibroin double-layer hydrogel prepared in Example 1-2 and Comparative Example 1-2 of the present application. DETAILED DESCRIPTION

[0029] Example 1

[0030] A method for preparing a photocured silk fibroin double-layer hydrogel, first preparing a pre-polymer solution of a lower hyaluronic acid hydrogel, an interface transition layer solution, and a pre-polymer solution of an upper silk fibroin hydrogel, respectively, then performing light curing step by step, to obtain the photocured silk fibroin double-layer hydrogel, prepared according to the following steps:

[0031] Step (1) Preparation of a pre-polymer solution of a lower hyaluronic acid hydrogel: first, 30.0 g of N,N-dimethylformamide was dissolved in 80.0 g of purified water, 1.0 g of hyaluronic acid was added, and purified water was added to 100.0 g, which was stirred at room temperature at a speed of 400 RPM; after the solution was dissolved, 3.0 g of methacrylic anhydride was added to the dissolved hyaluronic acid solution to 100.0 g, and sodium hydroxide was added to adjust the pH to 8.5, and reacted at 4°C for 24 hours; then 3 times the mass of anhydrous ethanol was added for product precipitation, the supernatant was removed, and the precipitate was centrifuged at a speed of 12000 RPM for 10 minutes, and the supernatant was removed; 3 times the mass of purified water was added to the precipitate to dissolve it, and deionized water dialysis was performed at 4°C for 3 days, with water changed three times a day; after dialysis, freeze-drying was performed to obtain a methacrylated hyaluronic acid powder; 4.0 g of the methacrylated hyaluronic acid powder was dissolved in 80.0 g of purified water, 0.1 g of lithium phenyl-2,4,6-trimethylbenzoylphosphinate was added after dissolution, and finally purified water was added to 100.0 g, and mixed to obtain a pre-polymer solution of a hyaluronic acid hydrogel.

[0032] Step (2) Preparation of an interface transition layer solution: 2.0 g of mussel mucin was dissolved in 80.0 g of purified water, and purified water was added to 100.0 g, and the pH was adjusted to 7.0 with 0.1 mol / L NaOH.

[0033] Step (3) Preparation of a pre-polymer solution of an upper silk fibroin hydrogel: silk was degummed with sodium carbonate, and after dissolved in a ternary solvent (calcium chloride-anhydrous ethanol-water), 0.15 wt% of methacrylic anhydride was added, and a 3500 kDa dialysis bag was dialyzed at 4°C for 3 days, with water changed three times a day; after dialysis, freeze-drying was performed to obtain a methacrylated silk fibroin powder. 7.0 g of the methacrylated silk fibroin powder and 0.2 g of lithium phenyl-2,4,6-trimethylbenzoylphosphinate were dissolved in 80.0 g of purified water, and purified water was added to 100.0 g, and mixed to obtain a pre-polymer solution of a silk fibroin hydrogel.

[0034] Step (4) Stepwise photocuring: ① Inject the pre-polymer solution of the lower layer of hyaluronic acid hydrogel into the mold, UV 365 nm (10 mW / cm²), irradiation for 6 seconds; ② Apply the interface transition layer solution on the surface of the lower layer which is not completely cured; ③ Cover the pre-polymer solution of the upper layer of silk fibroin hydrogel, UV 365 nm (10 mW / cm²), irradiation for 50 seconds. The picture of the prepared silk fibroin double-layer hydrogel is shown in Figure 2 .

[0035] Example 2

[0036] A method for preparing a photocured silk fibroin double-layer hydrogel, steps (1), (3), and (4) of this embodiment 2 are consistent with steps (1), (3), and (4) of embodiment 1, and are prepared according to the following steps:

[0037] Step (1) Preparation of the pre-polymer solution of the lower layer of hyaluronic acid hydrogel: First, dissolve 30.0 g of N,N-dimethylformamide into 80.0 g of purified water, add 1.0 g of hyaluronic acid, and add purified water to 100.0 g, stirring at room temperature at a speed of 400 RPM; after the solution is dissolved, take 3.0 g of methacrylic anhydride, add the dissolved hyaluronic acid solution to 100.0 g, adjust the pH to 8.5 with sodium hydroxide, and react at 4°C for 24 hours; then add 3 times the mass of anhydrous ethanol for product precipitation, remove the supernatant, and centrifuge the precipitate at 12000 RPM for 10 minutes, remove the supernatant; add 3 times the mass of purified water to the precipitate to dissolve it, and dialyze it in deionized water at 4°C for 3 days, changing the water three times a day; after dialysis, freeze-dry to obtain methyl methacrylate hyaluronic acid powder; dissolve 4.0 g of methyl methacrylate hyaluronic acid powder in 80.0 g of purified water, after dissolution, add 0.1 g of lithium phenyl-2,4,6-trimethylbenzoylphosphinate, and finally add purified water to 100.0 g, mix well to obtain the pre-polymer solution of the hyaluronic acid hydrogel.

[0038] Step (2) Preparation of the interface transition layer solution: Dissolve 1.5 g of mussel myosin in 80.0 g of purified water, add purified water to 100.0 g, and adjust the pH to 7.0 with 0.1 mol / L NaOH.

[0039] Step (3) Preparation of the upper layer silk fibroin hydrogel pre-polymer solution: degum silk with sodium carbonate, dissolve with ternary solvent (calcium chloride-absolute ethanol-water), add 0.15 wt% methacrylic anhydride, dialyze in a 3500 kDa dialysis bag at 4°C for 3 days, change water three times a day; after dialysis, freeze-dried to obtain methacrylated silk fibroin powder. Take 7.0 g of methacrylated silk fibroin powder and 0.2 g of lithium phenyl-2,4,6-trimethylbenzoylphosphinate and dissolve in 80.0 g of purified water, then add purified water to 100.0 g, mix well to obtain the pre-polymer solution of silk fibroin hydrogel.

[0040] Step (4) Step-by-step photocuring: ① Inject the lower layer hyaluronic acid hydrogel pre-polymer solution into the mold, UV 365 nm (10 mW / cm²), irradiate for 6 seconds; ② Coat the interface transition layer solution on the surface of the lower layer which is not completely cured; ③ Cover the upper layer silk fibroin hydrogel pre-polymer solution, UV 365 nm (10 mW / cm²), irradiate for 50 seconds. The picture of the prepared silk fibroin double-layer hydrogel is shown in Figure 2 .

[0041] Comparative Example 1

[0042] A method for preparing a photocured silk fibroin double-layer hydrogel, without mussel myoglobin interface layer in this comparative example 1, omitting mussel myoglobin coating, directly covering the upper layer pre-polymer solution after the lower layer is pre-cured, prepared according to the following steps:

[0043] Step (1) Preparation of the lower layer hyaluronic acid hydrogel pre-polymer solution: first dissolve 30.0 g of N,N-dimethylformamide in 80.0 g of purified water, add 1.0 g of hyaluronic acid, and add purified water to 100.0 g, stirring at room temperature at a speed of 400 RPM; after the solution is dissolved, take 3.0 g of methacrylic anhydride, add the dissolved hyaluronic acid solution to 100.0 g, adjust the PH to 8.5 with sodium hydroxide, and react at 4°C for 24 hours; then add 3 times the mass of absolute ethanol for product precipitation, remove the supernatant, and centrifuge the precipitate at 12000 RPM for 10 minutes to remove the supernatant; add 3 times the mass of purified water to dissolve the precipitate, dialyze in 4°C deionized water for 3 days, change water three times a day; after dialysis, freeze-dried to obtain methacrylated hyaluronic acid powder; take 4.0 g of methacrylated hyaluronic acid powder and dissolve in 80.0 g of purified water, after dissolution, add 0.1 g of lithium phenyl-2,4,6-trimethylbenzoylphosphinate, and finally add purified water to 100.0 g, mix well to obtain the pre-polymer solution of hyaluronic acid hydrogel.

[0044] Step (2) Preparation of the pre-polymer solution of the upper layer silk fibroin hydrogel: degum the silk with sodium carbonate, dissolve in the ternary solvent (calcium chloride-absolute ethanol-water), then add 0.15 wt% methacrylic anhydride, dialyze in a 3500 kDa dialysis bag at 4°C for 3 days, and change the water three times a day; after dialysis, freeze-dry to obtain methacrylated silk fibroin powder. Dissolve 7.0 g of methacrylated silk fibroin powder and 0.2 g of lithium phenyl-2,4,6-trimethylbenzoylphosphinate into 80.0 g of purified water, then add purified water to make up to 100.0 g, and mix well to obtain the pre-polymer solution of the silk fibroin hydrogel.

[0045] Step (3) Step-by-step photocuring: ① Inject the pre-polymer solution of the lower layer hyaluronic acid hydrogel into the mold, and irradiate with ultraviolet light 365 nm UV (10 mW / cm²) for 6 seconds; ② Cover the pre-polymer solution of the upper layer silk fibroin hydrogel on the surface of the lower layer that is not completely cured, and irradiate with ultraviolet light 365 nm UV (10 mW / cm²) for 50 seconds. Take out to obtain the silk fibroin double-layer hydrogel.

[0046] Comparative Example 2

[0047] In this comparative example 2, the silk fibroin hyaluronic acid hydrogel was prepared with a chemical crosslinking agent (EDC / NHS) according to the following steps:

[0048] Step (1) Degum the silk with sodium carbonate, dissolve in the ternary solvent (calcium chloride-absolute ethanol-water), then dialyze in a 3500 kDa dialysis bag at 4°C for 3 days, and change the water three times a day; after dialysis, freeze-dry to obtain silk fibroin powder.

[0049] Step (2) Dissolve 1.0 g of hyaluronic acid into 80.0 g of MES buffer solution (50 mM, PH 5.0), and add water to make up to 100.0 g, and stir until completely dissolved.

[0050] Step (3) Dissolve 1.0 g of EDC and 0.5 g of NHS into 80.0 g of purified water, respectively, and add water to make up to 100.0 g, and mix well.

[0051] Step (4) Dissolve 7.0 g of silk fibroin powder and 10.0 g of EDC / NHS solution of step (3) into 80.0 g of hyaluronic acid solution of step (2), and then add hyaluronic acid solution to make up to 100.0 g. Then, place in a constant temperature water bath at 37°C for 6 hours, and after the gel is formed, add 0.1 M glycine to terminate the reaction.

[0052] Performance test

[0053] Cell growth promoting test

[0054] The sterilized hydrogel samples of Example 1-2 and Comparative Examples 1-2 were immersed in a sterile test tube containing RPMI-1640 culture solution, and the test tube was placed in a 37°C incubator for 72 h, with the addition of culture solution at a concentration of 0.1 mL / g. 100 μL of L929 cell suspension was cultured in a 96-well plate at a density of 10 4 / mL, and after 24 h of incubation, the culture solution was removed, and the extract of the hydrogel was added to the well plate in place of the culture solution. The control group was still cultured with RPMI-1640 culture solution. After 1, 3, and 5 days of incubation, the cell morphology was observed under an inverted microscope and photographed, respectively. In addition, 50 μL of MTT solution was added to each well, and after 2 h of incubation at 37°C, the solution in the well plate was aspirated with a pipette, and 150 μL of dimethyl sulfoxide (DMSO) was added to continuously shake and dissolve the purple formazan precipitate at the bottom of the well plate. Finally, 100 μL of supernatant was aspirated, and the absorbance at 490 nm was measured with a spectrophotometer. The results are shown in Figure 3 , and the OD 490nm absorbance values of Example 1-2 were greater than those of the control group on the 1st, 3rd, and 5th days, and the OD 490nm absorbance values of Comparative Example 1 and the control group were close, but the OD 4 absorbance values of the silk fibroin double-layer hydrogel prepared by chemical crosslinking in Comparative Example 2 were significantly lower than those of the control group on the 1st, 3rd, and 5th days, indicating that Example 1-2 promoted the proliferation of L929 cells, Comparative Example 1 had no effect on the proliferation of L929 cells, but the silk fibroin double-layer hydrogel prepared in Comparative Example 2 inhibited the proliferation of L929 cells.

[0055] AO / EB Staining Test

[0056] The sterilized hydrogel samples of Example 1-2 and Comparative Examples 1-2 were immersed in a sterile test tube containing RPMI-1640 culture solution, and the test tube was placed in a 37°C incubator for 72 h, with the addition of culture solution at a concentration of 0.1 mL / g. 100 μL of L929 cell suspension was cultured in a 96-well plate at a density of 10 4 / mL, and after 24 h of incubation, the culture solution was removed, and the extract of the hydrogel was added to the well plate in place of the culture solution. The control group was still cultured with RPMI-1640 culture solution. After 24 h of incubation, the cells in the experimental group were stained with AO / EB (acridine orange / ethidium bromide), and the growth activity of the fibroblasts cultured with the extract of the hydrogel was observed under a fluorescence microscope and photographed. The results are shown in Figure 4 , and a small number of dead cells were observed for Example 1-2 and Comparative Example 1, but a large number of dead cells were observed for Comparative Example 2. This indicates that the silk fibroin double-layer hydrogel prepared in Example 1-2 and Comparative Example 1 had no cytotoxicity to L929 cells, but the silk fibroin double-layer hydrogel prepared in Comparative Example 2 had a toxic effect on L929 cells.

[0057] In vitro degradation test

[0058] The in vitro enzymatic degradation performance of the silk fibroin double-layer hydrogel was evaluated by placing the silk fibroin double-layer hydrogel in an enzyme degradation solution. At 25°C, silk fibroin double-layer hydrogel samples with a diameter of 10 mm*10 mm were prepared, the excess liquid on the surface was absorbed, and the mass was recorded as W0. They were placed in a 24-well plate, 3.0 mL of equal amount of hyaluronidase solution was added to each well, and the 24-well plate was placed in a 37°C constant temperature incubator. During the degradation period, the enzyme solution was replaced every four days. The samples were taken out on the 7th, 14th, 21st, 28th, and 35th days, respectively, the excess liquid on the surface of the gel samples was absorbed, and the mass of each group of samples was recorded as W t . Finally, the degradation curve was plotted according to the mass change, 3 parallel repeats were taken for each group of samples, the average value was taken, the remaining mass percentage (%) = (W t -W0) / W0*100%, and the experimental results are shown in Figure 5 Figure 5 As can be seen from the figure, the samples of Examples 1-2 and Comparative Examples 1-2 all showed a rapid degradation trend in the first week, and then the degradation rate was significantly slower and stable degradation, and the degradation rate and degradation rate of Examples 1-2 were lower than those of Comparative Examples 1-2, among which the degradation rate and degradation rate of Example 1 were the slowest.​

Claims

1. A photocurable silk fibroin bilayer hydrogel, characterized in that, It consists of an upper silk fibroin hydrogel, a lower hyaluronic acid hydrogel, and an interfacial transition layer of mussel adhesive protein; wherein: The upper silk fibroin hydrogel contains 6-8 wt% methacrylamide silk fibroin and 0.1-0.3 wt% photoinitiator. The lower hyaluronic acid hydrogel contains 2-5 wt% methacrylamide hyaluronic acid and 0.05-0.2 wt% photoinitiator; The interfacial transition layer has a mussel adhesive protein content of 1-3 wt% and a pH value of 6.0-7.

5.

2. The photocurable silk fibroin bilayer hydrogel according to claim 1, characterized in that: The photoinitiator is lithium phenyl-2,4,6-trimethylbenzoylphosphinate.

3. A method for preparing the photocurable silk fibroin bilayer hydrogel as described in claim 1, characterized in that... Includes the following steps: (1) Lower layer pre-curing: Inject the hyaluronic acid hydrogel prepolymer solution into the lower layer, irradiate with 365nm ultraviolet light for 5-8 seconds, with an irradiation intensity of 10mW / cm², to form a partially cross-linked gel substrate; (2) Interface treatment: Coat the incompletely cured lower surface with mussel adhesive protein solution; (3) Upper layer curing: Cover with silk fibroin hydrogel prepolymer solution, irradiate with 365nm ultraviolet light for 40-60 seconds, with an irradiation intensity of 10mW / cm², to trigger synchronous covalent cross-linking of the upper and lower layers.

4. The method for preparing the photocurable silk fibroin bilayer hydrogel according to claim 3, characterized in that, The specific preparation method of the hyaluronic acid hydrogel prepolymer solution is as follows: Hyaluronic acid is dissolved in 30 wt% N,N-dimethylformamide solution at a mass fraction of 1 wt%, and mixed well; then 2-4 wt% methacrylic anhydride is added, the pH of the solution is maintained at 8.5 with sodium hydroxide, and the reaction is carried out at 4℃ for 24 hours; then precipitation is carried out with 3 times the mass of anhydrous ethanol, the supernatant is removed after centrifugation, the solution is dissolved in water, dialyzed with deionized water for 3 days, and lyophilized to obtain methacrylamide hyaluronic acid; finally, methacrylamide hyaluronic acid is dissolved at a mass fraction of 2-5 wt%, and 0.05-0.2 wt% photoinitiator is added to dissolve it to obtain the hyaluronic acid hydrogel prepolymer solution.

5. The method for preparing the photocurable silk fibroin bilayer hydrogel according to claim 3, characterized in that, The specific preparation method of the mussel adhesive protein solution is as follows: dissolve mussel adhesive protein in an aqueous solution at a mass fraction of 1-3 wt%, and adjust the pH to 6.0-7.

5.

6. The method for preparing photocurable silk fibroin bilayer hydrogel according to claim 3, characterized in that, The specific preparation method of the silk fibroin hydrogel prepolymer solution is as follows: after degumming and dissolving silk, add 0.05-0.15 wt% methacrylic anhydride, dialyze with deionized water for 3 days, and freeze-dry to obtain methacrylamide silk fibroin; dissolve 6-8 wt% methacrylamide silk fibroin, add 0.1-0.3 wt% photoinitiator to dissolve, and then obtain the silk fibroin hydrogel prepolymer solution.

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