Methacrylated fibroin hybrid hydrogel and method for preparing the same

Abstract

The application relates to a methacrylated silk fibroin hybrid hydrogel and a preparation method thereof, wherein the methacrylated silk fibroin hybrid hydrogel is formed by a precursor system containing methacrylated silk fibroin, a Kartogenin-polyethylene glycol-maleimide prodrug, a thiol-containing crosslinking agent and a photoinitiator through light crosslinking treatment and beta-sheet induction treatment; the substitution degree of the methacrylated silk fibroin is 30% to 70%, the methacrylated silk fibroin forms a covalent crosslinking network through a light crosslinking reaction of a methacryl group, and forms a physical crosslinking network through a beta-sheet structure of a silk fibroin molecular chain segment. The Kartogenin-polyethylene glycol-maleimide prodrug is covalently fixed in the hydrogel network through a Michael addition reaction between a maleimide group and a thiol group in the thiol-containing crosslinking agent. The hybrid hydrogel has good structural stability, mechanical stability and Kartogenin sustained release capacity.

Description

Technical Field

[0001] This application relates to the field of biomedical materials technology, specifically to a methacrylamide silk fibroin hybrid hydrogel and its preparation method. Background Technology

[0002] Methacrylamide silk fibroin hydrogels have been used in tissue engineering scaffolds and drug delivery systems due to their good biocompatibility, biodegradability, and photocuring ability. For applications such as cartilage tissue repair, hydrogels not only need to rapidly form at the defect site but also need to possess certain mechanical stability and the ability to continuously release cartilage differentiation-promoting active substances. Kartogenin, a small molecule compound that can induce mesenchymal stem cells to differentiate into cartilage, is usually loaded into hydrogels through physical mixing or physical embedding. However, Kartogenin itself has poor water solubility and is prone to aggregation or uneven distribution in hydrogel precursor systems. Furthermore, due to the lack of stable binding between Kartogenin and the hydrogel network, early rapid release is common after hydrogel formation, leading to significant burst release and making it difficult to maintain a long-term stable effective drug concentration at the repair site. In addition, the methacrylamide silk fibroin hydrogels in related technologies mainly rely on photocrosslinking of methacryl groups to form a single covalent network. Although this type of network is beneficial for rapid molding, it has limited effect on improving the long-term structural stability and mechanical support of the hydrogel. If the crosslinking density is simply increased to enhance mechanical properties, it may affect the internal pore structure of the hydrogel and the release behavior of active substances. Summary of the Invention

[0003] The first aspect of this application provides a methacrylamide silk fibroin hybrid hydrogel, which can improve the structural stability and mechanical support of the hybrid hydrogel through a dual network structure, and can also improve the binding stability between Kartogenin and the hydrogel network.

[0004] The methacrylamide silk fibroin hybrid hydrogel provided in the first aspect of this application is formed by photocrosslinking and β-sheet induction treatment of a precursor system comprising methacrylamide silk fibroin, Kartogenin-polyethylene glycol-maleimide prodrug, a thiol-containing crosslinking agent and a photoinitiator; wherein the methacrylamide silk fibroin contains methacrylyl groups and the degree of substitution of the methacrylamide silk fibroin is 30% to 70%; The methacrylated silk fibroin forms a covalent cross-linked network through the photocross-linking reaction of the methacryl group, and forms a physical cross-linked network through the β-sheet structure of the silk fibroin molecular chain segments. The covalent cross-linked network and the physical cross-linked network together constitute a dual network structure. The Kartogenin-polyethylene glycol-maleimide prodrug comprises a Kartogenin group, a polyethylene glycol segment, and a maleimide group. The Kartogenin-polyethylene glycol-maleimide prodrug is covalently fixed in the hydrogel network of the hybrid hydrogel by a Michael addition reaction between the maleimide group and the thiol group in the thiol-containing crosslinking agent.

[0005] In one alternative embodiment, in the Kartogenin-polyethylene glycol-maleimide prodrug, the Kartogenin group is linked to the polyethylene glycol segment via an amide bond, and the number average molecular weight of the polyethylene glycol segment is 1000 Da to 5000 Da. The thiol-containing crosslinking agent is linked to the Kartogenin-polyethylene glycol-maleimide prodrug via maleimide-thiol addition bonds, thereby retaining the Kartogenin-polyethylene glycol-maleimide prodrug in the hydrogel network in a covalently bonded manner, thereby reducing the rapid diffusion and release of Kartogenin in the hydrogel.

[0006] In one alternative embodiment, the hybrid hydrogel has an interconnected three-dimensional porous structure with an average pore size of 70 μm to 150 μm and a porosity of not less than 85%; and the hybrid hydrogel has a compressive modulus of 10 kPa to 50 kPa and a storage modulus of 5 kPa to 30 kPa, thereby improving the mechanical stability of the hybrid hydrogel through the dual-network structure.

[0007] In one alternative embodiment, the Kartogenin is covalently immobilized in the hydrogel network in the form of a Kartogenin-polyethylene glycol-maleimide prodrug; the burst release rate of the Kartogenin in the in vitro release system is no higher than 20% in the first 24 hours, and the cumulative release time is no less than 14 days; when the hybrid hydrogel is used for three-dimensional culture of human bone marrow mesenchymal stem cells, the cell survival rate after 7 days of culture is no less than 90%.

[0008] In this methacrylamide silk fibroin hybrid hydrogel, methacrylamide silk fibroin can form a covalent cross-linked network through the photocrosslinking reaction of methacryl groups and a physical cross-linked network through the β-sheet structure of the silk fibroin molecular chain segments, resulting in a dual-network structure of covalent and physical cross-linked networks in the hybrid hydrogel. Simultaneously, Kartogenin is incorporated into the hydrogel network in the form of a Kartogenin-polyethylene glycol-maleimide prodrug. The polyethylene glycol segments improve the dispersion of Kartogenin in the hydrogel system, and the maleimide groups undergo a Michael addition reaction with the thiol groups in the thiol-containing crosslinking agent, covalently fixing the Kartogenin-polyethylene glycol-maleimide prodrug within the hydrogel network. Therefore, on the one hand, the dual-network structure enhances the structural stability and mechanical support of the hybrid hydrogel; on the other hand, it improves the binding stability between Kartogenin and the hydrogel network, reducing the early rapid release of Kartogenin, thus giving the hybrid hydrogel good molding stability, mechanical stability, and sustained Kartogenin release capability.

[0009] The second aspect of this application provides a method for preparing a methacrylamide silk fibroin hybrid hydrogel, used to prepare the methacrylamide silk fibroin hybrid hydrogel provided in the first aspect. The preparation method includes the following steps: S1. Degumming, dissolving, dialysis and freeze-drying of silkworm cocoons to obtain regenerated silk fibroin; S2. The regenerated silk fibroin is subjected to a methacrylation reaction with methacrylic anhydride to obtain methacrylated silk fibroin with a degree of substitution of 30% to 70%. S3. Kartogenin is coupled with amino-polyethylene glycol maleimide to obtain Kartogenin-polyethylene glycol-maleimide prodrug; S4. Mix the methacrylamide silk fibroin, the Kartogenin-polyethylene glycol-maleimide prodrug, the thiol-containing crosslinking agent and the photoinitiator to form a precursor solution; S5. The precursor solution is subjected to light treatment, so that the methacrylamide silk fibroin forms a covalent cross-linked network through the photocross-linking reaction of the methacrylamide group, and the Kartogenin-polyethylene glycol-maleimide prodrug is covalently fixed in the hydrogel network through the Michael addition reaction of the maleimide group with the thiol-containing cross-linking agent. S6. The hydrogel after light treatment is subjected to β-sheet induction treatment to induce the formation of a physical cross-linking network of silk fibroin molecular chain segments, thereby obtaining a methacrylamide silk fibroin hybrid hydrogel composed of the covalent cross-linking network and the physical cross-linking network.

[0010] In one alternative approach, step S1 specifically includes the following steps: After the silkworm cocoons are cut into pieces, they are boiled in a sodium carbonate solution to remove sericin and obtain silk fibroin fibers. The silk fibroin fibers were washed with deionized water and dried, then dissolved in lithium bromide solution to obtain a silk fibroin protein solution. The silk fibroin solution was placed in a dialysis bag with a molecular weight cutoff of 3500 Da, dialyzed in deionized water, and then freeze-dried to obtain the regenerated silk fibroin; wherein the silk fibroin fibers were dissolved in the lithium bromide solution at 60°C, and the dialysis was used to remove lithium bromide and obtain purified regenerated silk fibroin.

[0011] In one alternative approach, step S2 specifically includes the following steps: The regenerated silk fibroin was dissolved in a phosphate buffer solution with a preset pH to form a silk fibroin solution with a mass-volume concentration of 6%. Methacrylic anhydride is added to the silk fibroin solution to make the molar ratio of the methacrylic anhydride to the amino and / or tyrosine residues in the silk fibroin 0.5:1 to 2:1. The reaction is carried out at pH 7.4-8.0 for 1-4 hours to introduce methacryloyl groups onto the silk fibroin molecular chain; After the reaction was completed, unreacted methacrylic anhydride and byproducts were removed by dialysis, and the methacrylated silk fibroin was obtained by freeze drying.

[0012] In one alternative approach, step S3 specifically includes the following steps: Dissolve Kartogenin in an anhydrous organic solvent, add 1.0-1.5 equivalents of EDC and NHS, and activate the carboxyl groups in Kartogenin at room temperature for 2-6 hours. The activated Kartogenin was reacted with amino polyethylene glycol maleimide with a number average molecular weight of 1000 Da to 5000 Da at a pH of 7.2 to 7.6 for 8 to 24 hours, so that the Kartogenin was linked to the polyethylene glycol segment via amide bonds. After the reaction is complete, unreacted small molecule byproducts and organic solvents are removed by dialysis or high performance liquid chromatography, and then freeze-dried to obtain the Kartogenin-polyethylene glycol-maleimide prodrug.

[0013] In an optional embodiment, in step S4, the precursor solution is prepared using phosphate buffer or cell culture medium, and the concentration of the methacrylamide silk fibroin in the precursor solution is 5% to 15% w / v. The mass ratio of the methacrylamide silk fibroin to the Kartogenin-polyethylene glycol-maleimide prodrug is 5:1 to 20:1; the photoinitiator is LAP and / or Irgacure2959, and the concentration of the photoinitiator in the precursor solution is 0.1% to 1% w / v; The precursor solution is subjected to light-shielded stirring and degassing treatment before being used for photoforming to reduce the influence of oxygen on the photocrosslinking reaction and improve the uniformity of the precursor solution.

[0014] In one optional scheme, in step S5, a light source with a wavelength of 365nm~405nm and a light intensity of 5mW / cm²~20mW / cm² is used for the light treatment, and the irradiation time is 30S~2min, so that the precursor solution is photocrosslinked and formed. Step S6, when performing β-sheet induction treatment, includes: immersing the phototreated hydrogel in phosphate buffer or ethanol / water mixture to induce the formation of β-sheet structures in silk fibroin molecular chains; after the phototreatment and β-sheet induction treatment, the hybrid hydrogel forms a dual-network structure in which covalent cross-linked networks and physical cross-linked networks coexist, and the Kartogenin-polyethylene glycol-maleimide prodrug is covalently fixed in the hydrogel network.

[0015] The preparation method first introduces methacryloyl groups into the silk fibroin molecular chain through methacrylation modification, enabling the resulting methacryloylated silk fibroin to have photocrosslinking molding capabilities. Then, by coupling Kartogenin with amino-polyethylene glycol maleimide, a Kartogenin-polyethylene glycol-maleimide prodrug with polyethylene glycol segments and maleimide groups is obtained, allowing Kartogenin to enter the precursor solution in a well-dispersible prodrug form and to undergo Michael addition reaction with a thiol-containing crosslinking agent through the maleimide groups. Subsequently, the methacryloylated silk fibroin, Kartogenin-polyethylene glycol-maleimide prodrug, thiol-containing crosslinking agent, and photoinitiator are mixed to form a precursor solution, which is then phototreated to form a covalent crosslinking network and β-sheet induced to form a physical crosslinking network. Therefore, this preparation method can combine photocrosslinking molding, Kartogenin pre-drugation, maleimide-thiol covalent fixation, and β-sheet physical crosslinking in the same preparation process, so that the resulting hybrid hydrogel forms a dual network structure with both covalent crosslinking network and physical crosslinking network, and Kartogenin is stably bound in the hydrogel network, thereby obtaining a methacrylamide silk fibroin hybrid hydrogel with good molding stability, mechanical stability and continuous release ability of Kartogenin.

[0016] It should be understood that the above general description and the following detailed description are merely exemplary and do not limit this application. Detailed Implementation

[0017] To better understand the technical solution of this application, the following will describe this application in detail with reference to specific embodiments. It should be understood that the described embodiments are merely some embodiments of this application, and not all embodiments. All other technical solutions obtained by those skilled in the art based on the embodiments of this application without inventive effort are within the scope of protection of this application.

[0018] The terminology used in the embodiments of this application is for the purpose of describing particular embodiments only and is not intended to be limiting of this application. The singular forms “a,” “the,” and “the” used in the embodiments of this application and the appended claims are also intended to include the plural forms unless the context clearly indicates otherwise.

[0019] It should be understood that the term "and / or" used in this article is merely a description of the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A existing alone, A and B existing simultaneously, and B existing alone. Additionally, the character " / " in this article generally indicates that the preceding and following related objects have an "or" relationship.

[0020] The first aspect of this application provides a methacrylamide silk fibroin hybrid hydrogel, which is formed by photocrosslinking and β-sheet induction treatment of a precursor system comprising methacrylamide silk fibroin, Kartogenin-polyethylene glycol-maleimide prodrug, a thiol-containing crosslinking agent and a photoinitiator; the methacrylamide silk fibroin contains methacrylyl groups and the degree of substitution of the methacrylamide silk fibroin is 30% to 70%.

[0021] Methacrylamide silk fibroin forms a covalent cross-linked network through the photocross-linking reaction of the methacryloyl group, and a physical cross-linked network through the β-sheet structure of the silk fibroin molecular chain segments. The covalent cross-linked network and the physical cross-linked network together constitute a double network structure. The Kartogenin-polyethylene glycol-maleimide prodrug includes a Kartogenin group, a polyethylene glycol segment and a maleimide group. The Kartogenin-polyethylene glycol-maleimide prodrug is covalently fixed in the hydrogel network of the hybrid hydrogel through a Michael addition reaction between the maleimide group and the thiol group in the thiol-containing cross-linking agent.

[0022] It should be noted that the degree of substitution of methacrylamide silk fibroin is used to characterize the extent to which methacryl groups are introduced into the silk fibroin molecular chain that can participate in photocrosslinking reactions. When the degree of substitution is below 30%, the number of covalent crosslinking sites formed in the precursor system under light conditions is relatively small, and the network integrity and mechanical stability after hydrogel molding may be insufficient. When the degree of substitution is above 70%, the number of groups on the silk fibroin molecular chain that can be recognized by cells or maintain the native conformation decreases, and excessively high crosslinking density may reduce the pore connectivity and drug release controllability of the hydrogel. Therefore, controlling the degree of substitution of methacrylamide silk fibroin between 30% and 70% can achieve a good balance between photocuring ability, network stability, and biocompatibility.

[0023] Furthermore, thiol-containing crosslinking agents are used to provide thiol reaction sites for the maleimide groups to undergo addition reactions. These thiol-containing crosslinking agents can be dithiol polyethylene glycol, dithiothreitol, cysteine-derived crosslinking agents, or other crosslinking agents containing at least one thiol group and capable of dispersibility in aqueous precursor systems. To ensure the stable retention of the Kartogenin-polyethylene glycol-maleimide prodrug in the hydrogel network, a thiol-containing crosslinking agent containing two or more thiol groups is preferred. This allows one or part of the thiol group at one end to undergo Michael addition with the maleimide group of the Kartogenin-polyethylene glycol-maleimide prodrug, while the other end or remaining thiol group can bind to reactive groups or adjacent network structures in the hydrogel network, thereby improving the immobilization stability of the prodrug in the hydrogel network.

[0024] The maleimide-thiol Michael addition reaction can be carried out under near-neutral aqueous conditions, typically without requiring high temperatures or strong acid / base conditions, making it suitable for use with hydrogel precursor systems containing bioactive small molecules. In some embodiments, the molar ratio of maleimide groups to thiol groups can be 1:0.5 to 1:2, preferably 1:0.8 to 1:1.2. If the amount of thiol groups is too low, the covalent fixation of the Kartogenin-polyethylene glycol-maleimide prodrug may be insufficient; if the amount of thiol groups is too high, excessive thiol groups may affect the crosslinking uniformity of the precursor system or alter the release behavior of the hydrogel.

[0025] In some embodiments, the thiol-containing crosslinking agent can be added to the precursor system before phototreatment and pre-reacted with the Kartogenin-polyethylene glycol-maleimide prodrug for a certain period of time; alternatively, it can be mixed with methacrylamide silk fibroin, the Kartogenin-polyethylene glycol-maleimide prodrug, and a photoinitiator, allowing a maleimide-thiol addition reaction to occur simultaneously during the gelation process of the precursor system. The former is beneficial for improving the pre-binding degree between the prodrug and the thiol-containing crosslinking agent, while the latter simplifies the preparation steps.

[0026] In the methacrylamide silk fibroin hybrid hydrogel of this embodiment, the methacrylamide silk fibroin can form a covalent cross-linked network through the photocross-linking reaction of the methacryl group, and can also form a physical cross-linked network through the β-sheet structure of the silk fibroin molecular chain segments, so that the hybrid hydrogel forms a dual network structure with both covalent and physical cross-linked networks. At the same time, Kartogenin is set in the hydrogel network in the form of Kartogenin-polyethylene glycol-maleimide prodrug. The polyethylene glycol segment can improve the dispersion state of Kartogenin in the hydrogel system, and the maleimide group can undergo Michael addition reaction with the thiol group in the thiol-containing cross-linking agent, so that the Kartogenin-polyethylene glycol-maleimide prodrug is covalently fixed in the hydrogel network. Thus, on the one hand, it can improve the structural stability and mechanical support of the hybrid hydrogel through the dual network structure, and on the other hand, it can improve the binding stability between Kartogenin and the hydrogel network, reduce the early rapid release of Kartogenin, and enable the hybrid hydrogel to have good molding stability, mechanical stability and Kartogenin continuous release capability.

[0027] Specifically, the covalent cross-linking network is mainly used to endow the hybrid hydrogel with rapid molding and shape retention capabilities, while the β-sheet physical cross-linking network is mainly used to enhance the stability of the network structure through hydrogen bonding, hydrophobic interactions, and regular segment arrangement between silk fibroin molecular chains. The two networks interpenetrate and jointly restrict excessive movement of chain segments within the hydrogel, allowing the hybrid hydrogel to maintain a relatively intact network structure during water absorption, pressure, or drug release. Furthermore, the polyethylene glycol segment in the Kartogenin-polyethylene glycol-maleimide prodrug is located between the Kartogenin and maleimide groups. This improves the dispersibility of Kartogenin in the aqueous precursor system and forms a flexible space between the Kartogenin groups and the hydrogel network, allowing Kartogenin to retain some molecular mobility after being immobilized in the hydrogel network, which is beneficial for its gradual release during hydrogel degradation or network exchange.

[0028] In one specific embodiment, in the Kartogenin-polyethylene glycol-maleimide prodrug, the Kartogenin group is linked to a polyethylene glycol segment via an amide bond, and the number average molecular weight of the polyethylene glycol segment is 1000 Da to 5000 Da; a thiol-containing crosslinking agent is linked to the Kartogenin-polyethylene glycol-maleimide prodrug via a maleimide-thiol addition bond, so that the Kartogenin-polyethylene glycol-maleimide prodrug is retained in the hydrogel network in a covalently bonded manner, thereby reducing the rapid diffusion and release of Kartogenin in the hydrogel.

[0029] In this formulation, the Kartogenin group is linked to the polyethylene glycol (PEG) segment via an amide bond. The amide bond provides good aqueous stability, ensuring that Kartogenin maintains its connection with the PEG segment during precursor solution preparation, photoforming, and β-sheet induction. When the number-average molecular weight of the PEG segment is between 1000 Da and 5000 Da, it improves the water dispersibility of Kartogenin while preventing the prodrug molecule from becoming too large and affecting its uniform distribution in the hydrogel precursor system.

[0030] In some embodiments, the number-average molecular weight of the polyethylene glycol (PEG) segments can be 2000 Da, 3400 Da, or 5000 Da. Lower number-average PEG segments are advantageous for reducing the prodrug molecule size, while higher number-average PEG segments are advantageous for improving the hydrophilicity of the prodrug in aqueous systems. Those skilled in the art can select an appropriate molecular weight of amino-PEG maleimide based on the viscosity of the precursor solution, gelation rate, and Kartogenin release cycle.

[0031] In one specific embodiment, the hybrid hydrogel has an interconnected three-dimensional porous structure with an average pore size of 70 μm to 150 μm and a porosity of not less than 85%; and the hybrid hydrogel has a compressive modulus of 10 kPa to 50 kPa and a storage modulus of 5 kPa to 30 kPa, thereby improving the mechanical stability of the hybrid hydrogel through a dual-network structure.

[0032] The interconnected three-dimensional porous structure provides channels for the transport of water, nutrients, metabolites, and bioactive substances, and provides space for cell adhesion, extension, and proliferation within the hydrogel. When the average pore size is 70 μm to 150 μm, the hydrogel can balance pore connectivity and mechanical support; if the pore size is too small, internal mass transfer and cell activity space may be limited; if the pore size is too large, the hydrogel network support capacity may decrease.

[0033] In one specific embodiment, Kartogenin is covalently immobilized in a hydrogel network in the form of a Kartogenin-polyethylene glycol-maleimide prodrug; the burst release rate of Kartogenin in the in vitro release system is no higher than 20% in the first 24 hours, and the cumulative release time is no less than 14 days; when the hybrid hydrogel is used for three-dimensional culture of human bone marrow mesenchymal stem cells, the cell survival rate after 7 days of culture is no less than 90%.

[0034] The burst release rate in the first 24 hours was used to evaluate whether Kartogenin was rapidly released in the early stages of hydrogel formation, while the cumulative release time was used to evaluate whether Kartogenin could be released continuously over a longer period. Since the Kartogenin-polyethylene glycol-maleimide prodrug is covalently fixed in the hydrogel network via maleimide-thiol addition bonds, the release of Kartogenin no longer primarily depends on simple diffusion, but is jointly regulated by the binding state of the prodrug to the hydrogel network, the pore structure of the hydrogel network, and the gradual swelling or degradation process of the hydrogel. Therefore, early burst release can be reduced and the release period prolonged. A cell viability of at least 90% indicates that the network structure, residual reactant levels, and Kartogenin release state formed under the hybrid hydrogel preparation conditions meet the basic biocompatibility requirements for three-dimensional culture of human bone marrow mesenchymal stem cells.

[0035] The second aspect of this application provides a method for preparing a methacrylamide silk fibroin hybrid hydrogel, used to prepare the methacrylamide silk fibroin hybrid hydrogel of the first aspect embodiment. Specifically, the preparation method mainly includes the following steps: S1. Degumming, dissolving, dialysis and freeze-drying of silkworm cocoons to obtain regenerated silk fibroin; S2. Regenerated silk fibroin is subjected to a methacrylation reaction with methacrylic anhydride to obtain methacrylated silk fibroin with a degree of substitution of 30% to 70%. S3. Kartogenin is coupled with amino-polyethylene glycol maleimide to obtain Kartogenin-polyethylene glycol-maleimide prodrug; S4. Mix methacrylamide silk fibroin, Kartogenin-polyethylene glycol-maleimide prodrug, thiol-containing crosslinking agent and photoinitiator to form a precursor solution; S5. The precursor solution is subjected to light treatment, so that the methacrylamide silk fibroin forms a covalent cross-linked network through the photocross-linking reaction of the methacrylamide group, and the Kartogenin-polyethylene glycol-maleimide prodrug is covalently fixed in the hydrogel network through the Michael addition reaction between the maleimide group and the thiol group in the thiol-containing cross-linking agent. S6. The hydrogel after light treatment is subjected to β-sheet induction treatment to induce the formation of a physical cross-linking network of silk fibroin molecular chain segments, thereby obtaining a methacrylamide silk fibroin hybrid hydrogel composed of a covalent cross-linking network and a physical cross-linking network.

[0036] In the above preparation method, step S1 is used to obtain regenerated silk fibroin with good purity and reprocessability; step S2 is used to introduce photocrosslinkable methacryloyl groups onto the regenerated silk fibroin molecular chain; step S3 is used to convert the poorly water-soluble Kartogenin into a prodrug form with polyethylene glycol segments and maleimide reactive groups; step S4 is used to form a homogeneous hydrogel precursor solution; step S5 is used to form a covalent crosslinking network and complete the covalent fixation of Kartogenin-polyethylene glycol-maleimide prodrug in the hydrogel network; and step S6 is used to further induce the silk fibroin molecular chain segments to form a β-sheet physical crosslinking network. The steps are sequentially coordinated, enabling the resulting hybrid hydrogel to simultaneously possess photocurable molding capability, network structure stability, and sustained release capability of Kartogenin.

[0037] The preparation method first introduces methacryloyl groups into the silk fibroin molecular chain through methacrylation modification, enabling the resulting methacryloylated silk fibroin to have photocrosslinking molding capabilities. Then, by coupling Kartogenin with amino-polyethylene glycol maleimide, a Kartogenin-polyethylene glycol-maleimide prodrug with polyethylene glycol segments and maleimide groups is obtained, allowing Kartogenin to enter the precursor solution in a well-dispersible prodrug form and to undergo Michael addition reaction with a thiol-containing crosslinking agent through the maleimide groups. Subsequently, the methacryloylated silk fibroin, Kartogenin-polyethylene glycol-maleimide prodrug, thiol-containing crosslinking agent, and photoinitiator are mixed to form a precursor solution, which is then phototreated to form a covalent crosslinking network and β-sheet induced to form a physical crosslinking network.

[0038] Therefore, this preparation method can combine photocrosslinking molding, Kartogenin pre-drugation, maleimide-thiol covalent fixation, and β-sheet physical crosslinking in the same preparation process, so that the resulting hybrid hydrogel forms a dual network structure with both covalent crosslinking network and physical crosslinking network, and Kartogenin is stably bound in the hydrogel network, thereby obtaining a methacrylamide silk fibroin hybrid hydrogel with good molding stability, mechanical stability and continuous release ability of Kartogenin. It should be further clarified that the Kartogenin prodrugation, maleimide-thiol covalent fixation, and β-sheet induction treatment are not isolated steps. Kartogenin prodrugation improves the dispersion of Kartogenin in the aqueous precursor system and provides it with maleimide groups capable of addition reactions. Thiol-containing crosslinking agents react with the maleimide groups, preventing the Kartogenin-polyethylene glycol-maleimide prodrug from rapidly diffusing and leaching after hydrogel formation. β-sheet induction treatment enhances the physical interactions between silk fibroin segments, further limiting the rapid relaxation of the hydrogel network. Through the coordination of these steps, the hydrogel formation, reinforcement, and drug fixation processes can be completed within the same material system.

[0039] In one specific embodiment, step S1 specifically includes the following steps: cutting the silkworm cocoons into pieces and boiling them in a sodium carbonate solution to remove sericin and obtain silk fibroin fibers; Silk fibroin fibers were washed with deionized water and dried, then dissolved in lithium bromide solution to obtain a silk fibroin protein solution. The silk fibroin solution was placed in a dialysis bag with a molecular weight cutoff of 3500 Da, dialyzed in deionized water, and then freeze-dried to obtain regenerated silk fibroin. In this process, the silk fibroin fibers were dissolved in a lithium bromide solution at 60°C, and dialysis was used to remove the lithium bromide and obtain purified regenerated silk fibroin.

[0040] In this embodiment, sodium carbonate solution is used to remove sericin from silkworm cocoons to reduce the impact of residual sericin on subsequent dissolution, methacrylization reaction, and hydrogel biocompatibility. The concentration of sodium carbonate solution can be 0.01M~0.05M, and the boiling treatment time can be 20~60 minutes. If the treatment time is too short, sericin removal will be insufficient; if the treatment time is too long, the silk fibroin molecular chains may be excessively degraded. Lithium bromide solution is used to disrupt the stable intermolecular interactions in the silk fibroin fibers, transforming the silk fibroin fibers into a flowable silk fibroin solution. The concentration of lithium bromide solution can be 8M~10M; the dissolution temperature can be 50~70℃; and the dissolution time can be 1~4 hours. These conditions ensure dissolution efficiency while reducing excessive breakage of silk fibroin molecular chains.

[0041] Dialysis is used to remove lithium bromide, small molecule impurities, and residual salts. The molecular weight cutoff of the dialysis bag can be 3000 Da to 5000 Da; the dialysis time can be 2 to 4 days; during dialysis, deionized water can be changed every 6 to 12 hours to maintain dialysis drive and reduce salt residue. Freeze-drying can produce regenerated silk fibroin into a porous, sponge-like solid, facilitating subsequent weighing, dissolution, and methacrylylation reactions.

[0042] In one specific embodiment, step S2 specifically includes the following steps: dissolving regenerated silk fibroin in a phosphate buffer solution with a preset pH to form a silk fibroin solution with a mass-volume concentration of 6%. Add methacrylic anhydride to the silk fibroin solution so that the molar ratio of methacrylic anhydride to amino and / or tyrosine residues in the silk fibroin is 0.5:1 to 2:1. The reaction is carried out at pH 7.4-8.0 for 1-4 hours to introduce methacryloyl groups onto the silk fibroin molecular chain; After the reaction was completed, unreacted methacrylic anhydride and byproducts were removed by dialysis, and the product was freeze-dried to obtain methacrylamide silk fibroin.

[0043] Methacrylic anhydride can react with amino and / or tyrosine residues in the silk fibroin molecular chain, introducing methacryloyl groups onto the silk fibroin molecular chain. These methacryloyl groups can undergo free radical polymerization under photoinitiator and light irradiation conditions, thereby forming a covalently cross-linked network of methacrylated silk fibroin. By controlling the molar ratio of methacrylic anhydride to amino and / or tyrosine residues in silk fibroin, the degree of substitution of methacrylated silk fibroin can be adjusted, further regulating the gelation rate, cross-linking density, and mechanical properties of the hydrogel.

[0044] Maintaining the pH at 7.4–8.0 during the reaction helps preserve the stability of silk fibroin in the aqueous system and reduces silk fibroin aggregation caused by methacrylic anhydride hydrolysis or excessive acidity in the reaction system. The pH of the reaction system can be adjusted using sodium hydroxide solution or a phosphate buffer system. The reaction temperature can be 4°C–25°C, preferably room temperature; the reaction time can be 1–4 hours. After the reaction, unreacted methacrylic anhydride, methacrylic acid, and other small molecule byproducts are removed by dialysis, followed by freeze-drying to obtain methacrylamide silk fibroin.

[0045] In addition, in some embodiments, 1H NMR spectroscopy, Fourier transform infrared spectroscopy, the trinitrobenzenesulfonic acid method, or a combination thereof, can be used to characterize methacryloyl-modified silk fibroin. Specifically, the methacryloyl vinyl proton peak in 1H NMR spectroscopy can be used to determine whether a methacryloyl group has been introduced; the ester carbonyl characteristic peak in Fourier transform infrared spectroscopy can be used to assist in confirming the grafting reaction; and the trinitrobenzenesulfonic acid method can be used to determine the change in free amino content before and after the reaction, and thereby calculate the degree of substitution of methacryloyl-modified silk fibroin.

[0046] In one specific embodiment, step S3 specifically includes the following steps: dissolving Kartogenin in an anhydrous organic solvent, adding 1.0~1.5 equivalents of EDC and NHS, and activating the carboxyl groups in Kartogenin at room temperature for 2~6 hours; The activated Kartogenin was reacted with amino polyethylene glycol maleimide with a number average molecular weight of 1000 Da to 5000 Da at a pH of 7.2 to 7.6 for 8 to 24 hours, so that Kartogenin was linked to the polyethylene glycol segment through amide bonds. After the reaction was completed, unreacted small molecule byproducts and organic solvents were removed by dialysis or high performance liquid chromatography. The product was then freeze-dried to obtain the Kartogenin-polyethylene glycol-maleimide prodrug.

[0047] The anhydrous organic solvent can be anhydrous dimethyl sulfoxide or anhydrous N,N-dimethylformamide. The anhydrous organic solvent is used to dissolve Kartogenin, which has poor water solubility, and to reduce the influence of moisture on the carboxyl activation reaction. 1-Ethyl-(3-dimethylaminopropyl)carbodiimide is used to activate the carboxyl group in Kartogenin, and N-hydroxysuccinimide is used to form a more stable active ester intermediate, thereby improving the efficiency of the subsequent coupling reaction with the amino group in amino-polyethylene glycol maleimide. Amino-polyethylene glycol maleimide is a bifunctional polyethylene glycol derivative; one end of its amino group can form an amide bond with the activated Kartogenin carboxyl group, while the other end of its maleimide group can be retained as a reaction site for subsequent reactions with a thiol-containing crosslinking agent. Therefore, the resulting Kartogenin-polyethylene glycol-maleimide prodrug simultaneously possesses the active Kartogenin group, a polyethylene glycol segment that improves water dispersibility, and a maleimide group for covalent fixation.

[0048] The pH of the reaction system is controlled at 7.2–7.6, which is conducive to the amidation reaction between amino and activated carboxyl groups, while reducing the hydrolysis of maleimide groups under slightly alkaline conditions. The reaction is preferably conducted in the dark to minimize unintended changes in Kartogenin or maleimide groups under light exposure. After the reaction, dialysis can be performed using a dialysis bag with a molecular weight cutoff of approximately 1000 Da, or preparative high-performance liquid chromatography can be used for purification to remove unreacted Kartogenin, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, N-hydroxysuccinimide, and organic solvents; specific limitations are not specified in this paper.

[0049] In one specific embodiment, in step S4, a precursor solution is prepared using phosphate buffer or cell culture medium, and the concentration of methacrylamide silk fibroin in the precursor solution is 5%~15% w / v. The mass ratio of methacrylamide silk fibroin to Kartogenin-polyethylene glycol-maleimide prodrug is 5:1 to 20:1; the photoinitiator is LAP and / or Irgacure2959, and the concentration of the photoinitiator in the precursor solution is 0.1% to 1% w / v; The precursor solution is subjected to light-shielded stirring and degassing treatment before being used for photoforming to reduce the influence of oxygen on the photocrosslinking reaction and improve the uniformity of the precursor solution.

[0050] The concentration of methacrylamide silk fibroin in the precursor solution is used to adjust the network density, pore structure, and mechanical properties of the hydrogel. Lower concentrations of methacrylamide silk fibroin result in hydrogels with larger pore sizes and higher flexibility, but may reduce mechanical support. Higher concentrations result in denser hydrogel networks and higher compressive modulus, but may reduce cell activity space and drug release rate. Therefore, controlling the methacrylamide silk fibroin concentration at 5%–15% w / v achieves a good balance between structural stability, pore connectivity, and drug release performance. Furthermore, the mass ratio of methacrylamide silk fibroin to the Kartogenin-polyethylene glycol-maleimide prodrug is 5:1–20:1 to control the ratio between the prodrug loading and the main hydrogel network material.

[0051] Furthermore, the thiol-containing crosslinking agent can be added to the precursor solution after the Kartogenin-polyethylene glycol-maleimide prodrug, or it can be pre-reacted by mixing it with the Kartogenin-polyethylene glycol-maleimide prodrug in phosphate buffer before mixing it with methacrylamide silk fibroin and a photoinitiator. The amount of thiol-containing crosslinking agent added can be determined according to the molar ratio of maleimide groups to thiol groups, which can be 1:0.5 to 1:2. The pre-reaction conditions can be pH 6.8 to 7.6, room temperature to 37°C, and reaction in the dark for 5 to 60 minutes. These treatments can improve the binding degree between the Kartogenin-polyethylene glycol-maleimide prodrug and the thiol-containing crosslinking agent.

[0052] In some embodiments, when the thiol-containing crosslinking agent is dithiol polyethylene glycol, its number-average molecular weight can be 500 Da to 5000 Da; a lower number-average molecular weight is beneficial for improving crosslinking efficiency, while a higher number-average molecular weight is beneficial for improving network flexibility. When the thiol-containing crosslinking agent is dithiothreitol, its small molecular structure facilitates rapid reaction with maleimide groups. When the thiol-containing crosslinking agent is a cysteine-derived crosslinking agent, its thiol groups can participate in addition reactions, and its amino acid structure can improve the biocompatibility of the system. All of the above-mentioned thiol-containing crosslinking agents are materials that can be obtained by those skilled in the art and directly used in aqueous hydrogel systems.

[0053] In one specific embodiment, in step S5, a light source with a wavelength of 365nm~405nm and a light intensity of 5mW / cm²~20mW / cm² is used for the light irradiation treatment, and the irradiation time is 30S~2min, so that the precursor solution is photocrosslinked and formed. Step S6, during the β-sheet induction treatment, includes: immersing the phototreated hydrogel in phosphate buffer or an ethanol / water mixture to induce the formation of β-sheet structures in silk fibroin molecular chains; after phototreatment and β-sheet induction treatment, the hybrid hydrogel forms a dual-network structure in which covalent cross-linked networks and physical cross-linked networks coexist, and the Kartogenin-polyethylene glycol-maleimide prodrug is covalently fixed in the hydrogel network.

[0054] In step S5, the light treatment initiates free radical polymerization of the methacryloyl groups in methacryloyl silk fibroin, forming a covalent cross-linked network between the methacryloyl silk fibroin molecular chains. Simultaneously, the Kartogenin-polyethylene glycol-maleimide prodrug in the precursor solution undergoes a Michael addition reaction with the thiol groups in the thiol-containing cross-linking agent via the maleimide groups, thus immobilizing the Kartogenin-polyethylene glycol-maleimide prodrug within the hydrogel network during gelation. When the light wavelength is 365nm~405nm, the light intensity is 5mW / cm²~20mW / cm², and the irradiation time is 30s~2min, the photocross-linking of the precursor solution can be completed in a shorter time, reducing the potential impact of prolonged light exposure on Kartogenin and cell compatibility.

[0055] In step S6, the β-sheet induction treatment can be achieved through phosphate buffer equilibration, ethanol / water mixture treatment, or a combination thereof. The ethanol / water mixture reduces the hydration level around the silk fibroin molecular chains, promoting the orderly arrangement of silk fibroin segments and the formation of β-sheet structures; phosphate buffer equilibration removes unreacted small molecules and brings the hydrogel to a hydrated state suitable for subsequent applications. In some embodiments, the ethanol volume fraction in the ethanol / water mixture can be 20% to 60%, preferably 40%; the induction time can be 10 minutes to 60 minutes, preferably 30 minutes.

[0056] After phototreatment and β-sheet induction treatment, the covalent cross-linked network in the hybrid hydrogel mainly provides rapid molding and basic shape retention capabilities, while the physical cross-linked network enhances network stability and resistance to deformation through its β-sheet structure. The Kartogenin-polyethylene glycol-maleimide prodrug forms a stable bond with the hydrogel network through maleimide-thiol addition bonds. The combined effect of these structures enables the hybrid hydrogel to reduce early burst release of Kartogenin and maintain its release over a longer period during use.

[0057] The following will illustrate this application with a specific, tested, and verified case: Preparation of methacrylamide silk fibroin (SilMA): Silk fibroin extraction: Silkworm cocoons were cut into small pieces and boiled in 0.02M Na₂CO₃ solution for 30 minutes to remove sericin. The cocoons were then rinsed repeatedly with deionized water and dried at room temperature. The purified silk fibroin fibers were dissolved in 9.3M LiBr solution and heated in a 60°C water bath for 2 hours until completely dissolved. The solution was transferred to a dialysis bag (MWCO₃ 500 Da) and dialyzed against deionized water for 3 days, changing the water every 8 hours. After dialysis, the solution was freeze-dried to obtain white, spongy, regenerated silk fibroin.

[0058] Methacrylylation modification: Regenerated silk fibroin was dissolved in PBS (pH 7.4) to prepare a 6% (w / v) solution. Methacrylic anhydride was slowly added dropwise at a molar ratio of 1.5:1 to amino residues on the silk fibroin, while maintaining the pH between 7.4 and 8.0 with 1M NaOH. The reaction was carried out at room temperature for 2 hours. After the reaction was completed, the reaction solution was placed in a dialysis bag (MWCO 3500 Da) and dialyzed against deionized water for 3 days. SilMA was obtained by freeze-drying.

[0059] Structural characterization: ¹H NMR (D₂O, 400 MHz) showed characteristic peaks of the vinyl protons of the methacryloyl group at δ 5.3 and δ 5.7; FTIR (KBr pellet) showed a characteristic absorption peak of the ester carbonyl group (C=O) at 1720 cm⁻¹, confirming successful grafting of the methacryloyl group. The degree of amino substitution was determined by the trinitrobenzenesulfonic acid (TNBS) method, and the calculated degree of substitution was 58%.

[0060] Synthesis of Kartogenin-PEG-MAL (KGN-PEG-MAL) Activation of Kartogenin: Dissolve 10 mg of Kartogenin (approximately 0.032 mmol) in 2 mL of anhydrous DMSO, and add 6.1 mg of EDC (approximately 0.038 mmol, 1.2 equivalents) and 4.4 mg of NHS (approximately 0.038 mmol, 1.2 equivalents). Stir the reaction at room temperature for 4 hours to activate the carboxyl group.

[0061] Reaction with NH2-PEG-MAL: Dissolve 20 mg of NH2-PEG-MAL (molecular weight 3400, approximately 0.006 mmol) in 1 mL of PBS (pH 7.4). Add the activated Kartogenin solution dropwise to the PEG solution, and adjust the total volume to 4 mL with PBS to achieve a final pH of approximately 7.4. Stir the reaction mixture at room temperature in the dark for 12 hours.

[0062] Purification: The reaction solution was transferred to a dialysis bag (MWCO 1000 Da) and dialyzed in deionized water for 48 hours, changing the water every 8 hours to remove unreacted Kartogenin, EDC, NHS, and DMSO. After dialysis, the solution was freeze-dried to obtain a white powder, KGN-PEG-MAL.

[0063] Structural characterization: ¹H NMR (D₂O) showed a characteristic peak for the maleimide group at δ 6.7 and a characteristic peak for the newly formed amide bond at δ 7.9, indicating that KGN was successfully attached to the end of the PEG chain. FTIR spectra showed enhanced absorption at 1650 cm⁻¹ (amide I band) and 1550 cm⁻¹ (amide II band). The grafting rate was determined by UV spectrophotometry (KGN has a characteristic absorption at 279 nm), and the KGN content in each milligram of KGN-PEG-MAL was calculated to be approximately 8.2 μg.

[0064] Preparation of KGN-PEG-MAL-loaded SilMA hybrid hydrogels: Precursor solution preparation: Dissolve SilMA in sterile PBS to prepare a 10% w / v solution. Add KGN-PEG-MAL to make the mass ratio of SilMA to KGN-PEG-MAL 10:1. Add photoinitiator LAP to a final concentration of 0.5% w / v. Stir magnetically in the dark for 15 minutes and sonicate to degas for 5 minutes.

[0065] Photocrosslinking molding: The precursor solution is injected into a cylindrical mold (8 mm in diameter and 3 mm in height), placed under a 365 nm ultraviolet lamp (10 mW / cm²) and irradiated for 60 seconds to form a hydrogel.

[0066] Post-crosslinking enhancement (optional): The resulting hydrogel was immersed in 40% ethanol aqueous solution for 30 minutes, followed by washing with PBS 3 times to induce the formation of silk fibroin β-sheet structure and further enhance physical crosslinking.

[0067] Performance characterization of hybrid hydrogels: Microstructure: The cross-section of the freeze-dried hydrogel was observed using scanning electron microscopy. The results showed that the hydrogel has an interconnected pore structure with a pore size of approximately 80-120 μm (at a 10% SilMA concentration) and a porosity >85%.

[0068] Mechanical properties: Compression tests were conducted using a universal testing machine (compression speed 1 mm / min). The results showed that the compressive modulus of the hydrogel was 35 ± 5 kPa (n=5). Rheological tests (oscillation frequency 1 Hz) yielded a storage modulus G' = 15 ± 2 kPa and a loss modulus G'' = 1.5 ± 0.3 kPa, indicating that the material exhibits a predominantly elastic response.

[0069] Drug release performance: The hydrogel was immersed in PBS buffer (pH 7.4) containing 1% BSA and incubated with shaking at 37°C. The supernatant was collected periodically, and the cumulative release of KGN was determined by UV spectrophotometry (279 nm). Results showed that the burst release rate was 18% in the first 24 hours, the cumulative release rate was approximately 55% on day 7, and reached 85% on day 14, after which the release tended to plateau. This indicates that the present invention achieves long-term sustained release of KGN.

[0070] Cell compatibility: Human bone marrow mesenchymal stem cell (hBMSCs) suspension was mixed with an uncrosslinked precursor solution and then photocrosslinked. The mixture was cultured in DMEM medium containing 10% FBS for 7 days. Live / dead staining results showed that the cell viability in the hydrogel was >92%, and the cells exhibited good morphological extension. CCK-8 assays showed a continuous increase in cell number at 1, 3, and 7 days of culture, indicating that the hydrogel supports cell proliferation.

[0071] Comparison of hydrogel properties with different SilMA concentrations: Precursor solutions with SilMA concentrations of 5%, 10%, and 15% (w / v) were prepared, and hydrogels were prepared according to the above method. Key properties were tested. Based on the test results, considering mechanical properties, cell compatibility, and drug release behavior, a 10% SilMA concentration was selected as the preferred embodiment of the present invention.

[0072] Comparative example (SilMA hydrogel with physical loading of KGN): To verify the advantages of PEG modification and covalent bonding, a comparative example was set up: unmodified KGN was directly dispersed in a 10% SilMA solution (without PEG modification and covalent bonding), and hydrogels were prepared under the same photocrosslinking conditions. In vitro release experiments showed that the burst release rate of physically loaded KGN was as high as 65%, with over 80% released within 24 hours and almost complete release after 3 days. Cell viability tests showed that due to the burst release crystallization stimulation of KGN, cell viability in some areas was below 70%. This comparative example demonstrates that the PEG modification and covalent bonding strategy significantly improved the sustained-release performance and cell compatibility of KGN. The pore structure, mechanical properties, Kartogenin release behavior, and cell compatibility of the methacryloyl-acrylated silk fibroin hybrid hydrogel were also investigated, providing experimental support for technical indicators such as compressive modulus, storage modulus, burst release rate, cumulative release time, and cell viability.

[0073] The above are merely preferred embodiments of this application and are not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A methacrylamide silk fibroin hybrid hydrogel, characterized in that, It is formed by photocrosslinking and β-sheet induction treatment of a precursor system comprising methacrylamide silk fibroin, Kartogenin-polyethylene glycol-maleimide prodrug, thiol-containing crosslinking agent and photoinitiator; wherein the methacrylamide silk fibroin contains methacrylyl groups and the degree of substitution of the methacrylamide silk fibroin is 30% to 70%; The methacrylated silk fibroin forms a covalent cross-linked network through the photocross-linking reaction of the methacryl group, and forms a physical cross-linked network through the β-sheet structure of the silk fibroin molecular chain segments. The covalent cross-linked network and the physical cross-linked network together constitute a dual network structure. The Kartogenin-polyethylene glycol-maleimide prodrug comprises a Kartogenin group, a polyethylene glycol segment, and a maleimide group. The Kartogenin-polyethylene glycol-maleimide prodrug is covalently fixed in the hydrogel network of the hybrid hydrogel by a Michael addition reaction between the maleimide group and the thiol group in the thiol-containing crosslinking agent.

2. The methacrylamide silk fibroin hybrid hydrogel according to claim 1, characterized in that, In the Kartogenin-polyethylene glycol-maleimide prodrug, the Kartogenin group is connected to the polyethylene glycol segment via an amide bond, and the number average molecular weight of the polyethylene glycol segment is 1000 Da to 5000 Da. The thiol-containing crosslinking agent is linked to the Kartogenin-polyethylene glycol-maleimide prodrug via maleimide-thiol addition bonds, thereby retaining the Kartogenin-polyethylene glycol-maleimide prodrug in the hydrogel network in a covalently bonded manner, thereby reducing the rapid diffusion and release of Kartogenin in the hydrogel.

3. The methacrylamide silk fibroin hybrid hydrogel according to claim 1 or 2, characterized in that, The hybrid hydrogel has an interconnected three-dimensional porous structure with an average pore size of 70 μm to 150 μm and a porosity of not less than 85%. The hybrid hydrogel has a compressive modulus of 10 kPa to 50 kPa and a storage modulus of 5 kPa to 30 kPa, which improves the mechanical stability of the hybrid hydrogel through the dual network structure.

4. The methacrylamide silk fibroin hybrid hydrogel according to claim 1 or 2, characterized in that, The Kartogenin is covalently immobilized in the hydrogel network in the form of a Kartogenin-polyethylene glycol-maleimide prodrug; the burst release rate of the Kartogenin in the in vitro release system is no higher than 20% in the first 24 hours, and the cumulative release time is no less than 14 days; when the hybrid hydrogel is used for three-dimensional culture of human bone marrow mesenchymal stem cells, the cell survival rate after 7 days of culture is no less than 90%.

5. A method for preparing a methacrylamide silk fibroin hybrid hydrogel, characterized in that, The method for preparing the methacrylamide silk fibroin hybrid hydrogel according to any one of claims 1-4 comprises the following steps: S1. Degumming, dissolving, dialysis and freeze-drying of silkworm cocoons to obtain regenerated silk fibroin; S2. The regenerated silk fibroin is subjected to a methacrylation reaction with methacrylic anhydride to obtain methacrylated silk fibroin with a degree of substitution of 30% to 70%. S3. Kartogenin is coupled with amino-polyethylene glycol maleimide to obtain Kartogenin-polyethylene glycol-maleimide prodrug; S4. Mix the methacrylamide silk fibroin, the Kartogenin-polyethylene glycol-maleimide prodrug, the thiol-containing crosslinking agent and the photoinitiator to form a precursor solution; S5. The precursor solution is subjected to light treatment, so that the methacrylamide silk fibroin forms a covalent cross-linked network through the photocross-linking reaction of the methacrylamide group, and the Kartogenin-polyethylene glycol-maleimide prodrug is covalently fixed in the hydrogel network through the Michael addition reaction of the maleimide group with the thiol-containing cross-linking agent. S6. The hydrogel after light treatment is subjected to β-sheet induction treatment to induce the formation of a physical cross-linking network of silk fibroin molecular chain segments, thereby obtaining a methacrylamide silk fibroin hybrid hydrogel composed of the covalent cross-linking network and the physical cross-linking network.

6. The method for preparing the methacrylamide silk fibroin hybrid hydrogel according to claim 5, characterized in that, Step S1 specifically includes the following steps: After the silkworm cocoons are cut into pieces, they are boiled in a sodium carbonate solution to remove sericin and obtain silk fibroin fibers. The silk fibroin fibers were washed with deionized water and dried, then dissolved in lithium bromide solution to obtain a silk fibroin protein solution. The silk fibroin solution was placed in a dialysis bag with a molecular weight cutoff of 3500 Da, dialyzed in deionized water, and then freeze-dried to obtain the regenerated silk fibroin; wherein the silk fibroin fibers were dissolved in the lithium bromide solution at 60°C, and the dialysis was used to remove lithium bromide and obtain purified regenerated silk fibroin.

7. The method for preparing the methacrylamide silk fibroin hybrid hydrogel according to claim 5 or 6, characterized in that, Step S2 specifically includes the following steps: The regenerated silk fibroin was dissolved in a phosphate buffer solution with a preset pH to form a silk fibroin solution with a mass-volume concentration of 6%. Methacrylic anhydride is added to the silk fibroin solution to make the molar ratio of the methacrylic anhydride to the amino and / or tyrosine residues in the silk fibroin 0.5:1 to 2:

1. The reaction is carried out at pH 7.4-8.0 for 1-4 hours to introduce methacryloyl groups onto the silk fibroin molecular chain; After the reaction was completed, unreacted methacrylic anhydride and byproducts were removed by dialysis, and the methacrylated silk fibroin was obtained by freeze drying.

8. The method for preparing the methacrylamide silk fibroin hybrid hydrogel according to claim 5, characterized in that, Step S3 specifically includes the following steps: Dissolve Kartogenin in an anhydrous organic solvent, add 1.0-1.5 equivalents of EDC and NHS, and activate the carboxyl groups in Kartogenin at room temperature for 2-6 hours. The activated Kartogenin was reacted with amino polyethylene glycol maleimide with a number average molecular weight of 1000 Da to 5000 Da at a pH of 7.2 to 7.6 for 8 to 24 hours, so that the Kartogenin was linked to the polyethylene glycol segment via amide bonds. After the reaction is complete, unreacted small molecule byproducts and organic solvents are removed by dialysis or high performance liquid chromatography, and then freeze-dried to obtain the Kartogenin-polyethylene glycol-maleimide prodrug.

9. The method for preparing the methacrylamide silk fibroin hybrid hydrogel according to claim 5, characterized in that, In step S4, the precursor solution is prepared using phosphate buffer or cell culture medium, and the concentration of the methacrylamide silk fibroin in the precursor solution is 5%~15% w / v. The mass ratio of the methacrylamide silk fibroin to the Kartogenin-polyethylene glycol-maleimide prodrug is 5:1 to 20:1; the photoinitiator is LAP and / or Irgacure2959, and the concentration of the photoinitiator in the precursor solution is 0.1% to 1% w / v; The precursor solution is subjected to light-shielded stirring and degassing treatment before being used for photoforming to reduce the influence of oxygen on the photocrosslinking reaction and improve the uniformity of the precursor solution.

10. The method for preparing the methacrylamide silk fibroin hybrid hydrogel according to any one of claims 5-6 or 8-9, characterized in that, In step S5, a light source with a wavelength of 365nm~405nm and a light intensity of 5mW / cm²~20mW / cm² is used for the light treatment, and the irradiation time is 30S~2min, so that the precursor solution is photocrosslinked and formed. Step S6, when performing β-sheet induction treatment, includes: immersing the phototreated hydrogel in phosphate buffer or ethanol / water mixture to induce the formation of β-sheet structures in silk fibroin molecular chains; after the phototreatment and β-sheet induction treatment, the hybrid hydrogel forms a dual-network structure in which covalent cross-linked networks and physical cross-linked networks coexist, and the Kartogenin-polyethylene glycol-maleimide prodrug is covalently fixed in the hydrogel network.