Dual-network hyaluronic acid-silk fibroin gel sponge and preparation method thereof

Abstract

The invention provides a dual-network hyaluronic acid-silk fibroin gel sponge and a preparation method thereof, and belongs to the technical field of gel sponge materials. The preparation method comprises the following steps: mixing sulfhydrylation modified hyaluronic acid and a silk fibroin solution, and sequentially performing freezing and soft freezing treatment to form the hyaluronic acid-silk fibroin gel sponge with a good shape. In the freezing induction process, hyaluronic acid generates chemical crosslinking through disulfide bonds, silk fibroin forms physical crosslinking through freezing concentration, and finally the stable gel-sponge coexistence body with the double-network structure is formed. The gel sponge has double advantages of gel and sponge. Meanwhile, due to freeze concentration treatment, the gel sponge has a macroporous structure, which is beneficial to potentially promoting adhesion, proliferation and migration of cells; the prepared gel sponge does not introduce additional chemical reagents, the biological safety of the gel sponge is guaranteed, and the gel sponge is suitable for the fields of cartilage / skin repair, tissue filling, wound dressing and the like.

Description

Technical Field

[0001] The present invention relates to the technical field of gel sponge materials, and in particular to a double-network hyaluronic acid-silk fibroin gel sponge and a preparation method thereof. Background Art

[0002] Hyaluronic acid is a natural polysaccharide that is widely distributed in human connective tissue. With its good biocompatibility, water retention and biological activity, it has become an ideal material in the field of tissue engineering. It can provide cells with a moist growth environment, promote cell adhesion, proliferation and differentiation, and is particularly suitable for tissue repair such as cartilage and skin that have high requirements for water retention. In addition, hyaluronic acid can also be combined with bioactive molecules such as growth factors to enhance its ability to promote tissue repair and regeneration, and can be compounded with other biomaterials to expand its application range in bioengineering. However, the mechanical properties of hyaluronic acid hydrogels are relatively weak, and they are prone to rupture under mechanical stress, which limits their use in application scenarios that require higher mechanical strength.

[0003] Silk fibroin, a natural protein, also possesses excellent biocompatibility. Its surface is rich in hydrophilic amino acids, which promote cell adhesion and growth, providing an excellent living environment for cells. The sponge-like structure of silk fibroin also possesses excellent mechanical properties and toughness, making it suitable for the preparation of high-strength, high-elasticity scaffold materials for tissue engineering applications requiring high mechanical support, such as bone, ligaments, and tendons.

[0004] Currently, the combination of hyaluronic acid and silk fibroin is usually in a single gel or sponge state. It is difficult to combine the advantages of both structural materials at the same time and still has the limitation of singleness. Summary of the Invention

[0005] In view of the technical problems existing in the background technology, the present application provides a double-network hyaluronic acid-silk fibroin gel sponge and a preparation method thereof, aiming to solve the problem of structural uniformity of existing hyaluronic acid-silk fibroin composite materials.

[0006] In a first aspect, the present application provides a method for preparing a double-network hyaluronic acid-silk fibroin gel sponge, comprising the following steps: S1. Prepare silk fibroin solution; S2. preparing a thiolated hyaluronic acid solution; S3. mixing the silk fibroin solution and the thiolated hyaluronic acid solution and stirring to obtain a mixed solution; S4. The mixed solution is frozen at -20 to -80°C, then soft-frozen at -2 to -10°C, and then thawed to obtain a double-network hyaluronic acid-silk fibroin gel sponge.

[0007] In the technical solution of the embodiment of the present application, the present application mixes thiol-modified hyaluronic acid and silk fibroin solution, and sequentially undergoes freezing and soft freezing treatment to form a well-shaped hyaluronic acid-silk fibroin gel sponge. During the freezing induction process, hyaluronic acid first produces chemical crosslinks through disulfide bonds, and silk fibroin forms physical crosslinks through freeze concentration, eventually forming a gel-sponge coexistence body with a stable double-network structure. It has the dual advantages of both gel and sponge. When below the yield point, the gel sponge undergoes viscoelastic deformation and behaves as a rigid hydrogel; above the yield point, it behaves as a sponge that can be repeatedly squeezed, and energy dissipation is achieved by water diffusion. At the same time, the freeze concentration treatment makes the gel sponge have a macroporous structure, which is conducive to potentially promoting cell adhesion, proliferation and migration.

[0008] In some embodiments, in step S1, the molecular weight of the silk fibroin in the silk fibroin solution is less than or equal to 150 kDa; and the concentration of the silk fibroin solution is 0.5-5%.

[0009] In this embodiment, the silk fibroin with a lower molecular weight does not affect the preferential formation of the hyaluronic acid gel structure.

[0010] In some embodiments, in step S2, the molecular weight of hyaluronic acid in the thiolated hyaluronic acid solution is 500-1500 kDa.

[0011] In this embodiment, hyaluronic acid with a specific molecular weight can ensure good gel properties while not affecting the subsequent formation of a sponge-like silk fibroin structure.

[0012] In some embodiments, in step S2, the concentration of the thiolated hyaluronic acid solution is 0.5-5%.

[0013] In this embodiment, during the freezing process of the thiol-treated hyaluronic acid, the formation of ice crystals leads to local concentration of the thiol-treated hyaluronic acid and silk fibroin solution, prompting the oxidation of the thiol groups to disulfide bonds, thereby forming a stable covalent cross-linking network between the two.

[0014] In some embodiments, in step S3, the mass ratio of the silk fibroin in the silk fibroin solution to the thiolated hyaluronic acid in the thiolated hyaluronic acid solution is 1:3 to 3:1.

[0015] In this embodiment, the specific ratio of silk fibroin and thiolated hyaluronic acid makes the final gel sponge-like and has good mechanical properties and water retention properties.

[0016] In some embodiments, in step S4, the freezing treatment time is 6 to 12 hours.

[0017] In this embodiment, the mixed solution is frozen at -20~-80℃ for a period of time, and the thiolated hyaluronic acid preferentially forms a hyaluronic acid cross-linked network to form a gel; during the freezing process, the formation of ice crystals causes the thiolated hyaluronic acid and silk fibroin solution to be locally concentrated, prompting the oxidation of thiol groups to disulfide bonds, thereby forming a stable covalent cross-linked network; and the freeze-concentration treatment gives the gel sponge a macroporous structure.

[0018] In some embodiments, in step S4, the soft freezing treatment lasts for 0.25 to 4 days.

[0019] In this embodiment, the frozen mixed solution is subjected to a soft-freezing treatment at -2 to -10°C, and the silk fibroin gradually forms a silk I structure, thereby forming a sponge state.

[0020] In some embodiments, in step S4, the thawing temperature is 4-37°C.

[0021] In this embodiment, a composite material in a gel sponge state is obtained by thawing.

[0022] In the second aspect, the present application provides a double-network hyaluronic acid-silk fibroin gel sponge, which is prepared by the above-mentioned preparation method of the double-network hyaluronic acid-silk fibroin gel sponge; the double-network hyaluronic acid-silk fibroin gel sponge includes a hyaluronic acid gel matrix and a silk fibroin sponge structure distributed in the gel matrix.

[0023] In the technical solution of the embodiment of the present application, a hyaluronic acid-silk fibroin gel sponge with a double network structure is formed by combining dynamic covalent cross-linking and physical freezing molding. The gel sponge combines the advantages of gel and sponge, and has good flexibility and repeated compression characteristics.

[0024] The above description is only an overview of the technical solution of the present application. In order to more clearly understand the technical means of the present application, it can be implemented in accordance with the contents of the specification. In order to make the above and other purposes, features and advantages of the present application more obvious and easy to understand, the specific implementation methods of the present application are listed below. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] To more clearly illustrate the technical solution of this application, the following is a brief introduction to the drawings used in this application. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be derived from these drawings without inventive effort.

[0026] Figure 1 This is the SEM image of the hyaluronic acid-silk fibroin gel sponge prepared in Example 1.

[0027] Figure 2 This is a physical picture of the hyaluronic acid-silk fibroin gel sponge prepared in Example 1.

[0028] Figure 3 These are laser scanning confocal microscopy images of the materials prepared in Example 1 and Comparative Examples 1-2 co-cultured with human umbilical vein endothelial cells.

[0029] Figure 4 1 and 2 are far infrared images of the materials in Examples 1 to 3 and Comparative Examples 1 to 2.

[0030] Figure 5 2 are pressure-density diagrams of the materials prepared in Examples 1-3 and Comparative Examples 1-2.

[0031] Figure 6 These are pressure-strain diagrams of the materials prepared in Comparative Examples 1-3 and Comparative Examples 1-2. DETAILED DESCRIPTION

[0032] The following embodiments of the technical solution of the present application are described in detail. The following embodiments are only used to more clearly illustrate the technical solution of the present application and are therefore only used as examples and are not intended to limit the scope of protection of the present application.

[0033] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this application belongs; the terms "including" and "having" and any variations thereof used herein are only for the purpose of describing specific embodiments and are not intended to limit this application.

[0034] References herein to "embodiments" mean that a particular feature, structure, or characteristic described in connection with the embodiments may be included in at least one embodiment of the present application. The appearance of this phrase in various places in the specification does not necessarily refer to the same embodiment, nor does it constitute an independent or alternative embodiment that is mutually exclusive of other embodiments. It is understood, both explicitly and implicitly, by those skilled in the art that the embodiments described herein may be combined with other embodiments.

[0035] To solve the problem of single structure of the existing hyaluronic acid-silk fibroin composite material, the application provides a double-network hyaluronic acid-silk fibroin gel sponge and a preparation method thereof. The application mixes thiol-modified hyaluronic acid and a silk fibroin solution, and then sequentially performs freezing and soft freezing treatment, so that a hyaluronic acid-silk fibroin gel sponge with good morphology is formed. In the hyaluronic acid-silk fibroin gel sponge, the hyaluronic acid and the silk fibroin are respectively formed into a gel and a sponge state in steps, do not affect the formation of the respective states, and are respectively formed into chemical and physical cross-linking through dynamic covalent cross-linking and physical freezing forming, so as to form a stable double-network structure. The double-network gel sponge structure fully utilizes the advantages of the hyaluronic acid and the silk fibroin, overcomes the limitations of a single material, has certain mechanical properties and high water retention, has the function of super-strong water-rich gel at low strain, has the function of a repeatable compression sponge capable of quickly draining water at high strain, and has the characteristics of losing water at a yield point when filling a tissue in vivo, so that body fluid in the gel sponge can be discharged by extrusion, and the gel sponge can absorb fresh body fluid again after the stress is removed. The dynamic fluid exchange mechanism is beneficial to the rapid exchange of nutrients and oxygen and the effective discharge of metabolic end products, enhances cell infiltration of the gel sponge at an implantation site and subsequent cell-material response behavior, and the freezing concentration treatment makes the gel sponge have a large pore structure, which is beneficial to potentially promoting cell adhesion, proliferation and migration. The gel sponge prepared by the application is suitable for the fields of cartilage / skin repair, tissue filling and wound dressing.

[0036] The application provides a preparation method of a double-network hyaluronic acid-silk fibroin gel sponge, which comprises the following steps: S1. Preparing a silk fibroin solution; S2. Preparing a thiol-modified hyaluronic acid solution; S3. Mixing the silk fibroin solution and the thiol-modified hyaluronic acid solution, stirring, and obtaining a mixed solution; S4. Freezing the mixed solution at-20 to-80 DEG C, then soft freezing at-2 to-10 DEG C, and then thawing, to obtain a double-network hyaluronic acid-silk fibroin gel sponge.

[0037] Further, in some embodiments, the preparation method of the silk fibroin solution comprises the following steps: The silk is treated in 0.05% sodium carbonate, boiled at 100 DEG C for 30 min, repeated for 3 times, then washed with deionized water, and then the silk is dried at 60 DEG C. The treated degummed silk is dissolved in a 9.3 mol / L LiBr solution, treated at 60 DEG C for 1 h. Then the silk fibroin solution is dialyzed in a dialysis bag with a molecular weight cut-off of 9000 to 12000 Da in deionized water for 3 days, to obtain a purified silk fibroin solution, which is then diluted.

[0038] Furthermore, in some embodiments, the preparation of the thiolated hyaluronic acid comprises the following steps: Hyaluronic acid powder was dissolved in deionized water, and NHS and EDC were added. The pH was adjusted to 4.75 with HCl solution. CSA·HCl was then added to the reaction system, and the pH was adjusted to 4.75 with NaOH solution. After reacting at room temperature for 24 hours, the mixture was transferred to a dialysis bag and dialyzed with deionized water at room temperature for 3 days. The solution was then freeze-dried.

[0039] In the technical solution of the embodiment of the present application, the present application mixes thiol-modified hyaluronic acid and silk fibroin solution, and sequentially undergoes freezing and soft freezing treatment to form a well-shaped hyaluronic acid-silk fibroin gel sponge. During the freezing induction process, hyaluronic acid first produces chemical crosslinks through disulfide bonds, and silk fibroin forms physical crosslinks through freeze concentration, eventually forming a gel-sponge coexistence body with a stable double-network structure. It has the dual advantages of both gel and sponge. When below the yield point, the gel sponge undergoes viscoelastic deformation and behaves as a rigid hydrogel; above the yield point, it behaves as a sponge that can be repeatedly squeezed, and energy dissipation is achieved by water diffusion. At the same time, the freeze concentration treatment makes the gel sponge have a macroporous structure, which is conducive to potentially promoting cell adhesion, proliferation and migration.

[0040] Furthermore, in some embodiments, in step S1, the molecular weight of the silk fibroin in the silk fibroin solution is less than or equal to 150 kDa; and the concentration of the silk fibroin solution is 0.5-5%.

[0041] In the technical solution of the embodiment of the present application, the silk fibroin with a lower molecular weight will not affect the preferential formation of the hyaluronic acid gel structure.

[0042] Furthermore, in some embodiments, in step S2, the molecular weight of hyaluronic acid in the thiolated hyaluronic acid solution is 500-1500 kDa.

[0043] In the technical solution of the embodiment of the present application, hyaluronic acid with a specific molecular weight can ensure good gel performance while not affecting the subsequent formation of a sponge-like silk fibroin structure.

[0044] Furthermore, in some embodiments, in step S2, the concentration of the thiolated hyaluronic acid solution is 0.5-5%.

[0045] In the technical solution of the embodiment of the present application, during the freezing process of the thiol-treated hyaluronic acid, the formation of ice crystals leads to local concentration of the thiol-treated hyaluronic acid and silk fibroin solution, promoting the oxidation of the thiol groups to disulfide bonds, thereby forming a stable covalent cross-linking network between the two.

[0046] Further, in some embodiments, in step S3, the mass ratio of the silk fibroin in the silk fibroin solution and the thiolated hyaluronic acid in the thiolated hyaluronic acid solution is 1:3-3:1.

[0047] In the technical solution of the embodiments of the present application, the specific proportion of silk fibroin and thiolated hyaluronic acid enables the final obtained gel sponge to have certain mechanical properties and water retention properties.

[0048] Further, in some embodiments, in step S4, the freezing treatment time is 6-12 hours.

[0049] In the technical solution of the embodiments of the present application, the mixed solution is subjected to freezing treatment at-20--80℃ for a period of time, and the thiolated hyaluronic acid preferentially forms a hyaluronic acid crosslinking network to form a gel; during the freezing process, the formation of ice crystals causes the thiolated hyaluronic acid and the silk fibroin solution to be locally concentrated, which promotes the oxidation of thiol to disulfide bond, thereby forming a stable covalent crosslinking network; and the freezing concentration treatment enables the gel sponge to have a large pore structure.

[0050] Further, in some embodiments, in step S4, the soft freezing treatment time is 0.25-4 days.

[0051] In the technical solution of the embodiments of the present application, the mixed solution after freezing is subjected to soft freezing treatment at-2--10℃, and the silk fibroin gradually forms a silk I structure, thereby forming a sponge state.

[0052] Further, in some embodiments, in step S4, the thawing temperature is 4-37℃.

[0053] In the technical solution of the embodiments of the present application, the composite material in a gel sponge state is obtained through thawing.

[0054] In a second aspect, the embodiments of the present application provide a double-network hyaluronic acid-silk fibroin gel sponge, which is prepared by using the above-mentioned preparation method of the double-network hyaluronic acid-silk fibroin gel sponge; the double-network hyaluronic acid-silk fibroin gel sponge comprises a hyaluronic acid gel matrix and a silk fibroin sponge structure distributed in the gel matrix.

[0055] In the technical solution of the embodiments of the present application, by combining dynamic covalent crosslinking and physical freezing forming, a double-network hyaluronic acid-silk fibroin gel sponge is formed, which combines the advantages of gel and sponge and has good flexibility and repeated compression characteristics.

[0056] Some specific examples are listed below. It should be noted that the examples described below are exemplary and are used only to explain the present application and cannot be understood as a limitation of the present application. If the specific technology or condition is not specified in the examples, it is carried out according to the technology or condition described in the literature in the art or according to the product manual. If the reagent or instrument is not specified by the manufacturer, it is a conventional product that can be obtained by purchase.

[0057] Example 1 The present example provides a preparation method of a double-network hyaluronic acid-silk fibroin gel sponge, which specifically comprises the following steps: (1) Preparation of silk fibroin solution: silk is treated in 0.05% sodium carbonate, boiled at 100°C for 30 min, repeated 3 times, then washed with deionized water, and then the silk is dried at 60°C. The degummed silk after treatment is dissolved in a LiBr solution with a concentration of 9.3 mol / L, and treated at 60°C for 1 h. Then a dialysis bag with a molecular weight cut-off of 9000-12000 Da is immersed in deionized water for dialysis for 3 days to obtain a purified silk fibroin solution, which is then diluted to a concentration of 1.5%.

[0058] (2) Preparation of thiolated hyaluronic acid solution: 400 g of hyaluronic acid powder is dissolved in 80 mL of deionized water, then 230 mg of NHS and 575 mg of EDC are added, the pH is adjusted to 4.75 with HCl solution, then 342 mg of CSA·HCl is added to the reaction system, and then the pH is adjusted to 4.75 with NaOH solution, and the reaction is carried out at room temperature for 24 h. Then transfer to a dialysis bag and dialyze in deionized water at room temperature for 3 days, then freeze-dry the solution to obtain thiolated hyaluronic acid powder, then add the thiolated hyaluronic acid powder to deionized water to obtain a thiolated hyaluronic acid solution with a concentration of 1.5%.

[0059] (3) The above silk fibroin solution and thiolated hyaluronic acid solution are mixed in a mass ratio of 1:1, stirred in a blender for 5 min to obtain a mixed solution.

[0060] (4) The above mixed solution is frozen at -80°C for 24 h, then soft-frozen at -7°C for 3 days, and then thawed at 35°C to obtain a hyaluronic acid-silk fibroin gel sponge.

[0061] The SEM image and the actual image of the hyaluronic acid-silk fibroin gel sponge prepared in the present example are shown in Figure 1 and Figure 2 respectively.

[0062] As can be seen from the SEM image in Figure 1 , the prepared hyaluronic acid-silk fibroin gel sponge has a porous three-dimensional sponge structure.

[0063] Examples 2-3 Examples 2-3 provide a preparation method of a double-network hyaluronic acid-silk fibroin gel sponge, which differs from Example 1 in that the mass ratio of the silk fibroin solution and the thiolated hyaluronic acid solution is different. In Example 2, the mass ratio of the silk fibroin solution to the thiolated hyaluronic acid solution is 1:3, and in Example 3, the mass ratio of the silk fibroin solution to the thiolated hyaluronic acid solution is 3:1. The other steps are substantially the same as those of Example 1, and are not described here again.

[0064] Comparative Example 1 This comparative example provides a preparation method of a hyaluronic acid gel, which differs from Example 1 in that the treatment in step (3) is not performed, and the thiolated hyaluronic acid solution in step (2) is directly treated in step (4) to obtain a hyaluronic acid gel.

[0065] Comparative Example 2 This comparative example provides a preparation method of a silk fibroin sponge, which differs from Example 1 in that the treatment in step (3) is not performed, and the silk fibroin solution in step (1) is directly treated in step (4) to obtain a silk fibroin sponge.

[0066] The materials prepared in Example 1 and Comparative Examples 1-2 were subjected to in vitro biocompatibility testing: (1) Material sterilization: The materials prepared in Example 1 and Comparative Examples 1-2 were prepared into thin slices with a diameter of 15 mm and a thickness of 2 mm, soaked in alcohol for 1 h, and then placed in an ultraclean bench for ultraviolet sterilization for 1 h.

[0067] (2) Cell recovery: The cryopreserved seed cells were taken out of the liquid nitrogen tank, thawed in a 37°C water bath, centrifuged at 1000 rpm for 5 minutes, the supernatant was removed, complete culture medium was added, and the cells were blown and mixed evenly, then poured into a cell culture bottle, and finally complete culture medium was added.

[0068] (3) Cell passage: The cell culture bottle was first washed with PBS for 2 times, trypsin was added for digestion, the cells were observed under a microscope, the trypsin was discarded, complete culture medium was added, and the cells were blown and mixed, then centrifuged at 1000 rpm for 5 minutes, the supernatant was discarded, complete culture medium was added again, and the cells were blown and mixed evenly, then poured into a new culture bottle, and finally complete culture medium was added.

[0069] (4) Cell inoculation: After the cell density reached 80-90% confluence, the cells were digested with trypsin, centrifuged, and then blown and mixed evenly and counted, and then the cells were diluted with complete culture medium to 0.5×10 4cells / mL. 100 μL / well was inoculated on the surface of the materials prepared in Example 1 and Comparative Examples 1-2, 1 mL of complete culture medium was added, and the cells were placed in a CO2 incubator for culture, with the medium changed at intervals. Laser confocal imaging was performed at three time points: 1, 3, and 7 days. The results are shown in Figure 2. Figure 3 shown.

[0070] Depend on Figure 3 It can be seen that the cell numbers of the hyaluronic acid-silk fibroin gel sponge prepared in Example 1 at different time points are significantly more than that of the hyaluronic acid gel in Comparative Example 1, and are comparable to the cell numbers of the silk fibroin sponge in Comparative Example 2.

[0071] The far infrared images of the materials in Examples 1 to 3 and Comparative Examples 1 to 2 are as follows: Figure 4 shown.

[0072] Depend on Figure 4 It can be seen that disulfide bonds exist between hyaluronic acid and silk fibroin is mainly composed of Silk I structure.

[0073] The pressure-to-gravity curves of the materials in Examples 1 to 3 and Comparative Examples 1 to 2 were tested, and the results were as follows: Figure 5 shown.

[0074] Depend on Figure 5 It can be seen that pure thiolated hyaluronic acid gel has almost no yield point (strain value for water loss), which means it will not lose water even under great stress. The yield point of pure silk fibroin sponge is almost 0, which means it has almost no water retention effect, and its application scenarios will be limited. As the silk fibroin content increases, the yield point gradually decreases, and the appropriate ratio can be selected according to the needs of different scenarios.

[0075] The compressive stress-strain diagrams of the materials in Examples 1 to 3 and Comparative Examples 1 to 2 are as follows: Figure 6 shown.

[0076] Depend on Figure 6 It can be seen that the gel sponge in the present application has a certain compressive strength, and the strength decreases with the increase of silk fibroin content, while the compressive strength of pure silk fibroin sponge is almost 0 and its application scenarios will be limited.

[0077] In summary, the present application provides a double-network hyaluronic acid-silk fibroin gel sponge and its preparation method. The present application mixes thiol-modified hyaluronic acid and silk fibroin solution, and sequentially undergoes freezing and soft freezing treatment to form a hyaluronic acid-silk fibroin gel sponge with good morphology, wherein hyaluronic acid and silk fibroin form gel and sponge states respectively in steps, do not affect the formation of their respective states, and the two states are formed by dynamic covalent cross-linking and physical freezing molding, respectively forming chemical and physical cross-linking, forming a stable double-network structure. The double-network gel sponge structure gives full play to the advantages of hyaluronic acid and silk fibroin, overcomes the limitations of a single material, and has certain mechanical properties and high water retention. It has the function of a super-strong water-rich gel under low strain and the function of a reusable compressible sponge with rapid drainage under high strain. When performing tissue filling in the body, the water loss property of the gel sponge at the yield point can be utilized to discharge the body fluid in the gel sponge by squeezing, and fresh body fluid can be reabsorbed when the stress is released. This dynamic fluid exchange mechanism is conducive to the rapid exchange of nutrients and oxygen and the effective discharge of metabolic end products, enhancing the cell infiltration of the gel sponge at the implantation site and the subsequent cell-material response behavior. The freeze concentration treatment makes the gel sponge have a macroporous structure, which is conducive to potentially promoting cell adhesion, proliferation and migration. The gel sponge prepared in this application is suitable for cartilage / skin repair, tissue filling and wound dressing and other fields.

[0078] It should be noted that the present application is not limited to the above-mentioned embodiments. The above-mentioned embodiments are merely examples, and any embodiments having substantially the same structure and effect as the technical concept within the scope of the present application are all included in the technical scope of the present application. In addition, without departing from the scope of the present application, any other embodiments that can be conceived by those skilled in the art and that combine some of the constituent elements in the embodiments are also included in the scope of the present application.

Claims

1. A method for preparing a double-network hyaluronic acid-silk fibroin gel sponge, characterized in that: The following steps are involved: S1. Prepare silk fibroin solution; S2. preparing a thiolated hyaluronic acid solution; S3. mixing the silk fibroin solution and the thiolated hyaluronic acid solution and stirring to obtain a mixed solution; S4. The mixed solution is frozen at -20 to -80°C, then soft-frozen at -2 to -10°C, and then thawed to obtain a double-network hyaluronic acid-silk fibroin gel sponge.

2. The method for preparing the double-network hyaluronic acid-silk fibroin gel sponge according to claim 1, characterized in that: In step S1, in the silk fibroin solution, the molecular weight of the silk fibroin is less than or equal to 150 kDa.

3. The method for preparing the double-network hyaluronic acid-silk fibroin gel sponge according to claim 1, characterized in that: In step S1, the concentration of the silk fibroin solution is 0.5-5%.

4. The method for preparing the double-network hyaluronic acid-silk fibroin gel sponge according to claim 1, characterized in that: In step S2, the molecular weight of hyaluronic acid in the thiolated hyaluronic acid solution is 500-1500 kDa.

5. The method for preparing the double-network hyaluronic acid-silk fibroin gel sponge according to claim 1, characterized in that: In step S2, the concentration of the thiolated hyaluronic acid solution is 0.5-5%.

6. The method for preparing the double-network hyaluronic acid-silk fibroin gel sponge according to claim 1, characterized in that: In step S3, the mass ratio of the silk fibroin in the silk fibroin solution to the thiolated hyaluronic acid in the thiolated hyaluronic acid solution is 1:3 to 3:

1.

7. The method for preparing the double-network hyaluronic acid-silk fibroin gel sponge according to claim 1, characterized in that: In step S4, the freezing treatment time is 6 to 12 hours.

8. The method for preparing the double-network hyaluronic acid-silk fibroin gel sponge according to claim 1, characterized in that: In step S4, the soft-freeze treatment time is 0.25 to 4 days.

9. The method for preparing the double-network hyaluronic acid-silk fibroin gel sponge according to claim 1, characterized in that: In step S4, the thawing temperature is 4-37°C.

10. A double-network hyaluronic acid-silk fibroin gel sponge, characterized in that: The double-network hyaluronic acid-silk fibroin gel sponge is prepared by the preparation method of any one of claims 1 to 9; the double-network hyaluronic acid-silk fibroin gel sponge comprises a hyaluronic acid gel matrix and a silk fibroin sponge structure distributed in the gel matrix.

Images