A hyaluronic acid-based hydrogel precursor, a hyaluronic acid-based hydrogel, and a preparation method and application thereof

Abstract

The present application relates to a kind of hyaluronic acid-based hydrogel precursor, hyaluronic acid-based hydrogel and its preparation method and application.The hyaluronic acid-based hydrogel precursor includes A component and B component, the preparation method of the A component includes the following steps: S1.methacrylate group modified hyaluronic acid and furfurylamine are mixed, react, obtain furan-methacrylate modified hyaluronic acid;S2.furan-methacrylate modified hyaluronic acid and maleimide are mixed, react, obtain double-modified group hyaluronic acid;S3.double-modified group hyaluronic acid, photoinitiator and solvent, namely A component;The B component is mercapto polyethylene glycol solution.The hyaluronic acid-based hydrogel precursor has very high flowability, can be injected and used and jetted;It can also be slow self-healing, also can realize fast self-healing under ultraviolet irradiation;It also has good hemostatic capacity, bone defect repair ability and skin defect repair capacity.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, and more specifically, to a hyaluronic acid-based hydrogel precursor solution, a hyaluronic acid-based hydrogel, its preparation method, and its application. Background Technology

[0002] The maxillofacial region is an important area for normal physiological activities and the transmission of emotional information, and its bones and skin form the cornerstone of human aesthetics. The jawbone plays a role in the movement and support of the human body; the skin of the maxillofacial region, as an external barrier, reflects facial aesthetics while protecting internal tissues and organs from external physical and chemical damage and microbial invasion.

[0003] Due to the complexity of the maxillofacial structure, injuries to the maxillofacial region often result in simultaneous defects in the jawbone, skin, blood vessels, and other tissues. For example, missing teeth, trauma, and cleft lip and palate frequently cause jawbone defects. Traditional treatments for jawbone defects include distraction osteogenesis, guided tissue regeneration, and bone grafting. While these methods promote bone regeneration to some extent and reduce the difficulty of orthodontics or orthognathic surgery, they still have many limitations, such as complex procedures, poor fit, insufficient donors, immune rejection, and high costs. In recent years, significant breakthroughs have been made in the production of synthetic bone scaffold materials, but their poor osteoinductive properties remain a challenge. Although adding biological components (such as cultured cells and growth factors, BMP2 protein, RGD sequences, etc.) to bone scaffold materials has the potential to increase osteoinductive effects, these tissue-engineered materials containing biological components are subject to complex drug regulatory restrictions and have very high usage costs in clinical applications. Therefore, when it comes to experimental research, clinical translation, and commercial production, bone tissue scaffold materials that are free of biological components and possess strong osteoinductive capabilities, capable of recruiting the body's own cells for bone regeneration, will have significant advantages in bone tissue engineering.

[0004] Trauma, congenital malformations, and surgery can cause skin defects in the maxillofacial region, leading to scar hyperplasia, morphological abnormalities, and functional defects, severely impacting appearance and quality of life. While most dermal wounds can heal spontaneously, the healing process is slow and limited, failing to fully regenerate and rebuild functional skin tissue.

[0005] If a material could serve both as a bone scaffold material and induce bone regeneration, and as a dressing to promote skin repair, it would be highly valuable in the treatment of combined soft and hard tissue injuries of the maxillofacial region. A Chinese patent, titled "A Hyaluronic Acid Supramolecular Hydrogel for Three-Dimensional Culture of Chondrocytes and Its Preparation and Application," introduces temperature-responsive poly(2-(2-methoxyethoxy)methyl methacrylate (PDEGMA) and ureidopyrimidinone (UPy) functional groups onto the hyaluronic acid backbone chain. The resulting hyaluronic acid supramolecular hydrogel possesses the ability to promote cartilage formation; however, research on its mechanism of bone tissue repair is still insufficient, and it does not focus on skin repair, thus its application prospects in the treatment of combined soft and hard tissue injuries of the maxillofacial region are unclear.

[0006] In addition, in maxillofacial injuries caused by trauma (such as traffic accidents, wartime trauma, etc.), bleeding from large blood vessels often occurs, so it is especially important to achieve rapid hemostasis of the wound. Currently, hydrogels are commonly used in the treatment of trauma, but for scenarios such as bleeding from maxillofacial injuries, the application of hydrogels should have the following characteristics: (1) The hydrogel precursor solution has high fluidity and can be sprayed, so that it can directly cover the entire surface of the wound according to the condition of the maxillofacial wound (e.g., shape), making it convenient to use; (2) The hydrogel precursor solution can quickly form gel; (3) It has a low hemolysis rate and good hemostatic function. Currently, hydrogels possessing all these characteristics are still relatively rare. Among them, for point (2), the gelation time of the current hydrogel precursor solution (under direct ultraviolet light irradiation) is generally more than 10 seconds (e.g., XHLiu, ZSYao, WPXue, X.Li, Effect of Temperature and Accelerator on Gel Time and Compressive Strength of Resin Anchoring Agent, Adv. Polym. Technol. 2019 (2019), or QCZhu, XHZhou, YAZhang, D.Ye, K.Yu, WBCao, LWZhang, HWZheng, ZYSun, CCGuo, XQHong, Y.Zhu, YJZhang, Y.Xiao, TGValencak, TCRen, DXRen, White-light crosslinkable milkprotein bioadhesive with ultrafast gelation for first-aid wound treatment, Biomaterials Research). 27(1)(2023)), a longer gelation time is not conducive to rapid hemostasis of the wound.

[0007] Therefore, it is of great significance to develop materials that can be sprayed and quickly gelled to achieve hemostasis of wounds, while also having the ability to repair bone and skin defects, so as to provide an integrated treatment solution for complex injuries of the maxillofacial soft and hard tissues. Summary of the Invention

[0008] The primary objective of this invention is to overcome the technical problems mentioned in the prior art and to provide a hyaluronic acid-based hydrogel precursor solution.

[0009] A further objective of this invention is to provide a method for preparing the above-mentioned hyaluronic acid-based hydrogel precursor solution.

[0010] A further object of the present invention is to provide a hyaluronic acid-based hydrogel.

[0011] A further object of the present invention is to provide the application of the above-mentioned hyaluronic acid-based hydrogel precursor solution or hyaluronic acid-based hydrogel in the preparation of repair materials for treating maxillofacial complex injuries.

[0012] The above-mentioned objective of the present invention is achieved through the following technical solution:

[0013] A hyaluronic acid-based hydrogel precursor solution includes component A and component B, wherein the preparation method of component A includes the following steps:

[0014] S1. Hyaluronic acid modified with methacrylate groups and furfurylamine are mixed and reacted to obtain furan-methacrylate modified hyaluronic acid;

[0015] S2. Furan-methacrylate modified hyaluronic acid and maleimide are mixed and reacted to obtain hyaluronic acid with dual modified groups;

[0016] S3. Mix the dual-modified hyaluronic acid, photoinitiator, and solvent to obtain component A;

[0017] Component B is a mercapto-polyethylene glycol solution, and the mass ratio of component A to component B is 1:

[0018] (2.5~5.0).

[0019] The hyaluronic acid-based hydrogel precursor solution of this invention first modifies the hyaluronic acid in component A by grafting furan groups onto methacrylate groups, and then reacts the furan groups with maleimide to form norbornene-like groups, resulting in hyaluronic acid possessing both methacrylate and norbornene-like photocrosslinking groups, i.e., hyaluronic acid with dual modified groups. This hyaluronic acid-based hydrogel precursor solution (after mixing components A and B) exhibits extremely high fluidity, allowing for both injection and spray applications. Through the combination of hyaluronic acid with dual modified groups, a photoinitiator, and mercapto polyethylene glycol, it can achieve rapid self-healing gel formation in 0.5 seconds under direct ultraviolet light, a gel formation speed superior to existing hydrogel materials, and can also achieve slow self-healing gel formation under conditions without ultraviolet light. Furthermore, the hyaluronic acid-based hydrogel formed from this precursor solution possesses excellent mechanical and swelling properties.

[0020] The inventors of this invention further discovered through research that the hyaluronic acid-based hydrogel precursor solution and its gelled hydrogel possess a variety of biological properties. First, this hyaluronic acid-based hydrogel not only possesses the inherent physical guiding effect of hydrogels but also a biological induction effect, thus exhibiting excellent bone loss repair capabilities. Second, the hyaluronic acid-based hydrogel precursor solution or its gelled hydrogel can promote platelet adsorption and erythrocyte aggregation, activating exogenous and endogenous pathways in the coagulation cascade reaction, and forming a physical barrier through the transition from the liquid phase to the gel phase, achieving rapid hemostasis in vitro and in vivo. Third, this hyaluronic acid-based hydrogel promotes healing at various stages of skin defects, including regulating macrophage differentiation during the inflammatory phase, promoting angiogenesis during the proliferative phase, and promoting fibroblast secretion of extracellular matrix during the remodeling phase, thereby achieving immune regulation and promoting angiogenesis, and ultimately promoting the repair of skin defects.

[0021] Because the hyaluronic acid-based hydrogel precursor liquid of the present invention can be sprayed, has a fast gelation speed, and has hemostatic function, as well as the ability to repair bone defects and skin defects at the same time, it can provide an integrated treatment solution for complex soft and hard tissue injuries of the maxillofacial region, and has good application prospects.

[0022] It should be understood that the methacrylate-modified hyaluronic acid mentioned in step S1 refers to hyaluronic acid grafted with methacrylate groups after modification treatment.

[0023] Preferably, the degree of substitution of the methacrylate groups in the methacrylate-modified hyaluronic acid in step S1 is 25-35%.

[0024] It should be understood that the degree of substitution of the methacrylate group refers to the ratio of the peak area of ​​the methyl hydrogen in the methacrylate group to the peak area of ​​the methyl hydrogen on the six-carbon ring side chain of hyaluronic acid in the NMR spectrum of methacrylate-modified hyaluronic acid.

[0025] Preferably, the methacrylate-modified hyaluronic acid in step S1 is prepared by the following method: hyaluronic acid or its salt is dissolved in water, methacrylic anhydride is added first, the pH is adjusted to 8.45-8.55, the reaction is carried out for 20-24 hours, and then dialyzed to obtain the methacrylate-modified hyaluronic acid.

[0026] More preferably, the reaction is carried out at 35–40°C.

[0027] More preferably, the relative molecular weight of the hyaluronic acid is 90-100 kDa.

[0028] More preferably, the ratio of hyaluronic acid to methacrylic anhydride is 1 g: (2.8-3.2 mL).

[0029] More preferably, the dialysis bag for dialysis has a size of 3000-5000 Da.

[0030] Preferably, the specific process of step S1 is as follows: first, dissolve the methacrylate-modified hyaluronic acid in morpholine ethanesulfonic acid buffer, then add 4-(4,6-dimethoxytriazine)-4-methylmorpholine hydrochloride, then add furfurylamine, mix, react for 20-24 hours, and dialyze to obtain furan-methacrylate-modified hyaluronic acid.

[0031] Preferably, the reaction temperature in step S1 is 25–30°C.

[0032] More preferably, the pH of the morpholine ethanesulfonic acid buffer solution is 4.4 to 4.6.

[0033] More preferably, the mass ratio of the methacrylate-modified hyaluronic acid to 4-(4,6-dimethoxytriazine)-4-methylmorpholine hydrochloride is 1:(2.8-3.0).

[0034] More preferably, the ratio of the methacrylate-modified hyaluronic acid to furfurylamine is 1 g: (0.35-0.40 mL).

[0035] More preferably, the dialysis bag for dialysis has a size of 3000-5000 Da.

[0036] Preferably, the degree of substitution of the furan group in the furan-methacrylate modified hyaluronic acid in step S1 is 55-65%.

[0037] It should be understood that the degree of substitution of the furan group refers to the ratio of the sum of the peak areas of the three hydrogen atoms of the five-carbon ring of the furan group to the peak area of ​​the methyl hydrogen on the six-carbon ring side chain of the hyaluronic acid in the nuclear magnetic resonance spectrum of furan-methacrylate modified hyaluronic acid.

[0038] Preferably, the specific process of step S2 is as follows: first, furan-methacrylate modified hyaluronic acid is dissolved in water, maleimide is added, mixed, reacted for 20-24 hours, and dialyzed to obtain double-modified hyaluronic acid.

[0039] Preferably, the reaction temperature in step S2 is 25–30°C.

[0040] More preferably, the mass ratio of furan-methacrylate modified hyaluronic acid to maleimide is 1:(1.0-1.2).

[0041] More preferably, the dialysis bag for dialysis has a size of 3000-5000 Da.

[0042] Preferably, the concentration (mass-volume ratio) of the dual-modified hyaluronic acid in component A in step S3 is 20 mg / mL to 25 mg / mL.

[0043] Preferably, in step S3, the mass ratio of the dual-modified hyaluronic acid to the photoinitiator in component A is 1:(0.1-0.25).

[0044] Preferably, the photoinitiator in step S3 is lithium phenyl-2,4,6-trimethylbenzoylphosphinic acid.

[0045] Preferably, the solvent in step S3 is water, physiological saline, or PBS buffer.

[0046] Preferably, the mercapto polyethylene glycol solution is a four-arm mercapto polyethylene glycol solution.

[0047] Preferably, the concentration (mass-volume ratio) of the mercapto polyethylene glycol solution is 500–1500 mg / mL.

[0048] More preferably, the concentration (mass-volume ratio) of the mercapto polyethylene glycol solution is 500-1000 mg / mL.

[0049] Preferably, the volume ratio of component A to component B is 10:(1-2).

[0050] The preparation method of the above-mentioned hyaluronic acid-based hydrogel precursor solution includes the following steps:

[0051] Component A is prepared according to the above preparation method; mercapto polyethylene glycol is dissolved in a solvent to obtain component B; component A and component B are mixed before gelation.

[0052] A hyaluronic acid-based hydrogel is obtained by gelling the aforementioned hyaluronic acid-based hydrogel precursor solution or the hyaluronic acid-based hydrogel precursor solution.

[0053] The hyaluronic acid-based hydrogel of the present invention can be used either as a precursor solution or as a gel after gelation.

[0054] The application of the above-mentioned hyaluronic acid-based hydrogel in the preparation of repair materials for treating maxillofacial complex injuries is also within the scope of protection of this invention.

[0055] Preferably, the repair material is a bone defect repair material and / or a skin defect repair material.

[0056] More preferably, the repair material is a bone defect repair material and / or skin defect repair material that promotes ITGA6 expression.

[0057] Preferably, the repair material is a repair material with hemostatic function.

[0058] Compared with the prior art, the beneficial effects of the present invention are:

[0059] The hyaluronic acid-based hydrogel precursor solution of the present invention has extremely high fluidity and can be used by injection or spraying; it can heal slowly or rapidly under ultraviolet light irradiation; it also has good hemostatic ability, bone defect repair ability and skin defect repair ability, thus providing an integrated treatment solution for complex soft and hard tissue injuries of the maxillofacial region, with good application prospects. Attached Figure Description

[0060] Figure 1 This is a schematic diagram illustrating the synthesis process of the dual-modified hyaluronic acid in component A of the hyaluronic acid-based hydrogel precursor solution in Example 1.

[0061] Figure 2 The images show the nuclear magnetic resonance spectra of the intermediate products (HA, HM, HFM) and the final product (HFMM) of Example 1.

[0062] Figure 3 This is a schematic diagram of the transformation process of the hyaluronic acid-based hydrogel precursor solution from liquid to gel in Example 1.

[0063] Figure 4 a is a schematic diagram illustrating the injection and use of the hyaluronic acid-based hydrogel precursor solution from Example 1. Figure 4 b is a schematic diagram of the spraying application of the hyaluronic acid-based hydrogel precursor solution in Example 1.

[0064] Figure 5 This is a schematic diagram of the UV-induced gelation process of the hyaluronic acid-based hydrogel precursor solution in Example 1.

[0065] Figure 6 This is a schematic diagram of the process of forming a gel after the hyaluronic acid-based hydrogel precursor solution of Example 1 is sprayed onto a glass slide.

[0066] Figure 7 This is a schematic diagram of the process of forming a gel after the hyaluronic acid-based hydrogel precursor liquid of Example 1 is sprayed onto the glove.

[0067] Figure 8 This is a schematic diagram of the slow and rapid self-healing processes of the hyaluronic acid-based hydrogel precursor solution in Example 1.

[0068] Figure 9 Microscopic morphology images of hyaluronic acid-based hydrogels after freeze-drying under different light exposure times.

[0069] Figure 10 Line graph showing the compression modulus of hyaluronic acid-based hydrogels treated with different light exposure times.

[0070] Figure 11 The figure shows the swelling equilibrium water absorption rate of hyaluronic acid-based hydrogels treated with different light exposure times.

[0071] Figure 12 This is a diagram showing the experimental results of ALP staining in Example 5.

[0072] Figure 13 The figure shows the experimental results of mineralization detection of the alizarin red dye solution in Example 5.

[0073] Figure 14 This is a graph showing the changes in rBMSC transcription levels in Example 5.

[0074] Figure 15 The figure shows the experimental results of the hemolytic properties of the precursor solution in Example 6.

[0075] Figure 16 The graph shows experimental data on in vitro hemostasis of the precursor solution and hydrogel in Example 6.

[0076] Figure 17 This is a data graph of the hemostasis experiment of the rat femoral vein puncture model in Example 6.

[0077] Figure 18 This is a graph showing the hemostasis data from the rat lethal liver injury model in Example 6.

[0078] Figure 19 This is a diagram showing the experimental results of the hydrogel before gelation in the erythrocyte aggregation analysis experiment of Example 6.

[0079] Figure 20 This is a diagram showing the experimental results of the hydrogel gelation in the erythrocyte aggregation analysis experiment of Example 6.

[0080] Figure 21 This is a SEM image of the erythrocyte aggregation analysis experiment in Example 6.

[0081] Figure 22 This is a SEM image of the platelet aggregation analysis experiment in Example 6.

[0082] Figure 23 This is a graph showing the experimental data from the coagulation pathway activation experiment in Example 6.

[0083] Figure 24 The figure shows the experimental results of the HUVEC angiogenesis capacity detection experiment in Example 7.

[0084] Figure 25 This is a diagram showing the changes in adhesion or junctional protein genes after the hyaluronic acid-based hydrogel of Example 8 came into contact with rBMSC, Raw264.7, and HUVEC cells.

[0085] Figure 26 This is a diagram showing the experimental results of the inhibition of ITGA6 protein expression on the cell surface in rBMSC cells during the osteogenic differentiation process described in Example 8.

[0086] Figure 27 Figure A shows the experimental results of the inhibition of ITGA6 protein expression on the cell surface in Example 8, illustrating the change in the differentiation ability of Raw264.7 M2 cells. Figure 27 Figure A shows the experimental results of the inhibition of ITGA6 protein expression on the cell surface in HUVEC cells during the angiogenesis experiment in Example 8. Detailed Implementation

[0087] To more clearly and completely describe the technical solution of the present invention, the present invention will be further described in detail below through specific embodiments. It should be understood that the specific embodiments described herein are only for explaining the present invention and are not intended to limit the present invention. Various changes can be made within the scope of the claims of the present invention.

[0088] Example 1

[0089] This embodiment provides a hyaluronic acid-based hydrogel precursor solution, which includes component A and component B; the preparation method of component A includes the following steps:

[0090] 1.1 Preparation of methacrylate-modified hyaluronic acid

[0091] Weigh 5g of sodium hyaluronate (HA, 100kDa, Shanghai Yuanye) solid and add it to 500mL of deionized water. Stir at room temperature (25℃) for 2 hours until completely dissolved into a colorless, transparent liquid. Add 15mL of methacrylic anhydride (MA, Sigma-Aldrich) dropwise over 3 hours. Adjust the pH of the system to 8.5 using 5M sodium hydroxide solution and stir the reaction overnight in a 37℃ water bath. Dialyze the resulting mixture into a 3000Da dialysis bag, changing the peripheral deionized water every 8 hours for 3 days. Collect the solution and freeze-dry it into a white, flocculent solid, which is methacrylate-modified hyaluronic acid, denoted as HM.

[0092] 1.2 Preparation of furan-methacrylate modified hyaluronic acid

[0093] First, prepare a 100mM, pH 4.5 morpholine ethanesulfonic acid buffer (MES); take 1.2L of MES and place it in a 2L brown light-proof beaker, and dissolve it completely; then weigh 4g of HM and slowly add it to the MES with magnetic stirring at 1000rpm / min; seal the beaker with aluminum foil and continue stirring at room temperature for 2 hours until the HM is completely dissolved and a clear and transparent solution is formed.

[0094] Weigh 11.2 g of 4-(4,6-dimethoxytriazine)-4-methylmorpholine hydrochloride (DMTMM) and slowly add it to the aforementioned solution. Stir at room temperature for 1 h to activate the carboxyl functional groups on the hyaluronic acid backbone using DMTMM, resulting in a mixed solution. Add 1.5 mL of furfurylamine (Furan, AR, Sigma-Aldrich) dropwise to the above mixed solution over 5 min, seal the solution with aluminum foil, and continue stirring at room temperature for 24 h.

[0095] The reacted mixture was placed in a 3000 Da dialysis bag for dialysis, with the peripheral deionized water replaced every 8 hours for 3 days. The mixture was then collected and freeze-dried into a white flocculent solid, which is furan-methacrylate modified hyaluronic acid, denoted as HFM.

[0096] 1.3 Preparation of Hyaluronic Acid with Two Modified Groups

[0097] Weigh 3g of lyophilized HFM and add it to 1.2L of deionized water. Stir at room temperature for 2 hours until completely dissolved into a colorless, transparent liquid. Then add 3g of maleimide (Mal, AR, Sigma-Aldrich) and stir at room temperature for 1 day. Place the resulting mixture into a 3000Da dialysis bag for dialyzing, changing the peripheral deionized water every 8 hours for 3 days. Collect the solution and lyophilize it into a white, flocculent solid, which is the dual-modified hyaluronic acid, denoted as HFMM.

[0098] 1.4 Preparation of Component A

[0099] Weigh 0.4g of HFMM and 0.1g of photoinitiator phenyl-2,4,6-trimethylbenzoyl lithium phosphine (LAP), add them to 20mL of deionized water, stir and mix thoroughly to prepare a 2% (wt / v) HFMM solution, which is component A.

[0100] A schematic diagram of the synthesis process of dual-modified hyaluronic acid is shown below. Figure 1 .

[0101] In this embodiment, component B is an aqueous solution of four-armed mercapto polyethylene glycol (PEG, 2kDa, AR, Sigma-Aldrich) with a concentration of 500 mg / mL. When using the hyaluronic acid-based hydrogel precursor solution of this application, components A and B are mixed at a volume ratio of 10:1, and the hyaluronic acid-based hydrogel is obtained after gelation.

[0102] Example 2

[0103] This embodiment provides a hyaluronic acid-based hydrogel precursor solution, which differs from Example 1 in that: component B is an aqueous solution of tetra-arm thiol polyethylene glycol (PEG, 2kDa, AR, Sigma-Aldrich) with a concentration (wt / v) of 1000 mg / mL.

[0104] Example 3

[0105] This embodiment provides a hyaluronic acid-based hydrogel precursor solution, which differs from Example 1 in that: component B is an aqueous solution of four-arm thiol polyethylene glycol (PEG, 2kDa, AR, Sigma-Aldrich) with a concentration (wt / v) of 1500 mg / mL.

[0106] Example 4 Sample Characterization

[0107] In this embodiment, the intermediate and final products of Example 1 are characterized.

[0108] 4.1 Study on the chemical structure of each component

[0109] Weigh out 0.01 g of HA, HM, HFM, and HFMM respectively, and dissolve them in 0.5 mL of D2O until completely dissolved. Then, transfer the mixed solutions to clean NMR tubes for 1H NMR spectroscopy. The results are as follows: Figure 2 As shown. Figure 2 These are the nuclear magnetic resonance spectra (H NMR, 600 MHz) of each component, where i is HA, ii is HM, iii is HFM, and iv is HFMM; Figure 2 A and Figure 2 C are respectively Figure 2 Enlarged view of different chemical shift ranges of B.

[0110] from Figure 2 It can be known that:

[0111] The sharp peak at chemical shift 2.00 ppm in HA is a characteristic peak of the methyl hydrogen atom on the HA side chain. The appearance of the characteristic peaks of the methacrylate double bond at 6.22 ppm and 5.75 ppm and the characteristic peak of methyl hydrogen (from the methacrylate group) at 1.93 ppm in HM proves that the hydroxyl group of the hydroxymethyl group on the side chain of the hexacarbon sugar ring of HA undergoes a grafting reaction with methacrylic anhydride to form HM, and the methacrylate group is successfully grafted onto hyaluronic acid. Compared with other hydroxyl groups, the hydroxyl group in the hydroxymethyl group on the surface of hyaluronic acid is the farthest from the hyaluronic acid backbone and has the least steric hindrance, so it is most likely to undergo esterification reaction with the methacrylate group. The degree of substitution of the methacrylate group in HM refers to the ratio of the peak area of ​​the methyl hydrogen in the methacrylate group (Area (1.93 ppm)) to the peak area of ​​the methyl hydrogen on the side chain of the hexacarbon ring of hyaluronic acid (Area (2.00 ppm)) in the NMR spectrum of methacrylate-modified hyaluronic acid, and the calculation formula is as follows:

[0112]

[0113] Calculations show that the DS of the methacrylate groups in HM is approximately 25%.

[0114] HFM retains the characteristic peak of methyl hydrogen atom at 2.00 ppm, as well as methacrylate peaks at 6.22 ppm, 5.75 ppm, and 1.93 ppm, indicating that the methacrylate group remains stable during furan modification. New characteristic peaks appear at chemical shifts of 6.35 ppm, 6.48 ppm, and 7.42 ppm, corresponding to the three hydrogen atoms on the five-carbon ring of the furan group. This proves that an amidation reaction occurred between the carboxyl group on the side chain of the hyaluronic acid molecule and the amino group on the furfurylamine molecule, and the furan group was successfully grafted onto HM. The degree of substitution of the furan group in HFM refers to the ratio of the sum of the peak areas of the three hydrogen atoms on the five-carbon ring of the furan group (Area(6.35ppm) + Area(6.48ppm) + Area(7.42ppm)) to the peak area of ​​the methyl hydrogen atom on the six-carbon ring side chain of the hyaluronic acid (Area(2.00ppm)) in the NMR spectrum of furan-methacrylate modified hyaluronic acid. The calculation formula is as follows:

[0115]

[0116] Calculations show that the DS of furan groups in HFM is approximately 55%.

[0117] HFMM also retains the characteristic peak of methyl hydrogen atoms at 2.00 ppm, characteristic peaks of furan groups at 6.35 ppm, 6.48 ppm, and 7.42 ppm, and peaks of methacrylate at 6.22 ppm, 5.75 ppm, and 1.93 ppm, proving that the attachment of the maleimide group did not affect the modified groups on the aforementioned product. Comparison with the HFM spectrum reveals that the chemical shift intensity corresponding to the characteristic hydrogen atoms on the furan group is significantly weaker, and a new peak is generated at 5.29 ppm. This new peak is the characteristic peak of the double bond of the norbornene-like group generated through a DA click chemical reaction between maleimide and the furan group, indicating that the norbornene-like group was successfully modified onto the hyaluronic acid side chain. The degree of substitution of the norbornene-like group is mainly affected by the degree of substitution of the furan group. In the gel molecule, whether or not active groups are grafted, the type and position of the grafted active groups, all affect the properties and functional activity of the gel. The above NMR data show that the hyaluronic acid of the present invention, with its dual-modified group, simultaneously possesses two photocrosslinking groups on its hyaluronic acid side chain: a norbornene group and a methacrylate group.

[0118] Compared to pure hyaluronic acid, HFMM simultaneously grafts two photocrosslinking groups onto its side chains. Different photocrosslinking groups exhibit different steric hindrance effects. In this case, the grafting order may affect the success of grafting and the degree of substitution of the two groups, ultimately impacting the properties and functional activity of the hydrogel. In this invention, since the steric hindrance of the norbornene-like group is significantly greater than that of the methacrylate group, if the norbornene-like group is grafted first under MES conditions, although the degree of substitution of the norbornene-like group is ensured, the steric hindrance of the norbornene-like group will affect the contact between methacrylic anhydride and the hydroxyl groups on the hyaluronic acid surface, thus affecting the degree of substitution of the methacrylate group.

[0119] Furthermore, since methacrylated hyaluronic acid is grafted via hydroxyl groups on the side chain, the reaction itself is more difficult than amidation of carboxyl groups. Therefore, the DS of methacrylate groups in HM is lower than that of simply grafting methacrylate groups onto the gelatin surface. When grafting furan onto HM, the presence of methacrylate groups creates steric hindrance, resulting in a lower DS of furan groups in HFM compared to the case of simply grafting furan groups onto the hyaluronic acid surface. However, in the HFMM of this invention, it is still higher than 50%.

[0120] In addition, the HFMM solution was subjected to ultraviolet absorption spectroscopy. An absorption peak was observed at 217 nm, which was derived from the conjugated diene structure of the furan group in the norbornene group. An absorption peak was observed at 213 nm, which was derived from the conjugated structure of the maleimide carbonyl group in the norbornene group.

[0121] 4.2 Macroscopic morphology of hyaluronic acid-based hydrogels

[0122] a) Achieving the transformation of the hyaluronic acid-based hydrogel precursor solution from a liquid to a gel state through brief ultraviolet light irradiation, such as... Figure 3 As shown.

[0123] b) Mix component A and component B, as follows: Figure 4 As shown, the mixture can be used not only for injection ( Figure 4 a), and it can also be sprayed ( Figure 4 b) This indicates that the hyaluronic acid-based hydrogel precursor has high fluidity and wide application potential. The viscosity of the hyaluronic acid-based hydrogel precursor of this invention is only 0.07 Pa·s at 1Hz and 10% shear strain (the shear strain experiment was conducted on a Malvern Kinexus rheometer using parallel plates (P20 TiL, 40 mm in diameter). 0.5 mL of the precursor was dropped onto the plates with a 0.5 mm gap, at a test temperature of 25°C, with a 10% strain, and the frequency increasing from 0.01 to 10 Hz for a total increment time of 60 seconds), which is only 2 to 4 times that of anhydrous ethanol (0.02–0.03 Pa·s), allowing it to be sprayed before gel formation.

[0124] c) Figure 5 It is a gelation test performed under direct ultraviolet light irradiation. Figure 5 In the images, from 5a to 5b, the UV irradiation time was controlled by an electronic timer, and the hyaluronic acid-based hydrogel precursor was subjected to direct UV irradiation for 0.5 s. From 5b to 5c to 5d, a rapid photocrosslinking Thiol-ene reaction occurred between the norbornene-like and methacrylate groups of the dual-modified hyaluronic acid and the thiol groups of the tetra-arm thiol-based polyethylene glycol. This reaction showed gel formation and that the hydrogel sheet was completely lifted. Figure 5 It has been shown that the hyaluronic acid-based hydrogel precursor solution can be gelled in as little as 0.5 seconds of direct UV irradiation, a significantly faster gelation speed compared to currently reported hydrogels (which typically require more than 10 seconds). Furthermore, the hydrogel formed by rapid photocrosslinking after 0.5 seconds of UV irradiation is colorless and transparent. In addition, the UV irradiation gelation experiment also demonstrated that the hyaluronic acid-based hydrogel precursor solution can be prepared into shapes corresponding to the mold size using specific molds, indicating its effective ability to fill tissue defects and ensuring the regeneration and repair of damaged tissues.

[0125] d) The aforementioned experiments demonstrated that the hyaluronic acid-based hydrogel precursor has a rapid gelation rate and high fluidity, therefore it can be sprayed onto the desired surface and rapidly gels upon exposure to ultraviolet light. Specifically, Figure 6 and Figure 7 The experiments involved spraying a hyaluronic acid-based hydrogel precursor solution onto the surfaces of a glass slide and a glove. Figure 6 a~c and Figure 7a-c represent the control group, sprayed with PBS buffer. Figure 6 d~g and Figure 7 Groups d through g were experimental groups, sprayed with a hyaluronic acid-based hydrogel precursor solution. Figure 6 g and Figure 7 All g-sized hydrogel sheets can be lifted.

[0126] 4.3 Self-healing properties of hyaluronic acid-based hydrogels

[0127] The hyaluronic acid-based hydrogel precursor solution from Example 1 can be used to achieve rapid self-healing of the hydrogel under ultraviolet light via a rapid photocrosslinking reaction of Thiol-ene, as shown in [reference needed]. Figure 8 a~f; Slow self-healing of hydrogels can also be achieved using Michael chemical cross-linking in the absence of UV irradiation, see [reference needed]. Figure 8 The slow self-healing time is approximately 20 minutes, ranging from g to h. Figure 8 i is the final image after the self-healing process is complete. Figure 8 In the image, the hydrogel on the left is a fast-healing hydrogel, and the hydrogel on the right is a slow-healing hydrogel.

[0128] 4.4 Rheological properties and gelation time of hyaluronic acid-based hydrogels

[0129] The transformation process of hyaluronic acid-based hydrogel from a liquid precursor to a gel after gelation was detected using a rotational rheometer. The test temperature was 25℃, the strain was 10%, the frequency was 1Hz, and the duration was 60 seconds. Without UV irradiation, when cyclic vibrational strain was applied to the hyaluronic acid-based hydrogel precursor, the precursor exhibited shear thinning characteristics, with both the storage modulus (G') and loss modulus (G”) decreasing to some extent. When using a UV lamp (395nm wavelength, 50mW / cm²), the transformation was further investigated. 2 When light is shone through a gap of approximately 0.5 mm between the power rheometer plate and the test stage, the hyaluronic acid-based hydrogel precursor solution rapidly solidifies, exhibiting a rapid increase in storage modulus. Although the loss modulus also increases to some extent, the increase in storage modulus is even faster. Correspondingly, the time point at which the storage modulus exceeds the loss modulus is considered the gel point of the gel. This experiment (using a dynamic rheometer) shows that the gel point of the hyaluronic acid-based hydrogel is approximately 3.5 s, further demonstrating that the gelation rate of the hyaluronic acid-based hydrogel precursor solution of this invention is significantly faster than currently reported hydrogels (for example, the gel point of GelMA hydrogel measured using the same method is around 27 s).

[0130] 4.5 Microscopic morphological changes

[0131] Figure 9 These are images showing the microstructure of hyaluronic acid-based hydrogels after freeze-drying under different light exposure times. Figure 9a to g were subjected to UV irradiation for 1s, 2s, 5s, 10s, 20s, 30s, and 60s, respectively. After UV irradiation, the solids were removed, freeze-dried into a white flocculent solid, sectioned, and scanned by electron microscopy. Figure 9 It can be seen that when the illumination time is 1-2 seconds, the proportion of macropores in the lyophilized hydrogel network is relatively high, the proportion of micropores is relatively low, and the pore walls are relatively thin. As the illumination time increases, the proportion of micropores in the lyophilized hydrogel network gradually increases; after the illumination time exceeds 10 seconds, the porosity changes little, and the increase in crosslinking degree decreases, indicating that excessive light has little effect on the degree of UV crosslinking.

[0132] 4.6 Mechanical Properties

[0133] The mechanical compressive properties of the hydrogel were tested on a universal mechanical testing machine. The loading rate of the vertical compressive stress was 1 mm / min, and the final strain after loading was 90%. Figure 10 This is a line graph showing the compressive modulus of hyaluronic acid-based hydrogels treated with different light exposure times. From... Figure 10 It is known that the compressive modulus of hyaluronic acid-based hydrogels increases with increasing light exposure time. When the UV light exposure time is 1 s, the compressive modulus of the hyaluronic acid-based hydrogel is 80.18 kPa; when the UV light exposure time is 5 s, the compressive modulus is 423.08 kPa, reaching 81.83% of the maximum compressive modulus. When the UV light exposure time is 10 s, the compressive modulus of the hyaluronic acid-based hydrogel reaches 90% of the maximum compressive modulus; further increasing the UV light exposure time does not significantly improve the mechanical properties of the gel. Under 60 s of light exposure, the compressive modulus of the hyaluronic acid-based hydrogel of this invention reaches over 400 kPa, with a maximum of 517.02 kPa, far exceeding the compressive modulus of GelMA hydrogels (approximately 80 kPa).

[0134] 4.7 Swelling properties

[0135] The hyaluronic acid-based hydrogels irradiated with ultraviolet light were individually removed and freeze-dried into white, sponge-like solids, which were then lyophilized into samples. The original weight of each sample was recorded as M0. The lyophilized hydrogel samples were placed in six-well plates, and 5 mL of deionized water was added to completely immerse them. The samples were removed at regular intervals t (t = 1 h, 2 h, 4 h, 8 h, 12 h), and the surface water was gently blotted dry with filter paper. The weight was recorded as M. t Until M t The value no longer changes and is denoted as the weight M at equilibrium swelling. tmax Equilibrium swelling water absorption rate (ESR) = (M tmax -M0) / M0.

[0136] Figure 11This is a graph showing the swelling equilibrium water absorption rate of hyaluronic acid-based hydrogels treated with different light exposure times. From... Figure 11 It can be seen that the equilibrium water absorption rate of the hyaluronic acid-based hydrogel gradually decreases with increasing light exposure time. The hyaluronic acid-based hydrogel with a light exposure time of 1 second has the highest water absorption rate at swelling equilibrium, reaching 121.6%. The decrease in equilibrium water absorption rate is most significant within 5 seconds of light exposure. When the light exposure time exceeds 10 seconds, the decrease in equilibrium water absorption rate gradually diminishes. At a light exposure time of 10 seconds, the water absorption rate of the hyaluronic acid-based hydrogel is 69.2%. These results indicate that the hyaluronic acid-based hydrogel of the present invention has strong hydrophilicity and good water retention performance as a matrix material.

[0137] The hyaluronic acid-based hydrogel precursor solutions of Examples 2 and 3 have similar properties to those of Example 1. They can also form gels quickly, have high fluidity, can be used by injection and spraying, and have good self-healing properties, mechanical properties, and swelling properties.

[0138] Example 5: Bone Defect Repair Capacity of Hyaluronic Acid-Based Hydrogel

[0139] This embodiment is based on the hyaluronic acid-based hydrogel precursor solutions of Examples 1 and 2. The deionized water in component A was replaced with an equal volume of PBS, and then components A and B were mixed before the experiment. The hyaluronic acid-based hydrogel precursor solutions of Examples 1 and 2 are designated as HFMM / PEG1 hydrogel and HFMM / PEG2 hydrogel, respectively. Additionally, a 5% (wt / v) methacrylamide gelatin hydrogel (designated as GelMA, purchased from Suzhou Yongqinquan Intelligent Equipment Co., Ltd.) was used as a control group. The UV irradiation time for each group was 10 s, the UV wavelength was 395 nm, and the power was 50 mW / cm². 2 .

[0140] 5.1 Isolation and purification of rat bone marrow mesenchymal stem cells (rBMSCs)

[0141] Healthy SPF-grade male SD rats (approximately 80g in size) were anesthetized, euthanized by cervical dislocation, and immersed in 75% ethanol. In a clean bench, the skin was cut open, and bone marrow aspiration was performed from the tibia and femur. The medullary cavity was flushed with serum-free DMEM-F12 medium. rBMSCs were cultured in DMEM-F12 containing 10% fetal bovine serum using the whole bone marrow culture method, with a penicillin-antibody concentration of 50 IU / mL. The culture temperature was 37℃. The rBMSCs used in the experiment were cells from passage 2 to passage 6.

[0142] 5.2 Detection of cell viability and cell proliferation of rBMSCs after hydrogel co-culture

[0143] The viability of rBMSCs was detected using a CCK-8 assay kit. rBMSCs were digested with trypsin in culture flasks and seeded at 200,000 cells / well in 48-well plates. After 24 h of culture in DMEM-F12 containing 10% fetal bovine serum, pre-synthesized (i.e., gelled under UV light) HFMM / PEG1 and HFMM / PEG2 hydrogels were co-cultured in the wells. Control groups were prepared with equal volumes of PBS and GelMA. After 24 h of co-culture, 30 μL of CCK-8 assay solution was added to each well, and incubation continued for 3 h. 100 μL of the supernatant from each well was then transferred to a 96-well plate, and the absorbance was measured at 450 nm.

[0144] Experimental results showed that after co-culturing for 24 hours, the cell survival rate of PBS, GelMA, HFMM / PEG1 hydrogel, and HFMM / PEG2 hydrogel was greater than 100%, indicating that the hyaluronic acid-based hydrogel of the present invention is non-toxic.

[0145] The proliferation capacity of rBMSCs was detected using a CCK-8 assay kit. rBMSCs were digested with trypsin in culture flasks and seeded at 100,000 per well in 48-well plates. After 24 h of incubation in DMEM-F12 containing 10% fetal bovine serum, pre-synthesized HFMM / PEG1 and HFMM / PEG2 hydrogels were co-cultured in the wells. Control groups were treated with equal volumes of PBS and GelMA. At different proliferation time points, 30 μL of CCK-8 assay solution was added to each well, and after incubation for another 3 h, 100 μL of the supernatant from each well was transferred to a 96-well plate, and the absorbance was measured at 450 nm.

[0146] Experimental results showed that after 3 days of co-culture, rBMSCs exhibited good proliferation activity in the HFMM / PEG1 hydrogel and HFMM / PEG2 hydrogel co-culture systems, with cell proliferation rates 1.10 times and 1.12 times higher than those in the PBS group, respectively. GelMA itself did not promote rBMSC proliferation, indicating that the hyaluronic acid-based hydrogel of the present invention has a certain promoting effect on the proliferation of rBMSCs.

[0147] 5.3 Alkaline phosphatase (ALP) activity, ALP staining, and mineralization detection of rBMSCs after hydrogel co-culture

[0148] ALP expression in rBMSCs was detected using an ALP kit and a BCA protein quantification kit. rBMSCs were digested with trypsin and seeded at 100,000 cells / well in 48-well plates. After 24 h of culture in DMEM-F12 containing 10% fetal bovine serum, pre-synthesized HFMM / PEG1 and HFMM / PEG2 hydrogels were placed in the wells for co-culture. Control groups were treated with equal volumes of PBS and GelMA, respectively. At different time points, cells were lysed using 0.1% Triition-100, and 100 μL of lysis buffer was added to each well of a 96-well plate for ALP absorbance detection using an ALP kit. 25 μL of lysis buffer was transferred to each well of a 96-well plate for BCA protein quantification. Finally, the ALP absorbance values ​​for each well were normalized according to the BCA detection results.

[0149] The experimental results showed that on day 7, the PBS group had the lowest ALP activity, at 0.17 μg / μL. There was no statistically significant difference in ALP activity between the GelMA group and the PBS group. However, the HFMM / PEG1 hydrogel and HFMM / PEG2 hydrogel co-culture systems showed significantly higher relative ALP activity, which was 1.44 times and 1.61 times that of the GelMA group and the PBS group, respectively.

[0150] On day 14, there was no statistically significant difference in ALP activity between the GelMA group and the PBS group. Significant differences in ALP activity were observed in the remaining groups, with the ALP activities of the HFMM / PEG1 hydrogel and HFMM / PEG2 hydrogel being 2.59 times and 2.96 times that of the PBS group, respectively. These results indicate that the hyaluronic acid-based hydrogel of the present invention can promote osteogenic differentiation of rBMSCs.

[0151] ALP staining was performed using the Beyotime ALP staining kit. rBMSCs in culture flasks were digested with trypsin and seeded at 500,000 per well in 6-well plates. After culturing in DMEM-F12 containing 10% fetal bovine serum for 24 h, pre-synthesized HFMM / PEG1 and HFMM / PEG2 hydrogels were placed in the wells for co-culturing. Control groups were treated with equal volumes of PBS and GelMA, respectively. At different time points, fixation with paraformaldehyde for 15 min was performed, followed by washing three times with PBS. Then, 2 mL of ALP staining working solution was added to each well and incubated at room temperature for 30 min. After washing five times with PBS, the samples were observed under a stereomicroscope.

[0152] Experimental results are as follows Figure 12As shown, under the same culture time, the deep blue stained areas in the HFMM / PEG1 and HFMM / PEG2 hydrogels were significantly more numerous than those in the GelMA and PBS groups. At 14 days, calcified areas appeared in patches in the HFMM / PEG1 and HFMM / PEG2 groups, while calcified areas in the GelMA and PBS groups remained sporadic.

[0153] Mineralization was detected using Alizarin Red staining solution. rBMSCs in culture flasks were digested with trypsin and seeded at 500,000 per well in 6-well plates. After culturing in DMEM-F12 containing 10% fetal bovine serum for 24 h, pre-synthesized HFMM / PEG1 and HFMM / PEG2 hydrogels were placed in the wells for co-culturing. Control groups were treated with equal volumes of PBS and GelMA, respectively. At different time points, fixation with paraformaldehyde was performed for 15 min. After washing three times with PBS, 2 mL of Alizarin Red S (2% w / v) solution was added to each well and incubated at room temperature for 30 min. After washing five times with PBS, observation was performed under a stereomicroscope.

[0154] The result is as follows Figure 13 As shown, on day 7, large areas of calcified nodules stained red appeared in the HFMM / PEG2 hydrogel, small areas of calcified nodules appeared in the HFMM / PEG1 hydrogel, while the GelMA and PBS groups only showed cell outlines and sporadic calcified nodules. On day 14, calcified areas appeared in patches in the rBMSCs of the HFMM / PEG1 and HFMM / PEG2 groups, while calcified nodules still appeared sporadically in the GelMA and PBS groups.

[0155] ALP staining and alizarin red staining experiments further demonstrated that the hyaluronic acid-based hydrogel of the present invention can promote osteogenic differentiation of rBMSCs in vitro and form calcified nodules.

[0156] 5.4 Immunofluorescence detection of rBMSCs after hydrogel co-culture

[0157] rBMSCs were digested from culture flasks using trypsin and seeded at 500,000 cells / well in confocal dishes. After 24 hours of culture in DMEM-F12 containing 10% fetal bovine serum, pre-synthesized HFMM / PEG1 and HFMM / PEG2 hydrogels were placed in the wells for co-culture. Control groups were treated with equal volumes of PBS and GelMA. Cells were cultured for 7 and 14 days, respectively, fixed with paraformaldehyde for 15 minutes, and permeabilized with 0.1% Triton-100 for 15 minutes. BMP2 protein was stained green using Mouse-BMP2 primary antibody + Goat anti-mouse-AF488 secondary antibody; OCN protein was stained red using Rabbit-OCN primary antibody + Goat anti-rabbit-AF647 secondary antibody; and cell nuclei were stained blue using DAPI. Each staining step required washing with PBS 3-4 times to remove residual reagents. Finally, observation was performed using a laser confocal microscope.

[0158] The experimental results showed that after 7 days of co-culture, the expression of BMP2 and OCN in cells of both the PBS control group and the GelMA group was not significant, indicating that the number of osteogenic differentiated cells was relatively small in the PBS and GelMA groups. The expression of BMP2 and OCN in HFMM / PEG1 hydrogel and HFMM / PEG2 hydrogel was significantly higher than that in the PBS and GelMA groups, with HFMM / PEG2 showing the highest expression of both. Simultaneously, the cell morphology in both HFMM / PEG1 and HFMM / PEG2 hydrogel groups was more spindle-shaped, indicating a stronger tendency towards osteogenic differentiation. DAPI nuclear labeling revealed significant cell growth and aggregation in both HFMM / PEG1 and HFMM / PEG2 hydrogel groups, leading to cell cluster formation.

[0159] After 14 days of co-culture, BMP2 expression increased in both the PBS and GelMA groups. Cells expressing BMP2 were predominantly polygonal and spherical, exhibiting a relatively primitive morphology. BMP2 and OCN expression in the HFMM / PEG1 and HFMM / PEG2 hydrogel groups remained significantly higher than in the PBS and GelMA groups.

[0160] 5.5 Changes in the transcriptional level of rBMSCs after hydrogel co-culture

[0161] rBMSCs in culture flasks were digested with trypsin and seeded at 500,000 per well in 6-well plates. After 24 h of incubation in DMEM-F12 containing 10% fetal bovine serum, pre-synthesized HFMM / PEG1 and HFMM / PEG2 hydrogels were placed in the wells for co-culture. Control groups were treated with equal volumes of PBS and GelMA. Total RNA was extracted from the processed rBMSCs at different time points using an RNA extraction kit and a centrifugation column method. Total RNA quantification was performed using a Nanodrop RNA quantifier. This experiment was repeated three times.

[0162] The extracted total RNA was divided into two portions. One portion was placed on dry ice and sent for RNA-seq analysis. The other portion was reverse transcribed into cDNA using a Takara reverse transcription kit for subsequent RT-qPCR experiments.

[0163] Experimental results are as follows Figure 14 As shown in Figure A, at day 3, the Bmp2 gene expression levels in both the HFMM / PEG1 and HFMM / PEG2 hydrogel groups were significantly higher than those in the PBS control group and the GelMA group, with statistically significant differences. The Bmp2 gene expression level in the HFMM / PEG2 group was 1.85 times that of the control group. At day 7, the Bmp2 gene expression levels in the HFMM / PEG1 and HFMM / PEG2 hydrogel groups remained higher, while the Bmp2 expression level in the GelMA group showed a slight increase compared to the PBS group. At day 14, the Bmp2 gene expression levels in both the HFMM / PEG1 and HFMM / PEG2 hydrogel groups were significantly increased, reaching 2.96 times and 3.46 times that of the PBS group, respectively, and also 2.01 times and 3 times that of the GelMA group, with statistically significant differences between the groups.

[0164] Experimental results are as follows Figure 14As shown in Figure B, at day 3, the Ocn gene expression levels in both the HFMM / PEG1 and HFMM / PEG2 hydrogel groups were significantly higher than those in the PBS control group and the GelMA group, approximately twice that of the PBS group. There was no statistically significant difference in Ocn expression levels between the HFMM / PEG1 and HFMM / PEG2 hydrogel groups. At day 7, the Ocn gene expression levels in both the HFMM / PEG1 and HFMM / PEG2 hydrogel groups were 10-15 times higher than those in the PBS group. At this time, the Ocn expression level in the GelMA group showed a slight increase compared to the PBS group, consistent with the immunofluorescence results. At day 14, the Ocn gene expression levels in both the HFMM / PEG1 and HFMM / PEG2 hydrogel groups remained significantly higher than those in the PBS and GelMA groups, reaching 12.5 times and 20 times higher than those in the PBS group, respectively, with statistically significant differences between the groups. It is worth noting that the expression level of the Ocn gene in the GelMA group at 14 days was lower than that at 7 days, indicating that the hyaluronic acid-based hydrogel of the present invention plays an important role in enhancing and maintaining the expression of the Ocn gene.

[0165] 5.6 Changes in protein levels of rBMSCs after hydrogel co-culture

[0166] rBMSCs in culture flasks were digested with trypsin and seeded at 500,000 cells / well in 6-well plates. After culturing in DMEM-F12 containing 10% fetal bovine serum for 24 h, pre-synthesized HFMM / PEG1 and HFMM / PEG2 hydrogels were placed in the wells for co-culture. An equal volume of PBS and GelMA were added to the control group, respectively. At different time points, RIPA was added to lyse the cells, along with protease inhibitors and phosphatase inhibitors. Total protein was extracted from the treated rBMSCs. Protein quantification was performed using a BCA protein assay kit, with loading buffer added and incubated at 100°C for 10 min to denature proteins. Western blotting (WB) was then performed. This experiment was repeated three times.

[0167] The experimental results showed that, compared with the PBS control group, after co-culturing rBMSCs with HFMM / PEG1 hydrogel and HFMM / PEG2 hydrogel for 14 days, the levels of SMAD2 / 3, p-SMAD2 / 3, SMAD1 / 5 / 9, p-SMAD1 / 5 / 9 and TGFβ1 were significantly increased, suggesting that the hyaluronic acid-based hydrogel of the present invention activates the TGF-β pathway to promote osteoblast differentiation of rBMSCs.

[0168] Furthermore, this embodiment further demonstrates, through the construction of an SD rat skull defect model, that the hyaluronic acid-based hydrogel of the present invention can promote the repair of skull defects in rats, and the effect is significantly better than that of PBS and the hyaluronic acid-based hydrogel of the present invention.

[0169] In this embodiment, GelMA was used as the control group because: (1) GelMA hydrogel has similar structural characteristics to the hyaluronic acid-based hydrogel of the present invention; (2) GelMA hydrogel has similar physicochemical properties to the hyaluronic acid-based hydrogel of the present invention; and (3) the osteogenic efficacy of GelMA hydrogel has been widely verified. However, the present invention mainly studies the bio-induction effect of the hyaluronic acid-based hydrogel itself on rBMSCs, rather than the physical guiding effect of the scaffold material. Therefore, GelMA was used as the positive control in this experiment to exclude the physical guiding effect of the scaffold on bone defect repair.

[0170] Furthermore, the test data of this embodiment show that the hyaluronic acid-based hydrogel of the present invention significantly enhances the activity and proliferation of rBMSCs, increases the ALP activity of rBMSCs, increases the expression of osteogenic genes such as Ocn and Bmp2, increases the protein levels of OCN and BMP2, and promotes in vitro mineralization; increases the protein levels of BMP2, SMAD2 / 3, and SMAD1 / 5 / 9, increases the levels of p-SMAD2 / 3 and p-SMAD1 / 5 / 9, activates the TGF-β pathway, promotes osteogenic differentiation of rBMSCs, and inhibits osteoclast differentiation of rBMSCs; through the test data and its comparison with the GelMA control group, it is further shown that the hyaluronic acid-based hydrogel of the present invention has a biological induction effect on rBMSCs.

[0171] Example 6: Hemostatic ability of hyaluronic acid-based hydrogel

[0172] In this embodiment, the hyaluronic acid-based hydrogel precursor solutions from Examples 1 and 2 were used. The deionized water in component A was replaced with an equal volume of physiological saline. Then, components A and B were mixed and the experiment was conducted. The hyaluronic acid-based hydrogel precursor solutions from Examples 1 and 2 were designated as HFMM / PEG1 hydrogel and HFMM / PEG2 hydrogel, respectively. The UV irradiation time for each group was 10 seconds, the wavelength of the UV light was 395 nm, and the power was 50 mW / cm². 2 .

[0173] 6.1 Hemolytic properties of HFMM, four-arm thiol polyethylene glycol, and HFMM / PEG hydrogel precursor solution

[0174] Before the hydrogel photocures, the precursor solution remains in contact with the tissue; therefore, it is necessary to test the hemolysis of each component of the hydrogel and the precursor solution. Rabbit whole blood, anticoagulated with sodium citrate at a constant temperature of 37℃, was aliquoted into seven 2mL EP tubes, 0.2mL per tube. 0.8mL of deionized water, physiological saline, GelMA precursor solution, 2% HFMM solution, 500mg / mL four-arm thiol polyethylene glycol solution, HFMM / PEG1 hydrogel precursor solution, and HFMM / PEG2 hydrogel precursor solution were added to each EP tube, respectively. The material and blood were mixed thoroughly and incubated at 37℃ for 1 hour. After centrifugation at 3000rpm for 15 minutes, the supernatant was carefully aspirated, and the absorbance was measured at 562nm to calculate the hemolysis rate. This process was repeated three times.

[0175]

[0176] Experimental results showed that, using physiological saline as a negative control and deionized water as a positive control, the hemolysis rates of GelMA precursor solution, 2% HFMM solution, 500 mg / mL four-arm thiol polyethylene glycol solution, HFMM / PEG1 hydrogel precursor solution, and HFMM / PEG2 hydrogel precursor solution were all below 4%, significantly lower than the positive control. This indicates that the hyaluronic acid-based hydrogel precursor solution of the present invention has high safety for erythrocytes and a low possibility of inducing a hemolytic reaction. The hemolysis situation is shown in the figure below. Figure 15 As shown.

[0177] 6.2 Hemolytic Properties of Hydrogels

[0178] Take one 96-well plate and add 200 μL of deionized water, physiological saline, GelMA precursor solution, HFMM / PEG1 hydrogel precursor solution, and HFMM / PEG2 hydrogel precursor solution to the wells respectively, ensuring the material is evenly spread at the bottom of the plate. Irradiate with UV light for 10 s. Take rabbit whole blood anticoagulated with sodium citrate at 37℃ and add 200 μL to each well. Incubate at 37℃ for 1 h, centrifuge at 3000 rpm for 15 min, scan and record the erythrocyte sedimentation and hemolysis on the gel, and carefully aspirate the supernatant to measure the absorbance at 562 nm. Repeat three times. The hemolysis rate is calculated using the same formula as above.

[0179] Experimental results show that after the hydrogels solidify, the hemolysis rates of GelMA hydrogel, HFMM / PEG1 hydrogel, and HFMM / PEG2 hydrogel are all less than 5%, and the hemolysis rate of the hydrogels is lower than that of their precursor solutions.

[0180] 6.3. In vitro coagulation

[0181] Take 4 mL of rabbit whole blood anticoagulated with sodium citrate at a constant temperature of 37℃ and place it in a 15 mL centrifuge tube. Add 400 μL of 0.2 M CaCl2 solution (prepared with physiological saline) for activation. After mixing thoroughly by pipetting, quickly dispense 0.5 mL into six 1.5 mL EP tubes. Add 100 μL of physiological saline, GelMA precursor solution, 2% HFMM solution, four-arm mercapto polyethylene glycol solution, HFMM / PEG1 hydrogel precursor solution, and HFMM / PEG2 hydrogel precursor solution to each EP tube, respectively. Place the tube on a silent mixer and mix the materials with the blood thoroughly. Record the time when the blood in the tube stops flowing; this is the in vitro clotting time of the gel precursor solution and the process components. Repeat three times.

[0182] Experimental results are as follows Figure 16 As shown in Figure A, compared with the saline group, there was no statistically significant difference in in vitro coagulation time between the GelMA precursor solution group and the saline control group. The coagulation time of 2% HFMM solution, HFMM / PEG1 hydrogel precursor solution and HFMM / PEG2 hydrogel precursor solution was shortened by more than 50% compared with the GelMA group and the saline control group. Among them, HFMM / PEG2 hydrogel precursor solution had the best procoagulant effect, with a coagulation time of about 201s, which shortened the coagulation time by 72%.

[0183] Take one 96-well plate and add 100 μL of GelMA precursor solution, HFMM / PEG1 hydrogel precursor solution, and HFMM / PEG2 hydrogel precursor solution to the wells respectively, ensuring the material is evenly spread at the bottom of the plate. Irradiate with UV light for 10 seconds and carefully remove the plate. Take 2 mL of rabbit whole blood (anticoagulated with sodium citrate at 37℃) into four 4 mL EP tubes, add 60 μL of 0.1 M CaCl2 solution (prepared with physiological saline) for activation, mix thoroughly by pipetting, and then quickly add the prepared hydrogel. For the control group, add 50 μL of physiological saline. Place the tubes on a silent mixer and record the time it takes for the blood to stop flowing; this is the in vitro clotting time of the hydrogel. Repeat three times.

[0184] Experimental results are as follows Figure 16 As shown in B, GelMA hydrogel also did not promote coagulation after gelation; HFMM / PEG1 hydrogel and HFMM / PEG2 hydrogel also shortened the in vitro coagulation time after gelation. Among them, HFMM / PEG2 hydrogel shortened the coagulation time by 21% and had a better coagulation effect.

[0185] 6.4 Hemostasis Experiment for Rat Femoral Vein Puncture Model

[0186] Rats were anesthetized with Sutacetin, and the inner thigh and groin area were incised, taking care not to puncture the abdominal cavity. The femoral vein was carefully dissected. A 25-gauge syringe needle was used for femoral vein puncture; successful puncture was confirmed by blood aspiration. HFMM / PEG2 hydrogel was immediately sprayed, while the control group received physiological saline and GelMA (GelMA has low fluidity and was applied dropwise using a pipette). After UV curing, the immediate hemostasis effect was observed. The rat wound was then sutured layer by layer, and the rat survival rate was recorded postoperatively.

[0187] Experimental results are as follows Figure 17 As shown, Figure 17 The vertical axis of 'a' represents the amount of blood loss. Figure 17 The vertical axis of b represents clotting time. Figure 17 The ordinate of c represents the survival curve. The application of HFMM / PEG2 hydrogel after puncture resulted in the best hemostatic effect, with the highest survival rate of rats 12 hours post-surgery, significantly stronger than the GelMA group and the saline group. The hemostatic effect of the GelMA group was slightly stronger than that of the saline group, providing temporary hemostasis after puncture; however, touching the area around the puncture site easily induced rebleeding.

[0188] 6.5 Hemostasis experiment for a rat model of lethal liver injury

[0189] Under sedation with sutaric acid, the rat's abdomen was incised midline below the xiphoid process, taking care not to puncture the thoracic cavity. The liver was grasped with towel forceps, and a large portion of the liver was exposed by retracting the incision edge, with sterile filter paper placed underneath. The liver edge was removed using surgical scissors, with a cutting path length of approximately 2 cm. HFMM / PEG2 hydrogel was immediately sprayed on; the control group used physiological saline. After UV irradiation, the immediate hemostasis effect was observed. Blood loss in the rats was measured two minutes later. The liver tissue, along with the surgical materials, was then pushed back into the abdominal cavity, and the abdominal wound was sutured layer by layer. Postoperative survival of the rats was recorded.

[0190] Experimental results are as follows Figure 18 As shown, Figure 18 The vertical axis of 'a' represents the amount of blood loss. Figure 18 The vertical axis of b represents clotting time. Figure 18 The ordinate of c represents the survival curve. After establishing the rat lethal liver injury model, spraying HFMM / PEG2 hydrogel resulted in rapid hemostasis, with a significantly stronger hemostatic effect than the saline group. The rats had the highest survival rate at 12 weeks post-surgery.

[0191] 6.6 Investigation into the hemostatic mechanism

[0192] 6.6.1 Erythrocyte adsorption rate

[0193] Take one 96-well plate and add 50 μL of GelMA precursor solution, HFMM / PEG1 hydrogel precursor solution, and HFMM / PEG2 hydrogel precursor solution to the wells respectively, ensuring the material is evenly spread at the bottom of the plate. Irradiate with UV light for 10 s. Take rabbit whole blood anticoagulated with sodium citrate at 37℃, centrifuge at 3000 rpm for 15 min, and prepare a 5% red blood cell suspension (dissolved in physiological saline). Add 10 μL of 5% red blood cell suspension to the center of each well and let it stand for 60 min. Then gently add 100 μL of physiological saline, place on a shaker, and shake slowly for 5 min. Aspirate 20 μL of the supernatant and transfer it to a new 96-well plate. Add 100 μL of deionized water and shake slowly on a shaker for 10 min. Then measure the absorbance at a wavelength of 562 nm.

[0194] Calculate the red blood cell adsorption rate of the hydrogel. Repeat three times.

[0195]

[0196] Experimental results showed that the red blood cell adsorption rate of the HFMM / PEG1 hydrogel group was (27.54±2.72%), and the red blood cell adsorption rate of the HFMM / PEG2 hydrogel group was (36.58±4.61%), which were significantly higher than those of the GelMA group (9.87±1.57%), indicating that the hyaluronic acid-based hydrogel of the present invention can promote red blood cell adsorption.

[0197] 6.6.2 Erythrocyte aggregation analysis

[0198] Take one 96-well plate and add 50 μL of GelMA precursor solution, HFMM / PEG1 hydrogel precursor solution, and HFMM / PEG2 hydrogel precursor solution to the wells respectively. Incubate under UV light for 10 s to form GelMA gel, HFMM / PEG1 gel, and HFMM / PEG2 gel, respectively. Then add 50 μL of physiological saline to each well. Add 50 μL of physiological saline, GelMA precursor solution, 2% HFMM solution, 500 mg / mL four-arm thiol polyethylene glycol solution, HFMM / PEG1 hydrogel precursor solution, and HFMM / PEG2 hydrogel precursor solution to the other wells of the 96-well plate respectively. Take rabbit whole blood anticoagulated with sodium citrate at 37℃, centrifuge at 3000 rpm for 15 min to prepare a 5% erythrocyte suspension (dissolved in physiological saline), and quickly add 5 μL to each well of the 96-well plate. Shake for 15 s to mix thoroughly, and immediately observe the aggregation behavior of erythrocytes using an inverted microscope.

[0199] Before ultraviolet light irradiation, the experimental results are as follows: Figure 19 As shown. Figure 19 middle, Figure 19 a is physiological saline; Figure 19 b is a GelMA precursor solution; Figure 19 c is a 2% HFMM precursor solution; Figure 19 d represents PEG; Figure 19 e is the HFMM / PEG1 precursor solution; Figure 19 f represents the HFMM / PEG2 precursor solution; all scale bars represent 400 μm. From Figure 19 It was found that, compared to the saline group, diluted erythrocytes showed aggregation under both 2% HFMM and GelMA precursor solutions, indicating that both HFMM and GelMA have a certain erythrocyte aggregation effect. The erythrocyte aggregation was even more pronounced under HFMM / PEG1 and HFMM / PEG2 hydrogel precursor solutions.

[0200] After the gel was formed by ultraviolet light, the experimental results were as follows: Figure 20 As shown, Figure 20 middle, Figure 20 a is physiological saline; Figure 20 b is a GelMA hydrogel; Figure 20 c is an HFMM / PEG1 hydrogel; Figure 20 d represents HFMM / PEG2 hydrogel, and all scale bars represent 400 μm. From Figure 20 It can be seen that the erythrocyte aggregation effect on the surface of GelMA hydrogel is weak, while HFMM / PEG1 hydrogel and HFMM / PEG2 hydrogel still maintain a good surface erythrocyte aggregation effect.

[0201] Take one 96-well plate and add GelMA precursor solution, HFMM / PEG1 hydrogel precursor solution, and HFMM / PEG2 hydrogel precursor solution to the wells respectively, so that the material can be evenly spread on the bottom of the plate. Irradiate with UV light for 10s, and carefully remove the prepared hydrogel sheet. Take rabbit whole blood anticoagulated with sodium citrate at 37℃, centrifuge at 3000rpm for 15min to prepare a 5% red blood cell suspension (dissolved with physiological saline), and quickly add it dropwise onto the gel sheet, 50μL per sheet. After standing for 60min, soak the above sample in paraformaldehyde for 2h, dehydrate with ethanol gradient, dry, place on the sample stage, sputter-coated with gold, and observe with scanning electron microscopy.

[0202] Experimental results are as follows Figure 21 As shown in the SEM images, the aggregation of erythrocytes on the surface of HFMM / PEG hydrogel was significantly stronger than that on GelMA hydrogel; among them, HFMM / PEG2 hydrogel exhibited stronger aggregation of erythrocytes than HFMM / PEG1 hydrogel. Both of these groups showed stronger aggregation than GelMA hydrogel.

[0203] 6.6.3 Platelet Adsorption Rate and Aggregation Analysis

[0204] Rabbit blood anticoagulated with sodium citrate was centrifuged at 1500 rpm for 5 min, and the supernatant was collected to obtain platelet-rich plasma (PRP). One 96-well plate was used, and 50 μL of GelMA precursor solution, HFMM / PEG1 hydrogel precursor solution, and HFMM / PEG2 hydrogel precursor solution were added to the wells respectively. The plates were then exposed to UV light for 10 s to form GelMA gel, HFMM / PEG1 gel, and HFMM / PEG2 gel, respectively. Then, 20 μL of PRP was added to each well, and the plates were incubated at 37°C for 60 min. The gel surfaces were then carefully washed with physiological saline to remove unadhered platelets. After removing the saline, 50 μL of 1% Tritium X-100 saline solution was added to each well, and the plates were lysed for 60 min. 10 μL of the lysis buffer was then transferred to a new 96-well plate, and 100 μL of LDH working solution was added to each well. The absorbance was then measured at 450 nm to calculate the platelet adsorption rate of the hydrogel. PRP was used as a control. The experiment was repeated three times.

[0205]

[0206] The experimental results showed that the platelet adsorption capacity of HFMM / PEG1 hydrogel and HFMM / PEG2 hydrogel was significantly stronger than that of GelMA hydrogel. However, there was no statistically significant difference in platelet aggregation capacity between the two hyaluronic acid-based hydrogels, HFMM / PEG1 and HFMM / PEG2.

[0207] Take one 96-well plate and add GelMA precursor solution, HFMM / PEG1 hydrogel precursor solution, and HFMM / PEG2 hydrogel precursor solution to the wells respectively, so that the material can be evenly spread on the bottom of the plate. Irradiate with UV light for 10s and carefully remove the prepared hydrogel sheet. Take rabbit whole blood anticoagulated with sodium citrate at 37℃, centrifuge at 1500rpm for 5min to prepare platelet-rich serum (PRP), and quickly add 50μL of gel to each gel sheet. After standing for 60min, soak the above sample in paraformaldehyde for 2h, dehydrate with ethanol gradient, dry, place on the sample stage, sputter-coated with gold, and observe with scanning electron microscope.

[0208] Experimental results are as follows Figure 22 As shown in the SEM images, platelet aggregation on the HFMM / PEG hydrogel surface was significantly stronger than that on the GelMA hydrogel.

[0209] 6.6.4 Activation of the coagulation pathway

[0210] Prothrombin time (PT) and activated partial thromboplastin time (APTT) are important clinical indicators used to determine extrinsic and intrinsic coagulation pathways. PT is used for extrinsic coagulation, while APTT is used for intrinsic coagulation.

[0211] Fresh rabbit blood anticoagulated with sodium citrate was centrifuged at 3000 rpm for 15 min, and the supernatant was collected to obtain anemic platelet-rich plasma (PPP). 80 μL of PPP preheated to 37°C and 20 μL of each sample (containing physiological saline, GelMA precursor solution, 2% HFMM, four-arm mercapto polyethylene glycol, HFMM / PEG1 hydrogel precursor solution, HFMM / PEG2 hydrogel precursor solution, GelMA hydrogel block, HFMM / PEG1 hydrogel block, or HFMM / PEG2 hydrogel block, respectively) were added to 100 μL of ellagic acid solution. The mixture was kept in a 37°C water bath for 5 min, with intermittent mixing several times. Then, 100 μL of preheated 37°C CaCl2 (25 mM) solution was added, and the APTT timing was started immediately. During the test, the mixture was continuously agitated in the water bath, and the time to the appearance of fibrin filaments (initial coagulation with turbidity) was observed. This was repeated three times.

[0212] Experimental results are as follows Figure 23 As shown in Figure A, GelMA precursor solution, GelMA hydrogel block, and four-arm thiol polyethylene glycol did not shorten APTT. In fact, compared to the saline group, the four-arm thiol polyethylene glycol prolonged APTT by 8.9%, indicating that it slightly inhibited the intrinsic coagulation pathway. The 2% HFMM showed the most significant APTT reduction, effectively shortening plasma coagulation time by 60.5% compared to the saline group. HFMM / PEG2 hydrogel precursor solution, HFMM / PEG1 hydrogel precursor solution, HFMM / PEG2 hydrogel block, and HFMM / PEG1 hydrogel block also showed some degree of APTT reduction, by 49.2%, 46.0%, 25.8%, and 18.5%, respectively, indicating that these components can significantly activate the intrinsic coagulation pathway.

[0213] Fresh rabbit blood anticoagulated with sodium citrate was centrifuged at 3000 rpm for 15 min, and the supernatant was collected to obtain anemic platelet-rich plasma (PPP). 80 μL of PPP preheated to 37°C and 20 μL of each sample group (including physiological saline, GelMA precursor solution, 2% HFMM, four-arm mercapto polyethylene glycol, HFMM / PEG1 hydrogel precursor solution, HFMM / PEG2 hydrogel precursor solution, GelMA hydrogel block, HFMM / PEG1 hydrogel block, or HFMM / PEG2 hydrogel block, respectively) were added to 100 μL of PT reagent. The mixture was kept in a 37°C water bath for 5 min, with intermittent mixing several times. Then, 100 μL of preheated 37°C CaCl2 (25 mM) solution was added, and PT timing was started immediately. During the PT, the mixture was continuously agitated in the water bath, and the PPP coagulation time was calculated. This process was repeated three times.

[0214] Experimental results are as follows Figure 23As shown in B, compared with NS, the four-arm thiol polyethylene glycol shortened PT by about 3 seconds, indicating that it can activate the extrinsic coagulation pathway. There was no statistically significant difference in PT time among the HFMM / PEG2 hydrogel precursor solution, HFMM / PEG1 hydrogel, 2% HFMM, GelMA precursor solution, GelMA hydrogel block, HFMM / PEG1 hydrogel block and HFMM / PEG2 hydrogel block groups, indicating that these components are ineffective for the extrinsic pathway.

[0215] The hyaluronic acid-based hydrogel of this invention is grafted with methacrylate groups and norbornene-like groups. The hexasaccharide ring on its hyaluronic acid backbone still has abundant hydroxyl groups, which is beneficial to vasoconstriction. The hyaluronic acid-based hydrogel has a good adsorption capacity for platelets and red blood cells, thereby forming an initial blood clot. This is related to the structure of the methacrylate groups and norbornene-like groups. It is speculated that the positive charge of the methacrylate groups and norbornene-like groups adsorbs the negatively charged platelets and red blood cells.

[0216] The experimental results of this embodiment show that the hyaluronic acid-based hydrogel and its precursor solution of the present invention can achieve rapid hemostasis in vitro and in vivo by promoting platelet adsorption and erythrocyte aggregation, activating exogenous and endogenous pathways in the coagulation cascade reaction, and forming a physical barrier through the transformation from liquid phase to gel phase.

[0217] Example 7: Skin Defect Repair Capacity of Hyaluronic Acid-Based Hydrogel

[0218] In this embodiment, the hyaluronic acid-based hydrogel precursor solutions from Examples 1 and 2 were used. The deionized water in component A was replaced with an equal volume of physiological saline. Then, components A and B were mixed and the experiment was conducted. The hyaluronic acid-based hydrogel precursor solutions from Examples 1 and 2 were designated as HFMM / PEG1 hydrogel and HFMM / PEG2 hydrogel, respectively. The UV irradiation time for each group was 10 seconds, the wavelength of the UV light was 395 nm, and the power was 50 mW / cm². 2 .

[0219] 7.1 Cell proliferation detection of Raw264.7, HUVEC, and L929 cells after hydrogel co-culture

[0220] The cell proliferation capacity of Raw264.7, HUVEC, and L929 cells was assessed using a CCK-8 assay kit. Raw264.7 cells were scraped from culture flasks, and HUVEC and L929 cells were digested with trypsin. 100,000 cells / well were seeded into 48-well plates. Raw264.7 and L929 cells were cultured in DMEM containing 10% fetal bovine serum, while HUVEC cells were cultured in ECM endothelial medium. After 24 hours of culture, pre-synthesized HFMM / PEG hydrogels were added to the wells for co-culture. The control group received equal volumes of PBS and GelMA hydrogels. One day later, 30 μL of CCK-8 working solution was added to each well, and after incubation for another 2 hours, 100 μL of the supernatant was transferred to a 96-well plate, and the absorbance was measured at 450 nm.

[0221] Experimental results show that GelMA hydrogel has no proliferative toxicity to Raw264.7, HUVEC, and L929 in the short term; HFMM / PEG1 hydrogel and HFMM / PEG2 hydrogel can significantly enhance the proliferative activity of Raw264.7, HUVEC, and L929, indicating that the hyaluronic acid-based hydrogel of the present invention promotes the proliferation of cells (Raw264.7, HUVEC, and L929).

[0222] 7.2 Immunofluorescence detection of Raw264.7, HUVEC, and L929 after hydrogel co-culture

[0223] Raw264.7 cells were scraped from culture flasks and seeded at 50,000 cells / well in confocal dishes. The cells were cultured in DMEM containing 10% fetal bovine serum for 24 hours. Eight groups were formed: PBS group, GelMA group, HFMM / PEG1 hydrogel group, and HFMM / PEG2 hydrogel group. The effects of HFMM / PEG hydrogel on macrophage differentiation were investigated. The LPS group (1 μg / mL LPS treatment) was used to investigate the state of macrophages under LPS-simulated bacterial infection conditions; the LPS+HFMM / PEG2 group (1 μg / mL LPS and HFMM / PEG2 hydrogel added simultaneously to the cell culture system) was used to investigate the state of macrophages when LPS and HFMM / PEG2 acted together, simulating bacterial infection with HFMM / PEG2 in vitro; the FLPS+HFMM / PEG2 group (1 μg / mL LPS pretreatment for 3 h, 5 washes with PBS, then HFMM / PEG2 hydrogel added; FLPS refers to LPS pretreatment followed by interaction with the hydrogel) was used to investigate the state of macrophages after LPS treatment with HFMM / PEG2, simulating bacterial infection with HFMM / PEG2 in vitro; the IL-4 group (50 ng / mL) served as a positive control for M2 polarization. After culturing for another day, the cells were fixed with paraformaldehyde for 15 min and permeabilized with 0.1% Triton-100 for 15 min. The CD206 protein was stained green using Mouse-CD206-FITC direct-labeled primary antibody; the iNOS protein was stained red using Rabbit-iNOS primary antibody + Goat anti-Rabbit-AF647 secondary antibody; and the cell nucleus was stained blue using DAPI. Each staining step required washing with PBS 3-4 times to remove residual reagents from the previous step. Finally, observation was performed using a laser confocal microscope.

[0224] Experimental results showed that during co-culture of Raw264.7 with GelMA hydrogel, HFMM / PEG1 hydrogel, and HFMM / PEG2 hydrogel, the iNOS level did not increase significantly, indicating that GelMA hydrogel, HFMM / PEG1 hydrogel, and HFMM / PEG2 hydrogel do not directly induce M1 polarization of Raw264.7 and do not actively cause inflammatory responses. Compared with the PBS control group and GelMA hydrogel, the hyaluronic acid-based hydrogel of this invention can significantly increase the expression of CD206 in Raw264.7.

[0225] Furthermore, treatment with 1 μg / mL LPS successfully induced M1 polarization of Raw264.7, simulating the state of real skin defects. In the presence of HFMM / PEG2 hydrogel, iNOS expression was significantly downregulated in both the LPS-treated and LPS-pretreated groups, demonstrating that HFMM / PEG2 hydrogel effectively inhibits Raw264.7 M1 polarization. Even when HFMM / PEG2 hydrogel was applied later than LPS (FLPS+HFMM / PEG2 group), it still inhibited Raw264.7 M1 polarization. CD206 results showed that, in the presence of LPS, HFMM / PEG2 hydrogel significantly promoted Raw264.7 M2 polarization.

[0226] HUVEC cells in culture flasks were digested with trypsin, and 100,000 cells / well were seeded in confocal dishes. HUVECs were cultured in ECM endothelial medium for 24 hours. Afterward, pre-synthesized HFMM / PEG hydrogels were placed in confocal dishes for co-culture. The control group was treated with equal volumes of PBS and GelMA hydrogel. After another day of culture, cells were fixed with paraformaldehyde for 15 minutes and permeabilized with 0.1% Triton-100 for 15 minutes. PECAM protein was stained green using Mouse-PECAM-1 primary antibody and Goat anti-mouse-AF488 secondary antibody; cell nuclei were stained blue using DAPI. Each staining step required washing with PBS 3-4 times to remove residual reagents from the previous step. Finally, observation was performed using a laser confocal microscope.

[0227] The experimental results showed that, compared with the PBS group and the GelMA group, HUVECs in the HFMM / PEG hydrogel co-culture system expressed more PECAM-1 protein. Among them, the HFMM / PEG2 hydrogel had a stronger promoting effect on the expression of angiogenesis-related proteins than the HFMM / PEG1 hydrogel.

[0228] L929 cells in culture flasks were digested with trypsin, and 50,000 cells / well were seeded in confocal dishes. L929 cells were cultured in DMEM containing 10% fetal bovine serum for 24 hours. Seven groups were formed: PBS group, GelMA group, HFMM / PEG1 hydrogel group, and HFMM / PEG2 hydrogel group. The effects of HFMM / PEG hydrogel on L929 cell function and the effect of PEG content in the HFMM / PEG hydrogel on L929 cells were investigated. The TNFα group (treated with 2.5 ng / mL TNFα only) simulated the in vitro situation under inflammatory conditions without the use of hydrogel. The TNFα+HFMM / PEG2 group (2.5 ng / mL TNFα and HFMM / PEG2 hydrogel were added to the cell culture system simultaneously) was used to explore the macrophage state when TNFα and HFMM / PEG2 acted together, and was used to simulate the use of HFMM / PEG2 while under bacterial infection in vitro. The FTNFα+HFMM / PEG2 group (pretreated with 2.5 ng / mL TNFα for 3 h, pretreated for 3 h, washed 5 times with PBS, and then HFMM / PEG2 hydrogel was added) was used to explore the macrophage state after TNFα treatment with HFMM / PEG2, and was used to simulate the use of HFMM / PEG2 under bacterial infection in vitro. After culturing for another day, the cells were fixed with paraformaldehyde for 15 min and permeabilized with 0.1% Triton-100 for 15 min. Type I collagen was stained green using Mouse-COL1A1 primary antibody and Goat anti-mouse-AF488 secondary antibody; iNOS protein was stained red using Rabbit-iNOS primary antibody and Goat anti-rabbit-AF647 secondary antibody; and cell nuclei were stained blue using DAPI. Each staining step required washing with PBS 3-4 times to remove residual reagents from the previous step. Finally, observation was performed using a laser confocal microscope.

[0229] The experimental results showed that, compared with the PBS control group, the COL1A1 protein level in the GelMA hydrogel group was not significantly increased. After co-culturing L929 cells with GelMA hydrogel, HFMM / PEG1 hydrogel, and HFMM / PEG2 hydrogel, there was no significant difference in the iNOS protein level, indicating that GelMA hydrogel, HFMM / PEG1 hydrogel, and HFMM / PEG2 hydrogel did not actively activate the TNFα pathway in L929 cells to induce an inflammatory response. The significantly increased COL1A1 protein level after co-culturing L929 cells with HFMM / PEG1 hydrogel and HFMM / PEG2 hydrogel indicates that the hyaluronic acid-based hydrogel of this invention can significantly promote the secretion of type I collagen fibers by L929 cells.

[0230] Furthermore, treatment with 2.5 ng / mL TNFα successfully induced an inflammatory response in L929, significantly increasing iNOS protein levels. In the group treated with both TNFα and HFMM / PEG2 hydrogel, iNOS protein levels were significantly downregulated, indicating that the hyaluronic acid-based hydrogel of this invention effectively inhibits the L929 TNFα-induced inflammatory response. Even when the HFMM / PEG2 hydrogel was used later than TNFα, it still effectively inhibited the L929 TNFα-induced inflammatory response.

[0231] 7.3. Raw Flow Cytometry Analysis after Hydrogel Co-culture

[0232] Raw264.7 cells were scraped from culture flasks and seeded at 500,000 cells / well in 6-well plates. Cells were cultured in DMEM containing 10% fetal bovine serum for 24 hours. Cells were then divided into six groups: PBS group, HFMM / PEG2 hydrogel group, LPS group (treated with 1 μg / mL LPS), LPS+HFMM / PEG2 group (1 μg / mL LPS and HFMM / PEG2 hydrogel added simultaneously to the cell culture system), FLPS+HFMM / PEG2 group (pretreated with 1 μg / mL LPS for 3 hours, washed 5 times with PBS, and then added to HFMM / PEG2 hydrogel), and IL-4 group (treated with 50 ng / mL IL-4). Cells were cultured for another day. All subsequent procedures were performed on ice in the dark. Cells from each group were scraped, washed twice with cold PBS, centrifuged, and fixed with 100 μL paraformaldehyde for 15 min. After washing again, the membrane was perforated with Intracellular Straining and Premature Buffer for 20 min, centrifuged to remove the supernatant, and then perforated again for 20 min. Subsequently, the membrane was blocked with FCR blocking agent for 10 min. Rabbit anti-iNOS antibody and Mouse anti-CD206-FITC antibody were then diluted with antibody dilution buffer, and cells were stained for 30 min. After washing three times with Intracellular Straining and Premature Buffer, iNOS cells were stained with Goat anti-Rabbit antibody-AF647 secondary antibody for 30 min, washed three times with Intracellular Straining and Premature Buffer, resuspended, and analyzed. Simultaneously, single-staining groups for iNOS and CD206 and a blank control group were set up.

[0233] Experimental results show that after co-culturing with HFMM / PEG2 hydrogel, iNOS - / CD206 +The number of cells increased significantly, accounting for about 1.8% of the total number of cells. Although this was lower than the 3.5% of the positive control group (IL-4 group), it was still 1.62 times that of the PBS group.

[0234] LPS successfully induced M1 polarization in Raw264.7, and the iNOS of the LPS group + / CD206 - The cell count was highest, approximately 83%. When LPS and HFMM / PEG2 hydrogel were simultaneously applied to Raw264.7, the HFMM / PEG2 hydrogel exhibited an inhibitory effect on LPS-induced M1 polarization, iNOS + / CD206 - The cell count decreased significantly, from 83% to 81%. (This is due to) a decrease in iNOS. + / CD206 - Not all cells are converted to iNOS - / CD206 + Cells, more iNOS - / CD206 - The presence of cells indirectly indicates that the HFMM / PEG2 hydrogel plays a greater role in inhibiting the differentiation of Raw264.7 cells into M1 macrophages in the LPS environment.

[0235] Furthermore, in the FLPS+HFMM / PEG2 group, which was first induced by LPS and then co-cultured with HFMM / PEG2 hydrogel, iNOS + / CD206 - The cell count decreased to 66%, a reduction of nearly 20% compared to the LPS group, iNOS - / CD206 + The number of cells also increased significantly, suggesting that the HFMM / PEG2 hydrogel still has a strong M2 polarization-promoting effect on the LPS-induced Raw264.7 cells.

[0236] 7.4. Detection of angiogenic capacity of HUVECs after hydrogel co-culture

[0237] Corning Matrigel (Cat. 354234) was used at a concentration of 10 mg / mL. The Matrigel was thawed at 4°C. A PerkinElmer 96-well plate was placed on ice, and 80 μL of Matrigel was added to each well, ensuring all pipette tips were pre-cooled. Aseptic technique was maintained. The plate was gently shaken to evenly distribute the Matrigel at the bottom of each well. The plate was then incubated for 30 min to allow the Matrigel to gel completely. HUVEC cells in the culture flasks were digested with trypsin and cultured in ECM endothelial medium to dilute the cells, resulting in a final seeding density of 30,000 cells / well. The plates were immediately placed in a high-content instrument and incubated for 30 min. After all cells had settled, PBS, GelMA hydrogel, HFMM / PEG1 hydrogel, and HFMM / PEG2 hydrogel were added, and the plates were incubated for 24 h, with photographs taken every 0.5 h.

[0238] Experimental results are as follows Figure 24 As shown, compared to the PBS control group, GelMA hydrogel exhibited a transient angiogenesis-promoting effect within 4 hours, with the formed blood vessels rapidly undergoing apoptosis over time. In contrast, HFMM / PEG1 and HFMM / PEG2 hydrogels demonstrated a strong ability to promote HUVEC angiogenesis in vitro, maintaining the physiological activity of HUVECs and significantly inhibiting their apoptosis. After 24 hours, the blood vessels formed by HFMM / PEG1 and HFMM / PEG2 hydrogels remained well-preserved.

[0239] 7.5 Changes in transcriptional levels of Raw264.7, HUVEC, and L929 after hydrogel co-culture

[0240] Total RNA was extracted from Raw264.7, HUVEC, and L929 samples using an RNA extraction kit and a centrifugation column method. Total RNA quantification was performed using a Nanodrop One UV-Vis spectrometer. This experiment was repeated three times. Reverse transcription was performed using a Takara reverse transcription kit, and the resulting cDNA was used for subsequent RT-qPCR experiments.

[0241] The experimental results showed that HFMM / PEG1 and HFMM / PEG2 hydrogels significantly downregulated the expression levels of iNOS and Tnfα mRNA in Raw264.7, with iNOS mRNA expression levels being one-quarter that of the PBS and GelMA groups. HFMM / PEG1 and HFMM / PEG2 hydrogels also significantly upregulated the expression level of Cd206 mRNA in Raw264.7.

[0242] Furthermore, under conditions simulating real skin defects in vitro, 1 μg / mL LPS significantly upregulated the expression of Raw264.7 iNOS and significantly downregulated the expression of Cd206. In the same environment, HFMM / PEG hydrogel inhibited LPS-induced M1 polarization of Raw264.7, suppressed iNOS expression, significantly promoted Cd206 expression, and promoted M2 polarization. Even after pre-treatment with 1 μg / mL LPS, HFMM / PEG hydrogel still significantly inhibited M1 polarization of Raw264.7 and promoted M2 polarization.

[0243] Both HFMM / PEG1 and HFMM / PEG2 hydrogels significantly upregulated the expression levels of ICAM-1, VCAM-1, VEGFA, and IL-8 genes, promoting angiogenesis in HUVECs. The expression levels of ICAM-1, VCAM-1, VEGFA, and IL-8 genes in the HFMM / PEG1 hydrogel were 4.57-fold, 1.52-fold, 3.61-fold, and 3.93-fold higher than those in the PBS control group, respectively. The expression levels of ICAM-1, VCAM-1, VEGFA, and IL-8 genes in the HFMM / PEG2 hydrogel were 11.87-fold, 2.34-fold, 7.59-fold, and 8.96-fold higher than those in the PBS control group, respectively. There was no statistically significant difference in eNOS gene expression levels among the groups. There was no significant difference in the expression levels of vascular-related genes between the GelMA group and the PBS group.

[0244] Compared with the PBS group and the GelMA group, both HFMM / PEG1 hydrogel and HFMM / PEG2 hydrogel significantly downregulated the expression level of iNOS in L929 cells and significantly upregulated the expression level of Col1a1, with statistically significant differences between the groups.

[0245] TNFα at 2.5 ng / mL significantly upregulated L929 iNOS expression and significantly downregulated Col1a1 expression. In the group treated with both TNFα and HFMM / PEG2 hydrogel, HFMM / PEG2 hydrogel significantly inhibited TNFα-induced high expression of L929 iNOS, significantly promoted Col1a1 expression, and promoted extracellular matrix secretion. Even after pre-treatment with TNFα, HFMM / PEG2 hydrogel still significantly inhibited L929 iNOS expression and promoted Col1a1 expression. These results are consistent with the immunofluorescence results of L929 cells.

[0246] 7.6. Raw 264.7 after hydrogel co-culture; HUVEC transcriptome analysis.

[0247] Raw264.7 cells were scraped from culture flasks and seeded at 500,000 cells / well in 6-well plates. Cells were cultured in DMEM containing 10% fetal bovine serum for 24 hours. The cells were divided into five groups: PBS group, HFMM / PEG2 hydrogel group, LPS group (treated with 1 μg / mL LPS), LPS+HFMM / PEG2 group (1 μg / mL LPS and HFMM / PEG2 hydrogel were added simultaneously to the cell culture system), and FLPS+HFMM / PEG2 group (pretreated with 1 μg / mL LPS for 3 hours, washed 5 times with PBS, and then added to HFMM / PEG2 hydrogel). After culturing for another day, RNA-seq analysis was performed.

[0248] Experimental results showed that both HFMM / PEG2 hydrogel and LPS affected the immune system processes (GO:0002376) and defense functions (GO:0006952) of Raw264.7. After co-culturing with HFMM / PEG2 hydrogel alone, differentially expressed genes were more enriched in the innate immune response (GO:0045067), indicating that the hyaluronic acid-based hydrogel of this invention has a regulatory effect on the innate immune response of Raw264.7. When LPS and HFMM / PEG2 hydrogel were applied simultaneously to Raw264.7, the stimulatory effect of LPS on Raw264.7 remained; however, some differentially expressed genes were enriched in the negative regulation of biological processes (GO:0048519), indicating that the hyaluronic acid-based hydrogel of this invention has a certain inhibitory effect on the cell function of Raw264.7 after LPS induction. Furthermore, the expression of inflammatory factors and chemokine-related genes (Ccl2, Ccl3, Ccl4, Ccl5, Ccl7, Mmp9, Cxcl10, Tnf, Il-1b, Il-1a, etc.), NF-κB pathway-related genes (Nfkbia, etc.), TLR-NOD pathway-related genes (Nod1, Nod2, Irf7, etc.), and MAPK pathway-related genes (Mapk11, etc.) were significantly downregulated in the presence of HFMM / PEG hydrogel, indicating that HFMM / PEG hydrogel plays an important role in downregulating cellular inflammatory factors.

[0249] HUVEC cells in culture flasks were digested with trypsin, and 500,000 cells / well were seeded into 6-well plates. HUVECs were cultured in ECM endothelial medium for 24 h. Pre-synthesized HFMM / PEG hydrogels were then placed in the wells for co-culture, while an equal volume of PBS was added to the control group. After another day of culture, RNA-seq analysis was performed.

[0250] Experimental results showed that the expression of genes regulating cell adhesion (ICAM-1, COL1A1, TICAM-1, CALML4, etc.), genes related to the MAPK signaling pathway (MAPKAPK3, MAP3K5, etc.), and genes involved in angiogenesis differentiation in the PI3K-AKT pathway (VEGFA, AKT, SRC, RAC2, PTGS2, etc.) were significantly enhanced, demonstrating that the hyaluronic acid-based hydrogel of the present invention plays an important role in enhancing and maintaining the expression of HUVEC tube-forming genes.

[0251] 7.7 Changes in protein levels in Raw264.7 HUVECs after hydrogel co-culture

[0252] Cells from each group were lysed using RIPA, and protease and phosphatase inhibitors were added simultaneously. Total protein was extracted from Raw264.7 and HUVEC cells after treatment. Protein quantification was performed using a BCA protein assay kit, with loading buffer added and the proteins boiled at 100°C for 10 min. Western blotting (WB) was then performed. This experiment was repeated three times.

[0253] Experimental results showed that hyaluronic acid-based hydrogels can promote the phosphorylation of p-ERK and p-JNK, activate the MAPK pathway, and promote the proliferation of Raw264.7. Hyaluronic acid-based hydrogels alone did not significantly inhibit the JNK or NF-κB pathways. In the presence of LPS, hyaluronic acid-based hydrogels significantly inhibited the expression of p-ERK, p-MEK, and p-JNK in the MAPK pathway, significantly inhibited the expression of p65, p-p65, and p-IKBα in the NF-κB pathway, and significantly promoted the expression of IKBα.

[0254] In addition, hyaluronic acid-based hydrogels can promote HUVEC proliferation by activating the MAPK pathway through promoting p-ERK phosphorylation and ERK1 / 2 expression; and can promote HUVEC angiogenesis by activating the PI3K-AKT pathway through promoting p-AKT phosphorylation and AKT expression.

[0255] Furthermore, this embodiment further demonstrates that the hyaluronic acid-based hydrogel of the present invention has excellent skin defect repair capabilities by constructing an SD rat skin defect model.

[0256] In this embodiment, GelMA was used as a control group because hydrogel itself can act as a scaffold material and has a certain physical guiding effect on the repair of skin tissue defects. By using GelMA as a material control group, the physical guiding effect of this scaffold can be excluded during the experiment, so as to prove that the hyaluronic acid-based hydrogel of the present invention promotes the healing of skin defects at all stages.

[0257] The performance test data of this embodiment show that: the hyaluronic acid-based hydrogel of the present invention can significantly enhance the activity and proliferation of rBMSCs; significantly enhance the gene expression of Raw264.7 Cd206, reduce the gene expression of iNOS and Tnfα, increase the protein level of CD206, and reduce the protein level of iNOS; increase the gene expression of HUVEC ICAM-1, VCAM-1, VEGFA, and IL-8, and increase the protein levels of PECAM-1 and ICAM-1; increase the gene expression of L929 Col1a1, reduce TNFα-induced iNOS expression, increase the protein level of COL1A1, reduce the protein level of TNFα-induced iNOS, and inhibit TNFα-induced apoptosis of L929 cells; in the absence of LPS, it activates the classical MAPK pathway by promoting p-ERK expression, promotes macrophage proliferation, and promotes M2 polarization of macrophages; in the presence of LPS (simulating a bacterial environment), it significantly inhibits the p-ERK MAPK pathway through the TLR-NOD-NF-κB-MAPK axis. It significantly inhibited the expression of p-MEK and p-JNK, significantly suppressed the expression of p65, p-p65, and p-IKBα in the NF-κB pathway, significantly promoted the expression of IKBα, significantly inhibited M1 polarization of macrophages, and continuously stimulated M2 polarization of macrophages; it activated the MAPK pathway and promoted the proliferation of HUVECs by promoting the phosphorylation of p-ERK and the expression of ERK1 / 2; and it activated the PI3K-AKT pathway and promoted the proliferation and angiogenesis of HUVECs by promoting the phosphorylation of p-AKT and p-p85 and the expression of AKT.

[0258] Example 8

[0259] This embodiment utilizes transcriptomic analysis to study differential gene expression in cells after contact with the hydrogel of this invention, revealing key mechanisms. Pathway analysis showed that adhesion or junctional protein genes (such as integrins and cadherins) exhibit numerous changes during osteoogenesis, anti-inflammatory effects, and angiogenesis. Figure 25 A and Figure 25B showed that ITGA6 (integrin subunit α6) expression was most increased in rBMSCs and Raw264.7, while other molecules were downregulated or minimally increased to varying degrees. In HUVECs, ITGA7 (integrin subunit α7) expression was most increased, while ITGA6 also showed increased expression. ITGA6 and ITGA7 are paralogous genes encoding integrin pathway proteins that regulate the binding of protein-containing complexes and laminin binding, acting as laminin receptors in mesenchymal and epithelial cells, and involved in hemidesmosomes, IGF signaling pathways, and MAPK signaling pathways. The expression of ITGA6 and ITGA7 was further confirmed by RT-qPCR and Western blotting under HFMM / PEG hydrogel. Figure 25 C). ITGA6 levels were highly correlated with HFMM / PEG hydrogel treatment, suggesting that ITGA6 is a potential target.

[0260] Furthermore, by using an ITGA6 neutralizing antibody, a GRGDSP integrin inhibitor, and short interfering RNA (siRNA) to inhibit intracellular ITGA6 levels, the results were as follows: Figure 26 and Figure 27 As shown. Figure 26 and Figure 27 The results showed that after inhibiting ITGA6 in the hyaluronic acid-based hydrogel co-culture system, rBMSC cells ( Figure 26 The osteogenic differentiation / anti-inflammatory capacity of Raw264.77 cells ( Figure 27 A) / HUVEC cells' angiogenic capacity ( Figure 27 B) A significant decrease suggests that ITGA6 is a key molecule in hyaluronic acid-based hydrogels that promotes osteogenic differentiation / anti-inflammation / angiogenesis.

[0261] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. Those skilled in the art can make other variations or modifications based on the above description. It is neither necessary nor possible to exhaustively describe all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A hyaluronic acid-based hydrogel precursor solution, characterized in that, It includes component A and component B, and the preparation method of component A includes the following steps: S1. A mixture of methacrylate-modified hyaluronic acid and furfurylamine is reacted to obtain furan-methacrylate-modified hyaluronic acid; S2. Furan-methacrylate modified hyaluronic acid and maleimide are mixed and reacted to obtain dual-modified hyaluronic acid; the dual-modified hyaluronic acid is a hyaluronic acid that simultaneously possesses two photocrosslinking groups: methacrylate groups and norbornene-like groups; S3. Mix the dual-modified hyaluronic acid, photoinitiator, and solvent to obtain component A; Component B is a mercapto polyethylene glycol solution, and the mass ratio of the hyaluronic acid with dual modified groups in component A to the mercapto polyethylene glycol in component B is 1:(2.5~5.0); the mercapto polyethylene glycol solution is a four-arm mercapto polyethylene glycol solution. The degree of substitution of the methacrylate groups in the methacrylate-modified hyaluronic acid described in step S1 is 25-35%; The degree of substitution of the furan group in the furan-methacrylate modified hyaluronic acid described in step S1 is 55-65%.

2. The hyaluronic acid-based hydrogel precursor solution according to claim 1, characterized in that, The specific process of step S1 is as follows: First, dissolve the methacrylate-modified hyaluronic acid in morpholine ethanesulfonic acid buffer, then add 4-(4,6-dimethoxytriazine)-4-methylmorpholine hydrochloride, then add furfurylamine, mix, react for 20-24 hours, and dialyze to obtain furan-methacrylate-modified hyaluronic acid.

3. The hyaluronic acid-based hydrogel precursor solution according to claim 1, characterized in that, The specific process of step S2 is as follows: first, furan-methacrylate modified hyaluronic acid is dissolved in water, maleimide is added, mixed, reacted for 20-24 hours, and dialyzed to obtain double-modified hyaluronic acid.

4. A hyaluronic acid-based hydrogel, characterized in that, It is obtained by gelling the hyaluronic acid-based hydrogel precursor solution according to any one of claims 1 to 3.

5. The use of the hyaluronic acid-based hydrogel precursor liquid according to any one of claims 1 to 3 or the hyaluronic acid-based hydrogel according to claim 4 in the preparation of a repair material for treating maxillofacial complex injuries.

6. The application according to claim 5, characterized in that, The repair material is a bone defect repair material and / or a skin defect repair material.

7. The application according to claim 6, characterized in that, The repair material is a bone defect repair material and / or skin defect repair material that promotes ITGA6 expression.

8. The application according to claim 5, characterized in that, The repair material is a repair material with hemostatic function.

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