Joint microenvironment adjusting hydrogel and application

Abstract

The invention provides joint microenvironment adjusting hydrogel and application, and belongs to the technical field of medical and biomedical materials.The joint microenvironment adjusting hydrogel is prepared from methylacryloylated hydroformylated hyaluronic acid, RGDS grafted hydroformylated hyaluronic acid, epsilon-polylysine and GW1100, the raw materials are added into a buffer solution to be mixed, a gel precursor solution is obtained, the gel precursor solution is dried, and the joint microenvironment adjusting hydrogel is obtained. The gel precursor solution is subjected to light curing, and the joint microenvironment adjusting hydrogel is obtained. The joint microenvironment adjusting hydrogel is wide in raw material source, green, environmentally friendly and low in cost, the preparation method of the hydrogel is simple, and no organic solvent is left. Experimental results prove that the joint microenvironment adjusting hydrogel has good biocompatibility and biological activity, and has a good application prospect in the aspects of articular cartilage repair and tissue regeneration thereof.

Description

Technical Field

[0001] The invention belongs to the technical field of medical treatment and biomedical materials, and specifically relates to a joint microenvironment regulating hydrogel and application thereof. Background Art

[0002] Osteoarthritis, as a chronic degenerative disease, has become one of the most common musculoskeletal diseases. The initial cause of its onset is mainly due to the wear and tear of cartilage. Cartilage tissue has extremely low self-repair ability in the adult state and is almost stagnant, which means that once the cartilage is defective, it is difficult for the body to repair it through its own physiological mechanism. This characteristic makes the treatment of osteoarthritis particularly difficult. At present, although there are various clinical methods for cartilage repair in early osteoarthritis, they all have significant shortcomings. Although autologous cartilage transplantation can provide tissue similar to the defect site, the donor is limited and the operation is complicated; microfracture technology attempts to stimulate the bone marrow to generate new cartilage, but the effect is often not ideal, and the quality of the new tissue is difficult to guarantee; stem cell transplantation faces technical difficulties such as low cell survival rate and uncontrollable differentiation direction.

[0003] In recent years, with the deepening of stem cell research, people have discovered that stem cells do exist in cartilage tissue. These cells have the ability to self-replicate and multidirectionally differentiate, providing new possibilities for cartilage repair. In fact, stem cells have been widely used in the repair of tissues such as cartilage and meniscus, showing certain therapeutic effects. However, our research found that in the context of osteoarthritis, cartilage stem cells exhibit obvious aging characteristics. These cells not only lose their original repair ability, but may also aggravate inflammatory responses and further worsen the condition. This discovery undoubtedly brings new challenges to the application of stem cells in the treatment of osteoarthritis. How to overcome the aging problem of cartilage stem cells in the context of osteoarthritis has become a technical problem that needs to be solved urgently.

[0004] In addition to the problem of stem cells, cartilage repair and regeneration are also affected by many factors. Excessive mechanical stimulation may lead to aggravated damage to chondrocytes and affect the repair process; insufficient supply of nutrients may limit the growth and differentiation of chondrocytes; the presence of chronic inflammation may continue to damage cartilage tissue and hinder repair; and the cartilage differentiation ability of cells is directly related to the quality and quantity of new cartilage tissue. These factors are intertwined and jointly affect the effect of cartilage repair and regeneration. Therefore, to achieve effective cartilage repair, it is necessary not only to solve technical problems such as stem cell aging, but also to comprehensively consider factors such as mechanical stimulation, nutrient supply, inflammation control, and cell differentiation ability. Summary of the invention

[0005] In order to solve the problems existing in the prior art, the present invention provides a joint microenvironment regulating hydrogel and its application. The preparation process is simple, and the prepared hydrogel has good biocompatibility and biological activity, can regulate the joint microenvironment, and has important applications in cartilage repair and tissue regeneration.

[0006] To achieve the above-mentioned purpose, the present invention provides the following technical scheme: a joint microenvironment regulating hydrogel, the raw materials of which include methacrylylated aldehyded hyaluronic acid, RGDS-grafted aldehyded hyaluronic acid, ε-polylysine and GW1100, the above-mentioned raw materials are added into a buffer solution and mixed to obtain a gel precursor solution, the gel precursor solution is photocured to obtain the joint microenvironment regulating hydrogel.

[0007] Furthermore, by mass fraction, 5% of methacryloylated aldehyded hyaluronic acid, 7% of RGDS-grafted aldehyded hyaluronic acid, 0.5%~2% of ε-polylysine and 0.1% of GW1100 were added to the buffer solution, and after complete dispersion, a gel precursor solution was obtained. The gel precursor solution was photocured to obtain a joint microenvironment regulating hydrogel.

[0008] Furthermore, the structural formula of the methacrylylated aldehyde-modified hyaluronic acid is:

[0010] Further, the preparation method of methacrylylated aldehyded hyaluronic acid is as follows: 1. Preparation of aldehyde-modified hyaluronic acid Sodium periodate was added to the hyaluronic acid solution at a molar ratio of 1:3-5, and the mixture was reacted in the dark for 2.5h-5h to obtain a mixture 1; Adding ethylene glycol to mixture 1 to react to obtain mixture 2; The mixture 2 is dialyzed for 2 to 3 days in a light-proof environment to obtain aldehyde-modified hyaluronic acid; 2. Methacrylic acid-modified hyaluronic acid is obtained by reacting methacrylic anhydride with hyaluronic acid Adding methacrylic anhydride to the aldehyded hyaluronic acid solution at a molar ratio of 1:200-300, adjusting the pH value to 8-10, and reacting in an ice bath to obtain a mixture 3; The mixture 3 was dialyzed for 2 to 3 days in a light-proof environment to obtain methacrylylated aldehyde-modified hyaluronic acid. The dialysis pore size is .

[0011] Furthermore, the structural formula of the RGDS-grafted aldehyde-modified hyaluronic acid is:

[0013] Furthermore, the preparation method of RGDS-grafted aldehyde-modified hyaluronic acid is as follows: In a mass ratio of 100:1, RGDS oligopeptide was added to the activated aldehyded sodium hyaluronate solution, the pH value was adjusted to 8-10, and the mixture 4 was obtained by reaction; The mixture 4 was dialyzed for 2 to 3 days in a light-proof environment to obtain RGDS-grafted aldehyde-modified hyaluronic acid; The dialysis pore size is 10 kDa.

[0014] Furthermore, when the gel precursor solution is photocured, the methacryloylated aldehyded hyaluronic acid undergoes double bond free radical polymerization, and the aldehyde groups of the methacryloylated aldehyded hyaluronic acid and the RGDS-grafted aldehyded hyaluronic acid react with the amino groups of ε-polylysine to form a joint microenvironment regulating hydrogel encapsulating GW1100.

[0015] The present invention also provides a product for simulating natural cartilage tissue, comprising the above-mentioned joint microenvironment regulating hydrogel.

[0016] The present invention also provides a product for osteoarthritis cartilage repair, wherein a gel precursor solution matching the volume of the cartilage defect is injected into the cartilage defect site, and light curing is performed to form a joint microenvironment regulating hydrogel in situ of the cartilage defect site; The gel precursor solution is prepared by adding 5% of methacrylylated aldehyded hyaluronic acid, 7% of RGDS-grafted aldehyded hyaluronic acid, 0.5% to 2% of ε-polylysine and 0.1% of GW1100 to a buffer solution by mass fraction, and obtaining the gel precursor solution after complete dispersion.

[0017] Furthermore, the light curing is ultraviolet light curing for 50s to 80s.

[0018] Compared with the prior art, the present invention has at least the following beneficial effects: The present invention provides a joint microenvironment regulating hydrogel, wherein hyaluronic acid (HA) is functionally modified to obtain methacrylylated aldehyded hyaluronic acid and RGDS-grafted aldehyded hyaluronic acid, and further reacted with natural antibacterial materials ε-polylysine and GW1100 to obtain an anti-inflammatory and antibacterial hydrogel with a double cross-linking mode (free radical polymerization and Schiff base reaction), so that the structure of the hydrogel is more stable, has better mechanical properties and elasticity, is not easy to become brittle, has good biocompatibility and biological activity, can be well integrated with surrounding tissues, and promotes cell growth and repair. Therefore, the hydrogel can simulate the performance of natural cartilage tissue, and can also recruit autologous cartilage stem cells through loaded adhesion molecules (Gly(4)-Arg-Gly-Asp, RGDS), and improve the uptake of cartilage stem cells nutrients through base solution and lipid absorption regulatory factors, thereby reversing the living environment of cartilage stem cells, thereby activating endogenous repair of cartilage stem cells and treating osteoarthritis.

[0019] The present invention realizes the regulation of its mechanical properties by adjusting the monomer concentration and cross-linking time of the hydrogel, so that it simulates the performance of natural cartilage tissue, has a wide range of raw materials, is green and environmentally friendly, and has low cost. The preparation method of the hydrogel is simple, does not require complex equipment and steps, and has no organic solvent residue, ensuring the safety and reliability of the product; it can also customize the matching hydrogel products according to the specific conditions of the patient and the volume of the cartilage defect, realize personalized treatment, and simplify the surgical process by forming the hydrogel in situ, reducing the surgical trauma and recovery time of the patient. Experimental results show that this joint microenvironment regulating hydrogel has good biocompatibility and biological activity. The hydrogel has broad application prospects in the fields of joint repair, tissue regeneration, and osteoarthritis treatment, and provides new treatment methods and means for the medical field. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 A schematic diagram of synthesizing a joint microenvironment regulating hydrogel according to the present invention; Figure 2 is the structural formula of the methacryloylated aldehyde-modified hyaluronic acid of the present invention; Figure 3 FTIR spectrum of the aldehyde-modified hyaluronic acid prepared by the present invention; Figure 4 The methacrylylated aldehyde-modified hyaluronic acid prepared by the present invention is 1 H NMR spectrum; Figure 5 is the structural formula of the aldehyde-modified hyaluronic acid grafted with RGDS of the present invention; Figure 6 The injectable performance of the hydrogel prepared by the present invention is demonstrated; Figure 7Characterization of the self-healing properties of the hydrogel prepared by the present invention; Figure 8 Testing the antibacterial activity of the hydrogel prepared by the present invention; Fig. 9 The detection of the chondrogenic differentiation ability of cartilage stem cells by the effective ingredient GW1100 in the hydrogel prepared by the present invention; Fig.10 The hydrogel prepared by the present invention regulates the joint microenvironment and promotes the regeneration of cartilage tissue; Fig.11 This is a cytotoxicity test of the hydrogel prepared by the present invention. DETAILED DESCRIPTION

[0021] In order to better understand the present invention, the present invention is described in detail below with reference to the accompanying drawings and specific implementation modes, but the content of the present invention is not limited to the following embodiments.

[0022] like Figure 1 As shown, the present invention provides a method for preparing a joint microenvironment regulating hydrogel, comprising the following steps: S1 uses sodium periodate to formaldehyde hyaluronic acid (HA) to synthesize aldehyde hyaluronic acid (AHA): First, a certain amount of hyaluronic acid was dissolved in deionized water. After it was completely dissolved, sodium periodate was added, and the molar ratio of hyaluronic acid to sodium periodate was 1:3~5. Then the reaction was continued in the dark for 2.5h~5h, and then ethylene glycol was added and stirred. After the reaction was completed, dialyzed (3000 Da) in a dialysis bag in a dark environment for 2~3 days to obtain aldehyde-modified hyaluronic acid. The dialyzed aldehyde-modified hyaluronic acid was subjected to rotary evaporation, and finally freeze-dried and stored in a refrigerator.

[0023] S2 is obtained by reacting methacrylic anhydride with aldehyde-modified hyaluronic acid to obtain methacrylylated aldehyde-modified hyaluronic acid (AHA-MA): A certain amount of aldehyde-modified hyaluronic acid was dissolved in deionized water, and after it was completely dissolved, a certain amount of methacrylic anhydride was added, and the molar ratio of aldehyde-modified hyaluronic acid and methacrylic anhydride was AHA:Ma=1:200~300. Then, the pH value of the reaction system was adjusted to 8~10 with sodium hydroxide (1M), and then reacted in an ice bath (0~4℃) for 24h. After the reaction was completed, dialyzed (3000 Da) in a dialysis bag in a light-proof environment for 2~3 days to obtain methacrylylated aldehyde-modified hyaluronic acid, and the dialyzed methacrylylated aldehyde-modified hyaluronic acid was subjected to rotary evaporation, and finally freeze-dried and stored in a refrigerator.

[0024] Wherein: the structural formula of aldehyde hyaluronic acid is:

[0025] The structural formula of methacryloylated aldehyde hyaluronic acid is:

[0027] S3 obtains RGDS-grafted aldehyde-modified hyaluronic acid by reacting aldehyde-modified sodium hyaluronate with RGDS oligopeptide: Dissolve the aldehyded sodium hyaluronate in MES buffer (pH = 5-6) and stir to dissolve, activate with NHS and EDC, and after 10h-14h, add RGDS oligopeptide, adjust the pH of the reaction system to 8-10, and the reaction time is 24h-72h to obtain a mixture, in which the mass ratio of aldehyded sodium hyaluronate to RGDS oligopeptide is 100:1; finally, purify the reaction mixture with a dialysis tube (10kDa) for 2-3 days to obtain RGDS-grafted aldehyded hyaluronic acid. After freeze-drying, collect and store for further use; RGDS is an oligopeptide containing an arginine-glycine-aspartic acid (RGD) sequence, which is used to target integrin receptors and promote cell adhesion; The structural formula of RGDS-grafted aldehyde-modified hyaluronic acid is as follows:

[0029] S4 is a joint microenvironment regulating hydrogel prepared by reacting methacryloylated aldehyded hyaluronic acid with aldehyded hyaluronic acid modified with RGDS oligopeptide: Methacrylylated aldehyded hyaluronic acid and RGDS oligopeptide-modified aldehyded hyaluronic acid were dissolved in PBS buffer at a certain ratio, and then a certain amount of ε-polylysine (EPL) and GW1100 were added to form a precursor solution after complete dispersion, and a joint microenvironment regulating hydrogel was obtained through photocuring. Among them, the mass fractions of methacryloylated aldehyded hyaluronic acid and RGDS-grafted aldehyded hyaluronic acid in the hydrogel are 5% and 7% respectively, the amount of ε-polylysine (EPL) is 0.5%~2%, the amount of GW1100 is 0.1%, and the photocuring time is 50s~80s.

[0030] The application of the joint microenvironment regulating hydrogel provided by the present invention in achieving cartilage repair and tissue regeneration is as follows: First, the volume of the cartilage defect at the site of the cartilage defect was determined; Then, the gel precursor solution was prepared according to the volume of the cartilage defect, and the specific proportions were as follows: methacrylylated aldehyded hyaluronic acid and RGDS-grafted aldehyded hyaluronic acid were dissolved in PBS buffer at a mass fraction of 5% and 7%, respectively, and then ε-polylysine (EPL) and GW1100 were added at a mass fraction of 0.5%-2% and 0.1%, respectively, and after all the components were completely dispersed, a gel precursor solution was obtained; Finally, the gel precursor solution is injected into the cartilage defect and cured by ultraviolet light for 50s to 80s, preferably 60s, to form a joint microenvironment regulating hydrogel in situ.

[0031] Embodiment 1: 1) Preparation of aldehyde-modified hyaluronic acid: First, dissolve a certain amount of hyaluronic acid in deionized water, wait for it to completely dissolve, and then add sodium periodate (ratio 1:5). Then continue to react in the dark for 3 hours, then add 1 ml of ethylene glycol and continue stirring for 30 minutes. After the reaction is completed, dialyze in a dark environment for 3 days using a dialysis bag, and then perform rotary evaporation on the dialyzed aldehyde-modified hyaluronic acid, and finally freeze-dry and store in a refrigerator. Figure 2 is the structural formula of synthetic aldehyde-modified hyaluronic acid, Figure 3 This is the FTIR spectrum of the aldehyde-modified hyaluronic acid. It can be seen that there is a characteristic peak of aldehyde group (-CHO), which proves that the hyaluronic acid is successfully aldehyde-modified.

[0032] 2) Synthesis method of methacryloyl-formaldehyde hyaluronic acid: a certain amount of formaldehyde hyaluronic acid is dissolved in deionized water, and after it is completely dissolved, a certain amount of methacrylic anhydride is added, and then the pH value of the reaction system is adjusted to 8 with sodium hydroxide (1M), and then reacted in an ice bath for 24 hours. After the reaction is completed, dialyze in a light-proof environment for 3 days with a dialysis bag, and the dialyzed methacryloyl-formaldehyde hyaluronic acid is rotary evaporated, and finally freeze-dried and stored in a refrigerator. Figure 2 is the structural formula of the methacrylylated aldehyde-modified hyaluronic acid synthesized by the present invention, Figure 4 Methacryloylated aldehyde hyaluronic acid 1 From the H NMR spectrum, we can see the H on the double bond, which proves that the hyaluronan has been successfully aldehyded.

[0033] 3) Synthesis method of RGDS-grafted aldehyded hyaluronic acid: dissolve aldehyded sodium hyaluronate in MES buffer (pH = 5-6) and stir to dissolve, activate with NHS and EDC, add RGDS oligopeptide after 14 hours and continue stirring for 3 days, wherein the mass ratio of aldehyded sodium hyaluronate to RGDS oligopeptide is 100:1; finally, dialysis tube (10kDa) is used to purify the RGDS-grafted aldehyded hyaluronic acid obtained by the reaction for 3 days. After freeze-drying, collect and store for further use. The specific structural formula is as follows: Figure 5 As shown; 4) A method for preparing a joint microenvironment regulating hydrogel: Methacrylylated aldehyde-modified hyaluronic acid and RGDS-grafted aldehyde-modified hyaluronic acid are dissolved in a PBS buffer solution in a certain proportion, and then 2% ε-polylysine (EPL) and 0.1% GW1100 are added to form a precursor solution after complete dispersion, and the hydrogel is synthesized by photocuring, such as Figure 6 As shown, it can be pushed out through a syringe to form the desired shape. In addition, the hydrogel has self-healing properties, such as Figure 7 As shown in Figure 2, the hydrogel can heal itself within two minutes after being cut. At the same time, the hydrogel has good antibacterial activity, such as Figure 8 As shown, after the bacteria were cultured on the hydrogel surface for 2 hours, they were diluted 10 times with PBS and then applied to the LB plate. After 14-16 hours, no bacterial growth clones were found in the hydrogel treatment group, proving that it can effectively prevent the growth of three bacteria: E.coil, S. aureus and MRSA. In addition, we confirmed that the active ingredient GW1100 in the hydrogel can effectively promote the chondrogenic differentiation of cartilage stem cells ( Fig. 9 ). When cartilage induction factor and GW1100 were added to the high-glucose culture medium, the cartilage spheres formed by cartilage stem cells were the largest and most dense, and effectively promoted the expression of cartilage marker genes. COL2A1 and inhibit the expression of cartilage hypertrophy-related genes COL10A1 , cartilage degradation-related genes MMP13 , osteogenesis-related genes RUNX2 The precursor solution was used to fill the rabbit knee cartilage defect, which was a cylindrical defect with a diameter of 3 mm and a depth of 3 mm. A joint microenvironment regulating hydrogel was synthesized by photocuring. Samples were collected three months later and sections of the defect were stained. Fig.10 It can be seen that the cartilage defects in the AMAGE@G hydrogel group have been completely healed and the surface is smooth, proving that the hydrogel can promote the repair and regeneration of cartilage defects in rabbits.

[0034] Embodiment 2: 1) Preparation of aldehyde-modified hyaluronic acid: First, dissolve a certain amount of hyaluronic acid in deionized water, wait for it to completely dissolve, and then add sodium periodate (ratio 1:3). Then continue to react in the dark for 3 hours, then add 1 ml of ethylene glycol and continue stirring for 30 minutes. After the reaction is completed, dialyze in a dark environment for 2 days using a dialysis bag, and then perform rotary evaporation on the dialyzed aldehyde-modified hyaluronic acid, and finally freeze-dry and store in a refrigerator.

[0035] 2) Synthesis method of methacryloyl-formaldehyde hyaluronic acid: a certain amount of formaldehyde hyaluronic acid is dissolved in deionized water, and after it is completely dissolved, a certain amount of methacrylic anhydride is added, and then the pH value of the reaction system is adjusted to 10 with sodium hydroxide (1M), and then reacted in an ice bath for 24 hours. After the reaction is completed, dialyze in a light-proof environment for 2 days with a dialysis bag, and the dialyzed methacryloyl-formaldehyde hyaluronic acid is rotary evaporated, and finally freeze-dried and stored in a refrigerator.

[0036] 3) Synthesis method of RGDS-grafted aldehyded hyaluronic acid: dissolve aldehyded sodium hyaluronate in MES buffer (pH = 5-6) and stir to dissolve, activate with NHS and EDC, add RGDS oligopeptide after 14 hours and continue stirring for 2 days, wherein the mass ratio of aldehyded sodium hyaluronate to RGDS oligopeptide is 100:1; finally, dialysis tube (10kDa) is used to purify the aldehyded hyaluronic acid modified by GW1100 for 2 days. After freeze-drying, collect and store for further use; 4) A method for preparing a joint microenvironment regulating hydrogel: methacrylylated aldehyded hyaluronic acid and RGDS-grafted aldehyded hyaluronic acid are dissolved in PBS buffer at a certain ratio, and then 2% ε-polylysine (EPL) and 0.1% GW1100 are added to form a precursor solution after complete dispersion, and the hydrogel is synthesized by photocuring.

[0037] Embodiment 3: 1) Preparation of aldehyde-modified hyaluronic acid: First, dissolve a certain amount of hyaluronic acid in deionized water, wait for it to completely dissolve, and then add sodium periodate (ratio 1:5). Then continue to react in the dark for 6 hours, then add 1 ml of ethylene glycol and continue stirring for 30 minutes. After the reaction is completed, dialyze in a dark environment for 2.5 days using a dialysis bag, and then perform rotary evaporation on the dialyzed aldehyde-modified hyaluronic acid, and finally freeze-dry and store in a refrigerator.

[0038] 2) Synthesis method of methacryloyl-formaldehyde hyaluronic acid: a certain amount of formaldehyde hyaluronic acid is dissolved in deionized water, and after it is completely dissolved, a certain amount of methacrylic anhydride is added, and then the pH value of the reaction system is adjusted to 9 with sodium hydroxide (1M), and then reacted in an ice bath for 24 hours. After the reaction is completed, dialyze in a light-proof environment for 2.5 days with a dialysis bag, and the dialyzed methacryloyl-formaldehyde hyaluronic acid is rotary evaporated, and finally freeze-dried and stored in a refrigerator.

[0039] 3) Synthesis method of aldehyded hyaluronic acid modified by GW1100: Sodium aldehyded hyaluronate was dissolved in MES buffer (pH = 5-6) and stirred to dissolve, activated with NHS and EDC, and after 12 hours, RGDS oligopeptide was added and stirred for 2.5 days, wherein the mass ratio of sodium aldehyded hyaluronate to RGDS oligopeptide was 100:1; finally, the aldehyded hyaluronic acid modified by GW1100 was purified for 2.5 days using a dialysis tube (10 kDa). After freeze-drying, it was collected and stored for further use; 4) A method for preparing a joint microenvironment regulating hydrogel: methacrylylated aldehyded hyaluronic acid and RGDS-grafted aldehyded hyaluronic acid are dissolved in PBS buffer at a certain ratio, and then 2% ε-polylysine (EPL) and 0.1% GW1100 are added to form a precursor solution after complete dispersion, and the hydrogel is synthesized by photocuring.

[0040] Embodiment 4: 1) Preparation of aldehyde-modified hyaluronic acid: First, dissolve a certain amount of hyaluronic acid in deionized water, wait for it to completely dissolve, and then add sodium periodate (ratio 1:5). Then continue to react in the dark for 3 hours, then add 1 ml of ethylene glycol and continue stirring for 30 minutes. After the reaction is completed, dialyze in a dark environment for 3 days using a dialysis bag, and then perform rotary evaporation on the dialyzed aldehyde-modified hyaluronic acid, and finally freeze-dry and store in a refrigerator.

[0041] 2) Synthesis method of methacryloyl-formaldehyde hyaluronic acid: a certain amount of formaldehyde hyaluronic acid is dissolved in deionized water, and after it is completely dissolved, a certain amount of methacrylic anhydride is added, and then the pH value of the reaction system is adjusted to 8 with sodium hydroxide (1M), and then reacted in an ice bath for 24 hours. After the reaction is completed, dialyze in a light-proof environment for 3 days with a dialysis bag, and the dialyzed methacryloyl-formaldehyde hyaluronic acid is rotary evaporated, and finally freeze-dried and stored in a refrigerator.

[0042] 3) Synthesis method of RGDS-grafted aldehyded hyaluronic acid: dissolve aldehyded sodium hyaluronate in MES buffer (pH = 5-6) and stir to dissolve, activate with NHSRGDS oligopeptide and EDC, add RGDS oligopeptide and continue stirring for 3 days after 14 hours, wherein the mass ratio of aldehyded sodium hyaluronate to EDC is 100:1; finally, dialysis tube (10kDa) is used to purify the RGDS-grafted aldehyded hyaluronic acid obtained by the reaction for 3 days. After freeze-drying, collect and store for further use; 4) A method for preparing a joint microenvironment regulating hydrogel: methacrylylated aldehyded hyaluronic acid and RGDS-grafted aldehyded hyaluronic acid are dissolved in PBS buffer at a certain ratio, and then 1% ε-polylysine (EPL) and 0.1% GW1100 are added to form a precursor solution after complete dispersion, and the hydrogel is synthesized by photocuring.

[0043] Embodiment 5: 1) Preparation of aldehyde-modified hyaluronic acid: First, dissolve a certain amount of hyaluronic acid in deionized water, wait for it to completely dissolve, and then add sodium periodate (ratio 1:5). Then continue to react in the dark for 3 hours, then add 1 ml of ethylene glycol and continue stirring for 30 minutes. After the reaction is completed, dialyze in a dark environment for 3 days using a dialysis bag, and then perform rotary evaporation on the dialyzed aldehyde-modified hyaluronic acid, and finally freeze-dry and store in a refrigerator.

[0044] 2) Synthesis method of methacryloyl-formaldehyde hyaluronic acid: a certain amount of formaldehyde hyaluronic acid is dissolved in deionized water, and after it is completely dissolved, a certain amount of methacrylic anhydride is added, and then the pH value of the reaction system is adjusted to 8 with sodium hydroxide (1M), and then reacted in an ice bath for 24 hours. After the reaction is completed, dialyze in a light-proof environment for 3 days with a dialysis bag, and the dialyzed methacryloyl-formaldehyde hyaluronic acid is rotary evaporated, and finally freeze-dried and stored in a refrigerator.

[0045] 3) Synthesis method of RGDS-grafted aldehyded hyaluronic acid: dissolve aldehyded sodium hyaluronate in MES buffer (pH = 5-6) and stir to dissolve, activate with NHS and EDC, add RGDS oligopeptide after 14 hours and continue stirring for 3 days, wherein the mass ratio of aldehyded sodium hyaluronate to RGDS oligopeptide is 100:1; finally, dialysis tube (10kDa) is used to purify the RGDS-grafted aldehyded hyaluronic acid obtained by the reaction for 3 days. After freeze-drying, collect and store for further use; 4) A method for preparing a joint microenvironment regulating hydrogel: methacrylylated aldehyded hyaluronic acid and RGDS-grafted aldehyded hyaluronic acid are dissolved in PBS buffer at a certain ratio, and then 0.5% ε-polylysine (EPL) and 0.1% GW1100 are added to form a precursor solution after complete dispersion, and the hydrogel is synthesized by photocuring.

[0046] The present invention provides a joint microenvironment regulating hydrogel. The raw materials of the synthetic hydrogel used in the present invention are widely available, green and environmentally friendly, low in cost, and the preparation method of the hydrogel is simple, and there is no organic solvent residue. The experimental results show that the joint microenvironment regulating hydrogel, using the hydrogel extract and 10 times dilution to culture cells, shows no cytotoxicity (cell viability far exceeds 80%) ( Fig.11), proving that the hydrogel has good biocompatibility and bioactivity, and has good application prospects in articular cartilage repair and tissue regeneration.

[0047] The above contents are only for explaining the technical idea of ​​the present invention and cannot be used to limit the protection scope of the present invention. Any changes made on the basis of the technical solution in accordance with the technical idea proposed by the present invention shall fall within the protection scope of the claims of the present invention.

Claims

1. A joint microenvironment regulating hydrogel, characterized in that: The raw materials include methacrylylated aldehyded hyaluronic acid, RGDS-grafted aldehyded hyaluronic acid, ε-polylysine and GW1100. The raw materials are added into a buffer solution and mixed to obtain a gel precursor solution. The gel precursor solution is photocured to obtain the joint microenvironment regulating hydrogel.

2. A joint microenvironment regulating hydrogel according to claim 1, characterized in that: In terms of mass fraction, 5% of methacryloylated aldehyded hyaluronic acid, 7% of RGDS-grafted aldehyded hyaluronic acid, 0.5%-2% of ε-polylysine and 0.1% of GW1100 were added to the buffer solution and completely dispersed to obtain a gel precursor solution. The gel precursor solution was photocured to obtain a joint microenvironment regulating hydrogel.

3. A joint microenvironment regulating hydrogel according to claim 1 or 2, characterized in that: The structural formula of the methacrylylated aldehyde-modified hyaluronic acid is: 。 4. The joint microenvironment regulating hydrogel according to claim 3, characterized in that: The preparation method of methacrylylated aldehyde-modified hyaluronic acid is as follows: 1) Preparation of aldehyde-modified hyaluronic acid Sodium periodate was added to the hyaluronic acid solution at a molar ratio of 1:3-5, and the mixture was reacted in the dark for 2.5h-5h to obtain a mixture 1; Adding ethylene glycol to mixture 1 to react to obtain mixture 2; The mixture 2 is dialyzed for 2 to 3 days in a light-proof environment to obtain aldehyde-modified hyaluronic acid; 2) Methacrylic acid-formylated hyaluronic acid is obtained by reacting methacrylic anhydride with formylated hyaluronic acid Adding methacrylic anhydride to the aldehyded hyaluronic acid solution at a molar ratio of 1:200-300, adjusting the pH value to 8-10, and reacting in an ice bath to obtain a mixture 3; The mixture 3 was dialyzed for 2 to 3 days in a light-proof environment to obtain methacrylylated aldehyde-modified hyaluronic acid. The dialysis pore size is .

5. A joint microenvironment regulating hydrogel according to claim 1 or 2, characterized in that: The structural formula of the RGDS-grafted aldehyde-modified hyaluronic acid is: 。 6. The joint microenvironment regulating hydrogel according to claim 5, characterized in that: The preparation method of RGDS-grafted aldehyde-modified hyaluronic acid is as follows: In a mass ratio of 100:1, RGDS oligopeptide was added to the activated aldehyded sodium hyaluronate solution, the pH value was adjusted to 8-10, and the mixture 4 was obtained by reaction; The mixture 4 was dialyzed for 2 to 3 days in a light-proof environment to obtain RGDS-grafted aldehyde-modified hyaluronic acid; The dialysis pore size is 10 kDa.

7. A joint microenvironment regulating hydrogel according to claim 1 or 2, characterized in that: When the gel precursor solution is photocured, methacryloylated aldehyded hyaluronic acid, RGDS-grafted aldehyded hyaluronic acid and ε-polylysine form a gel, and GW1100 is encapsulated in the gel to obtain a joint microenvironment regulating hydrogel.

8. A product for simulating natural cartilage tissue, characterized in that: Comprising the joint microenvironment regulating hydrogel as described in claim 7.

9. A product for osteoarthritis cartilage repair, characterized in that: Inject a gel precursor solution that matches the volume of the cartilage defect into the cartilage defect site, and light-curing the solution to form a joint microenvironment regulating hydrogel in situ of the cartilage defect site; The gel precursor solution is prepared by adding 5% of methacrylylated aldehyded hyaluronic acid, 7% of RGDS-grafted aldehyded hyaluronic acid, 0.5% to 2% of ε-polylysine and 0.1% of GW1100 to a buffer solution by mass fraction, and obtaining the gel precursor solution after complete dispersion.

10. A product for osteoarthritis cartilage repair according to claim 9, characterized in that: The light curing is ultraviolet light curing for 50s to 80s.

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