Joint microenvironment-regulating hydrogel and application thereof

Abstract

The application provides a kind of joint microenvironment regulating hydrogel and application, belong to medical and biomedical material technical field, raw material includes methacrylated aldehyde-based hyaluronic acid, RGDS grafted aldehyde-based hyaluronic acid, epsilon-polylysine and GW1100, the above raw materials are added into buffer solution and mixed to obtain gel precursor solution, the gel precursor solution is photocured to obtain the joint microenvironment regulating hydrogel.The raw material of the joint microenvironment regulating hydrogel is widely sourced, green and environmentally friendly, low cost, the preparation method of the hydrogel is simple, and there is no organic solvent residue.The experimental results prove that the joint microenvironment regulating hydrogel has good biocompatibility and biological activity, and has good application prospect in joint cartilage repair and tissue regeneration.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of medical and biomedical materials, and particularly relates to a joint microenvironment regulating hydrogel and application thereof. BACKGROUND

[0002] Osteoarthritis, as a chronic degenerative disease, has become the most common one among skeletal muscle diseases. The primary cause of the disease is mainly due to the wear and tear and damage of the cartilage. The cartilage tissue has extremely low self-repairing ability in the adult state, and is almost in a state of stagnation, which means that once the cartilage is damaged, the body is difficult to repair through its own physiological mechanism. This feature makes the treatment of osteoarthritis particularly tricky. At present, although there are various clinical methods for repairing early osteoarthritis cartilage, there are still significant shortcomings. Autologous cartilage transplantation can provide similar tissue to the defect site, but the donor is limited and the operation is complex; microfracture technology attempts to stimulate bone marrow to generate new cartilage, but the effect is often unsatisfactory, and the quality of the new tissue is difficult to guarantee; stem cell transplantation faces technical problems such as low cell survival rate and uncontrollable differentiation direction.

[0003] In recent years, with the in-depth study of stem cells, it has been found that there are indeed stem cells in cartilage tissue, and these cells have the ability of self-replication and multi-directional differentiation, which provides a new possibility for cartilage repair. In fact, stem cells have been widely used in the repair of cartilage, meniscus and other tissues, and have shown certain therapeutic effect. However, our research has found that in the environment of osteoarthritis, cartilage stem cells show obvious signs of aging. These cells not only lose their original repair ability, but also may exacerbate the inflammatory response, further worsening 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 environment of osteoarthritis has become a technical problem to be solved.

[0004] In addition to the problem of stem cells, cartilage repair and regeneration is also affected by many factors. Excessive mechanical stimulation may lead to the aggravation of cartilage cell damage, affecting the repair process; insufficient supply of nutrients may limit the growth and differentiation of cartilage cells; the presence of chronic inflammation may continuously destroy cartilage tissue, hindering 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, not only the technical problems such as stem cell aging need to be solved, but also factors such as mechanical stimulation, nutrient supply, inflammation control and cell differentiation ability need to be considered comprehensively. SUMMARY

[0005] In order to solve the problems in the prior art, the application provides a joint microenvironment adjusting hydrogel and application, which has simple preparation process, good biocompatibility and biological activity, can adjust the joint microenvironment, and has important application in cartilage repair and tissue regeneration.

[0006] To achieve the above object, the application provides the following technical scheme: a joint microenvironment adjusting hydrogel, raw materials including methacrylated aldehyde-based hyaluronic acid, RGDS grafted aldehyde-based hyaluronic acid, epsilon-polylysine and GW1100, the raw materials are added into a buffer solution to mix, a gel precursor solution is obtained, and the gel precursor solution is subjected to light curing to obtain the joint microenvironment adjusting hydrogel.

[0007] Further, 5% of the methacrylated aldehyde-based hyaluronic acid, 7% of the RGDS grafted aldehyde-based hyaluronic acid, 0.5%-2% of the epsilon-polylysine and 0.1% of the GW1100 are added into the buffer solution, and the gel precursor solution is obtained after complete dispersion, and the gel precursor solution is subjected to light curing to obtain the joint microenvironment adjusting hydrogel.

[0008] Further, the structural formula of the methacrylated aldehyde-based hyaluronic acid is as follows:

[0009]

[0010] Further, the preparation method of the methacrylated aldehyde-based hyaluronic acid is as follows:

[0011] 1. Preparation of aldehyde-based hyaluronic acid

[0012] The sodium metaperiodate is added into the hyaluronic acid solution in a molar ratio of 1:3-5, and the mixture 1 is obtained after dark reaction for 2.5 h-5 h;

[0013] The ethylene glycol is added into the mixture 1, and the mixture 2 is obtained after reaction;

[0014] The aldehyde-based hyaluronic acid is obtained after dialysis of the mixture 2 in a dark environment for 2 days-3 days;

[0015] 2. The methacrylated aldehyde-based hyaluronic acid is obtained by reaction of methacrylic anhydride and aldehyde-based hyaluronic acid

[0016] The methacrylic anhydride is added into the aldehyde-based hyaluronic acid solution in a molar ratio of 1:200-300, the pH value is adjusted to 8-10, and the mixture 3 is obtained after reaction under ice bath condition;

[0017] The methacrylated aldehyde-based hyaluronic acid is obtained after dialysis of the mixture 3 in a dark environment for 2 days-3 days

[0018] Further, the structure of the RGDS grafted aldehyde hyaluronic acid is as follows:

[0019]

[0020] Further, the preparation method of the RGDS grafted aldehyde hyaluronic acid is as follows:

[0021] In the activated aldehyde hyaluronic acid sodium solution, the RGDS oligopeptide is added in a mass ratio of 100:1, the pH value is adjusted to 8-10, and a mixture 4 is obtained by reaction.

[0022] The mixture 4 is dialyzed for 2-3 days in a dark environment to obtain the RGDS grafted aldehyde hyaluronic acid.

[0023] The dialysis pore size is 10 kDa.

[0024] Further, when the gel precursor solution is subjected to photocuring, the methacrylated aldehyde hyaluronic acid is subjected to double bond radical polymerization, the aldehyde groups of the methacrylated aldehyde hyaluronic acid and the RGDS grafted aldehyde hyaluronic acid are subjected to Schiff base reaction with the amine groups of the epsilon-polylysine, and the joint microenvironment regulating hydrogel wrapping the GW1100 is formed.

[0025] The application further provides a product for simulating natural cartilage tissue, which comprises the joint microenvironment regulating hydrogel.

[0026] The application further provides a product for repairing osteoarthritis cartilage, wherein the gel precursor solution matching the volume of the cartilage defect is injected into the cartilage defect, and the joint microenvironment regulating hydrogel is formed in situ by photocuring.

[0027] The gel precursor solution is prepared by adding 5% of the methacrylated aldehyde hyaluronic acid, 7% of the RGDS grafted aldehyde hyaluronic acid, 0.5%-2% of the epsilon-polylysine and 0.1% of the GW1100 into a buffer, and the gel precursor solution is obtained after complete dispersion.

[0028] Further, the photocuring is ultraviolet photocuring for 50-80 s.

[0029] Compared with the prior art, the application has at least the following beneficial effects:

[0030] The application provides a kind of joint microenvironment regulating hydrogel, hyaluronic acid (HA) is functionally modified, and methacrylated aldehyde hyaluronic acid, RGDS grafted aldehyde hyaluronic acid are obtained, and further reacted with natural antibacterial material epsilon-polylysine, GW1100 to obtain an anti-inflammatory antibacterial hydrogel with double crosslinking mode (radical polymerization and Schiff base reaction), so that the structure of the hydrogel is more stable, has better mechanical properties and elasticity, and is not easy to be brittle, and has good biocompatibility and biological activity, can be well fused with surrounding tissue, promote the growth and repair of cells, so the hydrogel can simulate the performance of natural cartilage tissue, and the loaded adhesion molecule (Gly (4)-Arg-Gly-Asp, RGDS) can also recruit autologous chondrocyte stem cells, improve the uptake of chondrocyte stem cell nutrients through basal solution and lipid absorption regulating factors, and reverse the chondrocyte stem cell survival environment, thereby activating the endogenous repair of chondrocyte stem cells, and treating osteoarthritis.

[0031] The application regulates the mechanical properties of the hydrogel by adjusting the monomer concentration and crosslinking time, so that the performance of the hydrogel can simulate that of natural cartilage tissue, the raw materials are widely sourced, green and environmentally friendly, and low in 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; the hydrogel product can also be customized according to the specific condition of the patient and the volume of cartilage defect, realizing personalized treatment, and the in-situ formation of the hydrogel simplifies the surgical procedure and reduces the surgical trauma and recovery time of the patient. Experimental results prove that the joint microenvironment regulating hydrogel has good biocompatibility and biological activity, and has a wide application prospect in the fields of joint repair, tissue regeneration and osteoarthritis treatment, providing a new treatment method and means for the medical field. BRIEF DESCRIPTION OF DRAWINGS

[0032] Figure 1 A schematic diagram for synthesizing a joint microenvironment regulating hydrogel according to the application;

[0033] Figure 2 A structural formula of methacrylated aldehyde hyaluronic acid according to the application;

[0034] Figure 3 An FTIR spectrum of aldehyde hyaluronic acid prepared according to the application;

[0035] Figure 4 A H NMR spectrum of methacrylated aldehyde hyaluronic acid prepared according to the application; 1 H NMR spectrum;

[0036] Figure 5 A structural formula of RGDS grafted aldehyde hyaluronic acid according to the application;

[0037] Figure 6 The injectability of the hydrogel prepared in the present application is exhibited;

[0038] Figure 7 The self-healing property of the hydrogel prepared in the present application is characterized;

[0039] Figure 8 The antibacterial activity of the hydrogel prepared in the present application is detected;

[0040] Figure 9 The detection of the chondrogenic differentiation ability of the effective ingredient GW1100 in the hydrogel prepared in the present application on chondrocytes;

[0041] Figure 10 The results of the regulation of the joint microenvironment and the promotion of cartilage tissue regeneration by the hydrogel prepared in the present application;

[0042] Figure 11 The cytotoxicity detection of the hydrogel prepared in the present application. DETAILED DESCRIPTION

[0043] In order to better understand the present application, the present application will be described in detail below in combination with the drawings and specific embodiments, but the content of the present application is not limited to the following embodiments only.

[0044] As shown in Figure 1 , the present application provides a preparation method of a joint microenvironment regulating hydrogel, comprising the following steps:

[0045] S1 synthesizing aldehyde hyaluronic acid (AHA) by using sodium periodate to aldehyde hyaluronic acid (HA):

[0046] First, a certain amount of hyaluronic acid is dissolved in deionized water, and after it is completely dissolved, sodium periodate is added, and the molar ratio of hyaluronic acid to sodium periodate is 1:3~5. Then continue to react in the dark for 2.5h~5h, then add ethylene glycol and continue to stir. After the reaction is completed, dialysis bags are used to dialyze (3000 Da) in a dark environment for 2 days~3 days to obtain aldehyde hyaluronic acid. The aldehyde hyaluronic acid after dialysis is rotary evaporated, and finally freeze-dried and stored in the refrigerator.

[0047] S2 obtaining methacrylated aldehyde hyaluronic acid (AHA-MA) by reacting methacrylic anhydride and aldehyde hyaluronic acid:

[0048] A certain amount of aldehyde hyaluronic acid is dissolved in deionized water, and after it is completely dissolved, a certain amount of methacrylic anhydride is added, and the molar ratio of aldehyde hyaluronic acid and methacrylic anhydride is AHA:Ma=1:200~300, then the pH value of the reaction system is adjusted to 8~10 with sodium hydroxide (1M), and then the reaction is carried out under ice bath (0~4℃) for 24h. After the reaction is completed, dialysis (3000 Da) is carried out in a light-proof environment for 2~3 days, and methacrylated aldehyde hyaluronic acid is obtained. The methacrylated aldehyde hyaluronic acid after dialysis is rotary evaporated, and finally freeze-dried and stored in the refrigerator.

[0049] The structure of the aldehyde hyaluronic acid is:

[0050]

[0051] The structure of the methacrylated aldehyde hyaluronic acid is:

[0052]

[0053] S3 is obtained by reacting sodium aldehyde hyaluronic acid and RGDS oligopeptide to obtain RGDS grafted aldehyde hyaluronic acid:

[0054] Sodium aldehyde hyaluronic acid is dissolved in MES buffer (pH=5~6) and stirred to dissolve, and activated with NHS and EDC. After 10h~14h, the RGDS oligopeptide is added, the pH value of the reaction system is adjusted to 8~10, and the reaction time is 24h~72h, to obtain a mixture, wherein the mass ratio of sodium aldehyde hyaluronic acid to RGDS oligopeptide is 100:1; finally, the reaction mixture is purified with a dialysis tube (10kDa) for 2~3 days to obtain RGDS grafted aldehyde hyaluronic acid. After freeze-drying, it is collected and stored for further use;

[0055] RGDS is an oligopeptide containing arginine-glycine-aspartic acid (RGD) sequence, which is used to target integrin receptors and promote cell adhesion;

[0056] The structure of the RGDS grafted aldehyde hyaluronic acid is as follows:

[0057]

[0058] S4 is obtained by reacting methacrylated aldehyde hyaluronic acid and RGDS oligopeptide modified aldehyde hyaluronic acid to obtain a joint microenvironment regulating hydrogel:

[0059] The methacrylated aldehyde hyaluronic acid and the RGDS oligopeptide modified aldehyde hyaluronic acid are dissolved in PBS buffer at a certain ratio, then a certain amount of epsilon-polylysine (EPL) and GW1100 are added, and a precursor solution is formed after complete dispersion, and a joint microenvironment regulating hydrogel is obtained through photocuring;

[0060] The mass fraction of the methacrylated aldehyde hyaluronic acid and the RGDS grafted aldehyde hyaluronic acid in the hydrogel is 5% and 7% respectively, the amount of epsilon-polylysine (EPL) is 0.5%-2%, the amount of GW1100 is 0.1%, and the photocuring time is 50s-80s.

[0061] The joint microenvironment regulating hydrogel provided by the application has the following advantages:

[0062] Firstly, the volume of the cartilage defect is determined;

[0063] Then, the gel precursor solution is prepared according to the volume of the cartilage defect, and the specific ratio is as follows: the methacrylated aldehyde hyaluronic acid and the RGDS grafted aldehyde hyaluronic acid are dissolved in PBS buffer, and the mass fraction is 5% and 7% respectively, then epsilon-polylysine (EPL) and GW1100 are added, and the mass fraction is 0.5%-2% and 0.1% respectively, and the gel precursor solution is obtained after complete dispersion of all components;

[0064] Finally, the gel precursor solution is injected into the cartilage defect, and the ultraviolet light is cured for 50s-80s, preferably 60s, to form the joint microenvironment regulating hydrogel in situ.

[0065] Example 1:

[0066] 1) The preparation method of the aldehyde hyaluronic acid is as follows: firstly, a certain amount of hyaluronic acid is dissolved in deionized water, and the hyaluronic acid is completely dissolved, then sodium periodate is added (the ratio is 1:5). Then continue to react in the dark for 3h, then add 1ml of ethylene glycol and continue to stir for 30min. After the reaction is completed, the aldehyde hyaluronic acid after dialysis is freeze-dried and stored in the refrigerator. Figure 2 The structural formula of the synthesized aldehyde hyaluronic acid is as follows, Figure 3 The FTIR spectrum of the aldehyde hyaluronic acid is as follows, and the characteristic peak of the aldehyde group (-CHO) can be seen, which proves that the aldehyde group of the hyaluronic acid is successfully grafted.

[0067] 2) The method for synthesizing methacrylated aldehyde-hydrated hyaluronic acid: a certain amount of aldehyde-hydrated hyaluronic acid is dissolved in deionized water until it is completely dissolved, a certain amount of methacrylic anhydride is added, then the pH value of the reaction system is adjusted to 8 with sodium hydroxide (1M), and then the reaction is carried out under ice bath conditions for 24h. After the reaction is completed, the dialysis bag is dialyzed in the dark environment for 3 days, the methacrylated aldehyde-hydrated hyaluronic acid after dialysis is rotary evaporated, and finally freeze-dried and stored in the refrigerator. Figure 2 The structural formula of the methacrylated aldehyde-hydrated hyaluronic acid synthesized in the present application, Figure 4 The structural formula of the methacrylated aldehyde-hydrated hyaluronic acid synthesized in the present application, 1 The H NMR spectrum can be seen that the H on the double bond proves that the hyaluronic acid is successfully aldehyde-hydrated.

[0068] 3) The method for synthesizing RGDS grafted aldehyde-hydrated hyaluronic acid: aldehyde-hydrated hyaluronic acid sodium is dissolved in MES buffer solution (pH value = 5-6) and stirred to dissolve, activated with NHS and EDC, after 14h, RGDS oligopeptide is added and stirred for 3 days, wherein the mass ratio of aldehyde-hydrated hyaluronic acid sodium to RGDS oligopeptide is 100:1; finally, the RGDS grafted aldehyde-hydrated hyaluronic acid obtained by reaction is purified with a dialysis tube (10kDa) for 3 days. After freeze-drying, it is collected and stored for further use, and the specific structural formula is as shown in Figure 5

[0069] 4) A preparation method of a joint microenvironment regulating hydrogel: methacrylated aldehyde-hydrated hyaluronic acid and RGDS grafted aldehyde-hydrated hyaluronic acid are dissolved in PBS buffer solution according to a certain proportion, then 2% ε-polylysine (EPL) and 0.1% GW1100 are added, and after being completely dispersed, a precursor solution is formed, and a hydrogel is synthesized by photo-curing, as shown in Figure 6 , which can be pushed out by a syringe to form the required shape. In addition, the hydrogel has a self-healing property, as shown in Figure 7 , which can be self-healed within two minutes after being cut. At the same time, the hydrogel has good antibacterial activity, as shown in Figure 8 , after the bacteria are cultured on the surface of the hydrogel for 2h, they are diluted 10 times with PBS and then coated on LB plates, and after 14-16 hours, no bacterial growth clones are found in the hydrogel treatment group, which proves that it can effectively prevent the growth of E. coli, S. aureus and MRSA. In addition, we have confirmed that the effective ingredient GW1100 in the hydrogel can effectively promote the chondrogenic differentiation of chondrocyte stem cells ( Figure 9 ). In the high-sugar medium, the addition of chondrocyte induction factors and GW1100 can make the chondrocyte spheroids formed by chondrocyte stem cells have the largest volume and the most dense texture, effectively promote the expression of chondrocyte marker genes COL2A1 , and inhibit the chondrocyte hypertrophy related genes​COL10A1 , cartilage degradation related genes MMP13 , osteogenesis related genes RUNX2 . The precursor solution filled the rabbit knee cartilage defects, the defect size was 3mm in diameter and 3mm in depth, and a joint microenvironment regulating hydrogel was synthesized by photocuring. After three months, the defect site was sectioned and stained, and the expression of Figure 10 It can be seen that the cartilage defects in the AMAGE hydrogel group have completely healed and the surface is smooth, which proves that the hydrogel can promote the repair and regeneration of rabbit cartilage defects.

[0070] Example 2:

[0071] 1) Preparation method of aldehyde hyaluronic acid: first, a certain amount of hyaluronic acid is dissolved in deionized water, and after it is completely dissolved, sodium periodate is added (the ratio is 1:3). Then continue to react in the dark for 3h, then add 1ml of ethylene glycol and continue to stir for 30min. After the reaction is complete, dialysis bag is used to dialyze in the dark environment for 2 days, and the aldehyde hyaluronic acid after dialysis is rotary evaporated, finally freeze-dried and stored in the refrigerator.

[0072] 2) Synthesis method of methacrylated aldehyde hyaluronic acid: a certain amount of aldehyde hyaluronic acid is dissolved in deionized water, and after it is completely dissolved, a certain amount of methacrylic anhydride is added, then the pH value of the reaction system is adjusted to 10 with sodium hydroxide (1M), and then reacted in ice bath for 24h. After the reaction is complete, dialysis bag is used to dialyze in the dark environment for 2 days, and the methacrylated aldehyde hyaluronic acid after dialysis is rotary evaporated, finally freeze-dried and stored in the refrigerator.

[0073] 3) Synthesis method of RGDS grafted aldehyde hyaluronic acid: aldehyde hyaluronic acid sodium is dissolved in MES buffer (pH value = 5~6) and stirred to dissolve, activated with NHS and EDC, after 14h, add RGDS oligopeptide and continue to stir for 2 days, wherein the mass ratio of aldehyde hyaluronic acid sodium and RGDS oligopeptide is 100:1; finally, the GW1100 modified aldehyde hyaluronic acid obtained by reaction is purified with dialysis tube (10kDa) for 2 days. After freeze-drying, collect and store for further use;

[0074] 4) Preparation method of a joint microenvironment regulating hydrogel: methacrylated aldehyde hyaluronic acid and RGDS grafted aldehyde hyaluronic acid are dissolved in PBS buffer according to a certain proportion, then 2% ε-polylysine (EPL) and 0.1% GW1100 are added, and after completely dispersed, a precursor solution is formed, and a hydrogel is synthesized by photocuring.

[0075] Example 3:

[0076] 1) Preparation method of aldehyde hyaluronic acid: first, a certain amount of hyaluronic acid is dissolved in deionized water, and after complete dissolution, sodium periodate is added (the ratio is 1:5). Then continue to react in the dark for 6h, then add 1ml of ethylene glycol and continue to stir for 30min. After the reaction is completed, dialysis bag is used to dialyze in the dark environment for 2.5 days, and the aldehyde hyaluronic acid after dialysis is rotary evaporated, finally freeze-dried and stored in the refrigerator.

[0077] 2) Synthesis method of methacrylated aldehyde hyaluronic acid: a certain amount of aldehyde hyaluronic acid is dissolved in deionized water, and after complete dissolution, a certain amount of methacrylic anhydride is added, then the pH value of the reaction system is adjusted to 9 with sodium hydroxide (1M), and then reacted in ice bath for 24h. After the reaction is completed, dialysis bag is used to dialyze in the dark environment for 2.5 days, and the methacrylated aldehyde hyaluronic acid after dialysis is rotary evaporated, finally freeze-dried and stored in the refrigerator.

[0078] 3) Synthesis method of GW1100 modified aldehyde hyaluronic acid: aldehyde hyaluronic acid sodium is dissolved in MES buffer (pH value = 5~6) and stirred to dissolve, activated with NHS and EDC, after 12h, RGDS oligopeptide is added and stirred for 2.5 days, wherein the mass ratio of aldehyde hyaluronic acid sodium to RGDS oligopeptide is 100:1; finally, the GW1100 modified aldehyde hyaluronic acid obtained by reaction is purified with dialysis tube (10kDa) for 2.5 days. After freeze-drying, collect and store for further use;

[0079] 4) Preparation method of joint microenvironment regulating hydrogel: methacrylated aldehyde hyaluronic acid and RGDS grafted aldehyde hyaluronic acid are dissolved in PBS buffer according to a certain proportion, then 2% ε-polylysine (EPL) and 0.1% GW1100 are added, and after complete dispersion, a precursor solution is formed, and the hydrogel is synthesized by photocuring.

[0080] Example 4:

[0081] 1) Preparation method of aldehyde hyaluronic acid: first, a certain amount of hyaluronic acid is dissolved in deionized water, and after complete dissolution, sodium periodate is added (the ratio is 1:5). Then continue to react in the dark for 3h, then add 1ml of ethylene glycol and continue to stir for 30min. After the reaction is completed, dialysis bag is used to dialyze in the dark environment for 3 days, and the aldehyde hyaluronic acid after dialysis is rotary evaporated, finally freeze-dried and stored in the refrigerator.

[0082] 2) The method for synthesizing methacrylated aldehyde hyaluronic acid: a certain amount of aldehyde hyaluronic acid is dissolved in deionized water until it is completely dissolved, a certain amount of methacrylic anhydride is added, then the pH value of the reaction system is adjusted to 8 with sodium hydroxide (1M), and then the reaction is carried out under ice bath conditions for 24h. After the reaction is completed, the dialysis bag is dialyzed in the dark environment for 3 days, the methacrylated aldehyde hyaluronic acid after dialysis is rotary evaporated, and finally freeze-dried and stored in the refrigerator.

[0083] 3) The method for synthesizing RGDS grafted aldehyde hyaluronic acid: aldehyde hyaluronic acid sodium is dissolved in MES buffer (pH value = 5~6) and stirred to dissolve, activated with NHSRGDS oligopeptide and EDC, after 14h, RGDS oligopeptide is added and stirred for 3 days, wherein the mass ratio of aldehyde hyaluronic acid sodium to is 100:1; finally, the RGDS grafted aldehyde hyaluronic acid obtained by reaction is purified with a dialysis tube (10kDa) for 3 days. After freeze-drying, collect and store for further use;

[0084] 4) A method for preparing a joint microenvironment regulating hydrogel: methacrylated aldehyde hyaluronic acid and RGDS grafted aldehyde hyaluronic acid are dissolved in PBS buffer according to a certain proportion, then 1% ε-polylysine (EPL) and 0.1% GW1100 are added, and a precursor solution is formed after complete dispersion, and the hydrogel is synthesized by photocuring.

[0085] Example 5:

[0086] 1) The method for preparing aldehyde hyaluronic acid: a certain amount of hyaluronic acid is dissolved in deionized water until it is completely dissolved, and sodium periodate is added (the ratio is 1:5). Then continue to react in the dark for 3h, then add 1ml of ethylene glycol and continue to stir for 30min. After the reaction is completed, the dialysis bag is dialyzed in the dark environment for 3 days, the dialyzed aldehyde hyaluronic acid is rotary evaporated, and finally freeze-dried and stored in the refrigerator.

[0087] 2) The method for synthesizing methacrylated aldehyde hyaluronic acid: a certain amount of aldehyde hyaluronic acid is dissolved in deionized water until it is completely dissolved, a certain amount of methacrylic anhydride is added, then the pH value of the reaction system is adjusted to 8 with sodium hydroxide (1M), and then the reaction is carried out under ice bath conditions for 24h. After the reaction is completed, the dialysis bag is dialyzed in the dark environment for 3 days, the methacrylated aldehyde hyaluronic acid after dialysis is rotary evaporated, and finally freeze-dried and stored in the refrigerator.

[0088] 3) The method for synthesizing RGDS grafted aldehyde hyaluronic acid: Aldehyde hyaluronic acid sodium was dissolved in MES buffer (pH = 5~6) and stirred to dissolve, activated by NHS and EDC, after 14h, RGDS oligopeptide was added and stirred for 3 days, the mass ratio of aldehyde hyaluronic acid sodium and RGDS oligopeptide was 100:1; finally, the RGDS grafted aldehyde hyaluronic acid obtained by reaction was purified by dialysis tube (10kDa) for 3 days. After freeze-drying, it was collected and stored for further use;

[0089] 4) The preparation method of a joint microenvironment regulating hydrogel: Methylacrylated aldehyde hyaluronic acid and RGDS grafted aldehyde hyaluronic acid were dissolved in PBS buffer according to a certain proportion, then 0.5% epsilon-polylysine (EPL) and 0.1% GW1100 were added, and a precursor solution was formed after complete dispersion, and the hydrogel was synthesized by photocuring.

[0090] The application provides a joint microenvironment regulating hydrogel, raw materials for synthesizing the hydrogel are widely sourced, the hydrogel is 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 regulating hydrogel has no cytotoxicity (cell viability is far more than 80%) (cell viability is far more than 80%) when cells are cultured by using a hydrogel extract and a 10-fold diluent, and the hydrogel has good biocompatibility and bioactivity, and has a good application prospect in joint cartilage repair and tissue regeneration. Figure 11

[0091] The above content only illustrates the technical idea of the application, and cannot limit the protection scope of the application, and any modification made according to the technical idea of the application on the basis of the technical scheme falls within the protection scope of the claims of the application.

Claims

1. An articular microenvironment modulating hydrogel, characterized in that, The raw materials include methyl acrylated aldehyde-based hyaluronic acid, RGDS grafted aldehyde-based hyaluronic acid, epsilon-polylysine and GW1100, the raw materials are added into a buffer solution to mix to obtain a gel precursor solution, and the gel precursor solution is subjected to photocuring to obtain the joint microenvironment regulating hydrogel. 5% of the methyl acrylated aldehyde-based hyaluronic acid, 7% of the RGDS grafted aldehyde-based hyaluronic acid, 0.5%-2% of the epsilon-polylysine and 0.1% of the GW1100 are added into a buffer solution in terms of mass fraction, and a gel precursor solution is obtained after complete dispersion, and the gel precursor solution is subjected to photocuring to obtain the joint microenvironment regulating hydrogel.

2. The joint microenvironment-modulating hydrogel of claim 1, wherein, The structural formula of the methyl acrylated aldehyde-based hyaluronic acid is as follows:

3. The joint microenvironment-modulating hydrogel of claim 2, wherein, The preparation method of the methyl acrylated aldehyde-based hyaluronic acid is as follows: 1) Preparation of aldehyde-based hyaluronic acid Sodium periodate is added into a hyaluronic acid solution in a molar ratio of 1:3-5, and reaction is carried out in the dark for 2.5-5 hours to obtain a mixture 1; Ethylene glycol is added into the mixture 1 to obtain a mixture 2; The mixture 2 is dialyzed in a dark environment for 2-3 days to obtain aldehyde-based hyaluronic acid. 2) Preparation of methyl acrylated aldehyde-based hyaluronic acid by reacting methyl acrylate and aldehyde-based hyaluronic acid Methyl acrylate is added into an aldehyde-based hyaluronic acid solution in a molar ratio of 1:200-300, the pH value is adjusted to 8-10, and a mixture 3 is obtained under ice bath conditions; The mixture 3 is dialyzed in a dark environment for 2-3 days to obtain methyl acrylated aldehyde-based hyaluronic acid.

4. The joint microenvironment-modulating hydrogel of claim 1, wherein, The structural formula of the RGDS grafted aldehyde-based hyaluronic acid is as follows:

5. The joint microenvironment-modulating hydrogel of claim 4, wherein, The preparation method of the RGDS grafted aldehyde-based hyaluronic acid is as follows: RGDS oligopeptide is added into an activated aldehyde-based hyaluronic acid sodium solution in a mass ratio of 100:1, the pH value is adjusted to 8-10, and a mixture 4 is obtained; The mixture 4 is dialyzed in a dark environment for 2-3 days to obtain RGDS grafted aldehyde-based hyaluronic acid. The dialysis pore size is 10 kDa.

6. The joint microenvironment-modulating hydrogel of claim 1, wherein, When the gel precursor solution is subjected to photocuring, the methyl acrylated aldehyde-based hyaluronic acid, the RGDS grafted aldehyde-based hyaluronic acid and the epsilon-polylysine form a gel, and the GW1100 is wrapped in the gel to obtain the joint microenvironment regulating hydrogel.

7. A product for simulating natural cartilage tissue, characterized by, The joint microenvironment regulating hydrogel of claim 1 is included.

8. A product for osteoarthritic cartilage repair, characterized by, The gel precursor solution matching the volume of the cartilage defect is injected into the cartilage defect, and the joint microenvironment regulating hydrogel is formed in situ by photocuring; The gel precursor solution is obtained by adding 5% of the methyl acrylated aldehyde-based hyaluronic acid, 7% of the RGDS grafted aldehyde-based hyaluronic acid, 0.5%-2% of the epsilon-polylysine and 0.1% of the GW1100 into a buffer solution in terms of mass fraction.

9. A product for the repair of osteoarthritic cartilage according to claim 8, wherein The photocuring is ultraviolet photocuring for 50-80 seconds.

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