CN111499889A

CN111499889A - Chondroitin sulfate magnesium hyaluronic acid hydrogel, preparation method and application thereof, and gel product

Info

Publication number: CN111499889A
Application number: CN202010378644.XA
Authority: CN
Inventors: 林荔军; 唐斌; 李思敬; 马粉波; 莫晓琼
Original assignee: Southern Medical University Zhujiang Hospital
Current assignee: Southern Medical University Zhujiang Hospital
Priority date: 2020-05-07
Filing date: 2020-05-07
Publication date: 2020-08-07
Anticipated expiration: 2040-05-07
Also published as: CN111499889B

Abstract

The invention provides chondroitin sulfate magnesium hyaluronic acid hydrogel, a preparation method and application thereof, and a gel product, and belongs to the technical field of biomedicine. The preparation method comprises the following steps: mixing a first mixed solution containing hyaluronic acid and magnesium chondroitin sulfate with a crosslinking agent to load the magnesium chondroitin sulfate on the crosslinked hyaluronic acid gel. The method is simple and convenient to operate and suitable for industrial production. The prepared chondroitin sulfate magnesium hyaluronic acid hydrogel has good biocompatibility, good slow release effect and long action time. It can be used for preparing gel product with effects of inhibiting inflammation, filling articular cartilage defect, delaying cartilage aging and promoting articular cartilage repair. The gel product containing the chondroitin sulfate magnesium hyaluronic acid hydrogel is beneficial to the repair of articular cartilage of osteoarthritis patients.

Description

Chondroitin sulfate magnesium hyaluronic acid hydrogel, preparation method and application thereof, and gel product

Technical Field

The invention relates to the technical field of materials, and particularly relates to chondroitin sulfate magnesium hyaluronic acid hydrogel, a preparation method and application thereof, and a gel product.

Background

Magnesium chondroitin sulfate (MgCS) is a novel chondroitin sulfate complex, and has anti-inflammatory, chondrocyte apoptosis inhibiting, anti-aging, and collagen synthesis promoting effects.

The effect of directly using the traditional Chinese medicine composition for treating the articular cartilage damage is short in time and needs to be improved.

In view of this, the present application is specifically made.

Disclosure of Invention

The first purpose of the invention comprises providing a preparation method of chondroitin sulfate magnesium hyaluronic acid hydrogel, which is simple and convenient to operate and suitable for industrial production.

The second purpose of the invention comprises providing the chondroitin sulfate magnesium hyaluronic acid hydrogel prepared by the preparation method, wherein the chondroitin sulfate magnesium hyaluronic acid hydrogel has good biocompatibility, good slow release effect and long action time.

The third objective of the present invention includes providing an application of the chondroitin sulfate magnesium hyaluronic acid hydrogel, such as preparing a gel product with effects of suppressing inflammation, resisting apoptosis, delaying cartilage aging and promoting articular cartilage repair.

The fourth purpose of the invention comprises providing a gel product with the chondroitin sulfate magnesium hyaluronic acid hydrogel, which is beneficial to the repair of articular cartilage of osteoarthritis patients.

The technical problem to be solved by the invention is realized by adopting the following technical scheme:

the invention provides a preparation method of chondroitin sulfate magnesium hyaluronic acid hydrogel, which comprises the following steps: mixing a first mixed solution containing hyaluronic acid and magnesium chondroitin sulfate with a crosslinking agent to load the magnesium chondroitin sulfate on the crosslinked hyaluronic acid gel.

In some embodiments, the first mixed solution and the cross-linking agent are reacted for 2 to 6 hours at a temperature of 30 to 50 ℃.

In some embodiments, the first mixed solution is incubated with the crosslinking agent at 40 ℃ for 6 hours.

In some embodiments, the first mixed solution is mixed with the crosslinking agent under alkaline conditions, preferably in NaOH solution.

In some embodiments, the NaOH solution has a NaOH concentration of 1 to 1.5 wt%, for example, the NaOH solution has a NaOH concentration of 1 wt%.

In some embodiments, the volume ratio of the first mixed solution to the NaOH solution is 1: 0.7-0.9.

In some embodiments, the crosslinking agent comprises 1, 4-butanediol diglycidyl ether or divinyl sulfone, preferably 1, 4-butanediol diglycidyl ether.

In some embodiments, the crosslinking agent is present in the chondroitin sulfate magnesium hyaluronic acid hydrogel in an amount of < 2 ppm.

In some embodiments, the preparing of the first mixed solution comprises: mixing the hyaluronic acid solution with the chondroitin sulfate magnesium.

In some embodiments, chondroitin sulfate powder is added to the hyaluronic acid solution.

In some embodiments, the hyaluronic acid solution is mixed with the chondroitin sulfate magnesium at 30-50 ℃, preferably under stirring at 50 ℃.

In some embodiments, the concentration of hyaluronic acid in the hyaluronic acid solution is 15-20 wt%.

In some embodiments, the amount of magnesium chondroitin sulfate is 1-3g per liter of hyaluronic acid solution.

In some embodiments, the hyaluronic acid solution has a hyaluronic acid concentration of 17 wt% and the amount of magnesium chondroitin sulfate is 1.44g per liter of hyaluronic acid solution.

Further, the method comprises the steps of crushing the hyaluronic acid gel loaded with the chondroitin sulfate magnesium, adjusting the pH value to be neutral or weakly alkaline, and filtering to obtain undersize products.

In some embodiments, the pH is adjusted with an acid, more preferably, HCl.

In some embodiments, the filtration is through a 150-250 mesh screen, preferably a 200 mesh screen.

Further, drying the undersize product.

In some embodiments, freeze-drying is employed.

In some embodiments, drying is carried out in a freeze dryer for 12-24 hours, preferably for 12 hours.

In addition, the invention also provides a chondroitin sulfate magnesium hyaluronic acid hydrogel prepared by the preparation method.

In addition, the invention also provides application of the chondroitin sulfate magnesium hyaluronic acid hydrogel in preparing a gel product for filling articular cartilage defects.

In some embodiments, the chondroitin sulfate magnesium hyaluronate hydrogel is used for preparing a gel product for inhibiting chondrocyte apoptosis or aging.

In some embodiments, the chondroitin sulfate magnesium hyaluronic acid hydrogel is used for preparing a gel product for promoting articular cartilage repair, and more preferably, a gel product for promoting collagen type ii production.

In some embodiments, the chondroitin sulfate magnesium hyaluronate hydrogel is used to prepare an anti-inflammatory gel product.

In addition, the invention also provides a gel product which contains the chondroitin sulfate magnesium hyaluronic acid hydrogel.

The application provides a chondroitin sulfate magnesium hyaluronic acid hydrogel, a preparation method and an application thereof, and a gel product has the beneficial effects that:

according to the application, the first mixed solution containing hyaluronic acid and chondroitin sulfate magnesium is crosslinked with the crosslinking agent, so that the hyaluronic acid gel with higher molecular weight than the uncrosslinked hyaluronic acid gel is obtained, a better slow release effect is achieved, and the action time is prolonged. The chondroitin sulfate magnesium is loaded on the hyaluronic acid gel obtained by crosslinking, and has better effect on the injury of arthritic cartilage and the like than pure chondroitin sulfate magnesium. The obtained chondroitin sulfate magnesium hyaluronic acid hydrogel can be used for preparing gel products with the effects of inhibiting inflammation, filling articular cartilage defects, delaying cartilage aging and promoting articular cartilage repair. The gel product containing the chondroitin sulfate magnesium hyaluronic acid hydrogel is beneficial to the repair of articular cartilage of osteoarthritis patients.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are required to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.

FIG. 1 is an SEM topographic map of a hyaluronic acid hydrogel of comparative example 1 in (one) of the experimental examples of the present application;

FIG. 2 is an SEM topographic map of the hyaluronic acid hydrogel of comparative example 2 in (one) of the experimental examples of the present application;

FIG. 3 is an SEM topographic map of a magnesium chondroitin sulfate hyaluronic acid hydrogel of example 3 in (one) of the experimental examples of the present application;

FIG. 4 is an SEM topography of a magnesium chondroitin sulfate hyaluronic acid hydrogel of example 4 in (one) of experimental examples of the present application;

FIG. 5 is a surface element spectrum analysis chart of the chondroitin sulfate magnesium hyaluronic acid hydrogel of example 1 in (two) of the experimental examples of the present application;

FIG. 6 is a surface element spectrum analysis chart of the chondroitin sulfate magnesium hyaluronic acid hydrogel of example 2 in (two) of the experimental examples of the present application;

FIG. 7 is a surface elemental energy spectrum analysis chart of the hyaluronic acid hydrogel of comparative example 1 in test example (II) of the present application;

FIG. 8 is a graph showing the results of proliferation of OA chondrocytes in each treatment group of (III) (1) in the test example of the present application;

FIG. 9 is a graph showing the results of proliferation of OA chondrocytes in each treatment group of (III) and (2) in the test example of the present application;

FIGS. 10 to 12 are graphs showing the results of the inhibition of apoptosis of OA chondrocytes by each treatment group In (IV) of the test example of the present application;

FIG. 13 is a graph showing the results of the rate of apoptosis of OA chondrocytes corresponding to each treatment group in test example (V) of the present application;

FIG. 14 is a graph showing the results of expression of OA chondrocyte inflammatory gene in each treatment group in test example (VI) of the present application;

FIG. 15 is a graph showing the results of OA chondrocyte apoptosis and senescence gene expression in each treatment group in (VI) of the test examples of the present application.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

The chondroitin sulfate magnesium hyaluronic acid hydrogel provided by the application, the preparation method and the application thereof, and a gel product are specifically described below.

The preparation method of the chondroitin sulfate magnesium hyaluronic acid hydrogel comprises the following steps: mixing a first mixed solution containing hyaluronic acid and magnesium chondroitin sulfate with a crosslinking agent to load the magnesium chondroitin sulfate on the crosslinked hyaluronic acid gel.

The gel is a gel using water as a dispersion medium, can swell in water and retain a large amount of water, and is not soluble in water. Hydrogels are more hydrophilic and have a lower surface tension, which can reduce their adsorption of proteins in body fluids. In addition, the hydrogel also has better water vapor transmission rate and proper gas transmission rate.

Common hydrogels are in the form of blocks, films, and liquids. Hydrogels can be classified as natural hydrogels and synthetic hydrogels, depending on the source. They can be classified into chemical hydrogels and physical hydrogels according to the manner of crosslinking.

Hyaluronic Acid (HA), a chain-like polymeric mucopolysaccharide formed by repeatedly and alternately linking D-glucuronic acid and N-acetylglucosamine with β -1,3 and β -1,4 glycosidic bonds, is one of glycosaminoglycans, belongs to acidic mucopolysaccharides, is widely distributed in various parts of the human body, and HAs the following structure:

according to the application, the gel is prepared by matching chondroitin sulfate magnesium with hyaluronic acid, so that the gel has a better effect on arthritis cartilage injury and the like than pure chondroitin sulfate magnesium.

It should be noted that, the inventors found that a gel obtained by merely combining magnesium chondroitin sulfate with hyaluronic acid is a non-crosslinked hyaluronic acid gel, and has the disadvantages of faster degradation rate and short time for maintaining the filling effect, so that the first mixed solution containing the hyaluronic acid solution and magnesium chondroitin sulfate is crosslinked with the crosslinking agent in the present application, thereby obtaining a hyaluronic acid gel having a higher molecular weight than the non-crosslinked hyaluronic acid gel, and having a better sustained release effect and a longer action time.

For reference, the first mixed solution and the cross-linking agent can be reacted for 2-6h (e.g., 2h, 3h, 4h, 5h, or 6h, etc.) under the condition of 30-50 ℃ (e.g., 30 ℃, 35 ℃, 40 ℃, 45 ℃, or 50 ℃, etc.). In some preferred embodiments, the first mixed solution is reacted with the crosslinking agent at 40 ℃ for 6 hours. The first mixed solution and the cross-linking agent are reacted under the above preferred conditions to effectively cross-link the first mixed solution and the cross-linking agent in a short time.

In the present application, the first mixed solution and the crosslinking agent may be mixed under alkaline conditions, for example, in a NaOH solution. The first mixed solution and the crosslinking agent are mixed under an alkaline condition for the reasons including: the viscosity of hyaluronic acid in alkaline solution, especially NaOH solution, is reduced, the solubility is increased, thereby being beneficial to mixing and compatibility with chondroitin sulfate magnesium, and further enabling the chondroitin sulfate magnesium to be fully loaded on the crosslinked hyaluronic acid gel in the crosslinking process with the crosslinking agent.

Alternatively, the NaOH concentration in the NaOH solution may be 1-1.5 wt% (e.g., 1 wt%, 1.2 wt%, 1.5 wt%, etc.), preferably, the NaOH concentration in the NaOH solution is 1 wt%. The mixing effect of hyaluronic acid and chondroitin sulfate magnesium in the NaOH solution with the concentration is better. In some embodiments, the volume ratio of the first mixed solution to the NaOH solution may be 1: 0.7-0.9, such as 1: 0.7, 1: 0.75, 1: 8. 1: 0.85 or 1: 0.9, etc.

Alternatively, the crosslinking agent may include 1, 4-butanediol diglycidyl ether or divinyl sulfone. Since the cross-linking agent generally has strong biological toxicity, 1, 4-butanediol diglycidyl ether with lower toxicity is preferably included to ensure the biological safety of the gel. It is worth noting that the amount of the crosslinking agent in the chondroitin sulfate magnesium hyaluronic acid hydrogel of the present application is < 2 ppm.

In some embodiments, the preparing of the first mixed solution may include, for example: mixing the hyaluronic acid solution with the chondroitin sulfate magnesium. During operation, chondroitin sulfate magnesium powder can be added into the hyaluronic acid solution, namely, the hyaluronic acid is dissolved into the hyaluronic acid solution, so that the solubility of the hyaluronic acid is improved, and then the hyaluronic acid solution is mixed with the chondroitin sulfate magnesium powder, so that the compatibility among all substances can be promoted.

In some embodiments, the hyaluronic acid solution is mixed with the chondroitin sulfate magnesium at 30-50 ℃, for example, stirred at 50 ℃.

By reference, the hyaluronic acid solution of the present application may have a concentration of hyaluronic acid of 15-20 wt%, such as 15 wt%, 16 wt%, 17 wt%, 18 wt%, 19 wt%, or 20 wt%, etc. magnesium chondroitin sulfate may be used in an amount of 1-3g per liter of hyaluronic acid solution, in some preferred embodiments, the hyaluronic acid solution has a concentration of hyaluronic acid of 17 wt%, and magnesium chondroitin sulfate is used in an amount of 1.44 g/L, i.e., 1.44g per liter of hyaluronic acid solution.

Further, the preparation method comprises crushing the hyaluronic acid gel loaded with chondroitin sulfate magnesium, adjusting pH to neutral or alkalescence (pH 7-7.35), and filtering to obtain undersize product. It is to be noted that the obtained chondroitin sulfate magnesium-loaded hyaluronic acid gel is jelly-like and is not favorable for injection, and by crushing the gel, the crushed material can be made into particles, and an injectable form can be realized. The gel is acidic or weakly alkaline by adjusting the pH value, is more suitable for the internal environment of a human body, and avoids symptoms such as inflammation caused by acidity.

In some embodiments, where the pH is adjusted with an acid, the pH is preferably adjusted with HCl.

In some embodiments, the filtration can be through a 150-250 mesh screen, such as a 200 mesh screen (75 μm pore size) to improve injectability of the gel.

Further, drying the undersize product may be included.

In some embodiments, the drying is performed by freeze-drying. By the method, the appearance and the property of chondroitin sulfate magnesium in the chondroitin sulfate magnesium hyaluronic acid hydrogel can be maintained, and the structural damage of the chondroitin sulfate magnesium hyaluronic acid hydrogel belonging to polysaccharide substances in a heating state is avoided.

The freeze-drying may, by reference, be a drying in a freeze-dryer for 12-24h, preferably 12 h.

The chondroitin sulfate magnesium hyaluronic acid hydrogel prepared by the preparation method has good biocompatibility, good slow release effect and long action time. The chondroitin sulfate magnesium hyaluronic acid hydrogel is an injectable reagent, has smaller gastrointestinal tract reaction compared with an oral medicament, has higher molecular weight hyaluronic acid in an uncrosslinked state through crosslinking with a crosslinking agent, and has the advantage of more clinical curative effect in a long-term treatment process.

In addition, the invention also provides an application of the chondroitin sulfate magnesium hyaluronic acid hydrogel, for example, the chondroitin sulfate magnesium hyaluronic acid hydrogel can be used for preparing a gel product for filling articular cartilage defects, or a gel product for inhibiting chondrocyte apoptosis or senescence, or a gel product for promoting articular cartilage repair, such as a gel product for promoting type II collagen generation, or a gel product for inhibiting inflammation.

In addition, the present invention also provides a gel product, which contains the chondroitin sulfate magnesium hyaluronic acid hydrogel, and the gel product may also contain other medically acceptable chemical components (refer to the prior art), which are not described herein again.

The features and properties of the present invention are described in further detail below with reference to examples.

Example 1

The preparation steps of the chondroitin sulfate magnesium hyaluronic acid hydrogel of the embodiment are as follows:

(1) 0.5g of hyaluronic acid was dissolved in deionized water to obtain a hyaluronic acid solution having a hyaluronic acid concentration of 17 wt%, and then 1.44 g/L g of magnesium chondroitin sulfate was added thereto and uniformly stirred to obtain a first mixed solution.

(2) Mixing the first mixed solution with a NaOH solution with the concentration of 1.0 wt% according to the volume ratio of 1: 0.8, then adding 1, 4-butanediol diglycidyl ether 36u L, carrying out heat preservation reaction at 50 ℃ for 6 hours to obtain hyaluronic acid gel loaded with chondroitin sulfate magnesium, crushing the gel, adjusting the pH value to be neutral by HCl, and filtering the gel by a 200-mesh filter screen to obtain the injectable chondroitin sulfate magnesium hyaluronic acid hydrogel.

Example 2

(1) 0.5g of hyaluronic acid was dissolved in deionized water to obtain a hyaluronic acid solution having a hyaluronic acid concentration of 17 wt%, and then 2.88 g/L g of magnesium chondroitin sulfate was added thereto and uniformly stirred to obtain a first mixed solution.

Example 3

(2) Mixing the first mixed solution with a NaOH solution with the concentration of 1.0 wt% according to the volume ratio of 1: 0.8, then adding 1, 4-butanediol diglycidyl ether 72u L, carrying out heat preservation reaction at 50 ℃ for 6 hours to obtain hyaluronic acid gel loaded with chondroitin sulfate magnesium, crushing the gel, adjusting the pH value to be neutral by HCl, and sieving by a 200-mesh sieve to obtain the injectable chondroitin sulfate magnesium hyaluronic acid hydrogel.

Example 4

Comparative example 1

This comparative example provides a hyaluronic acid hydrogel which differs from example 1 only in that chondroitin sulfate magnesium is not contained in the raw material.

Comparative example 2

This comparative example provides a hyaluronic acid hydrogel which differs from example 3 only in that chondroitin sulfate magnesium is not contained in the raw material.

Comparative example 3

This comparative example provides a hyaluronic acid hydrogel which differs from example 1 in that chondroitin sulfate magnesium is not contained in the raw material, and 0.25g of divinyl sulfone is used as a crosslinking agent.

Comparative example 4

This comparative example provides a hyaluronic acid hydrogel which differs from example 1 in that chondroitin sulfate magnesium is not contained in the raw material, and 0.5g of divinyl sulfone is used as a crosslinking agent.

Test examples

(A)

The hyaluronic acid hydrogels obtained in comparative examples 1 and 2 were subjected to SEM morphology observation, and the results are shown in fig. 1 and 2, respectively.

As can be seen from fig. 1 and 2, as the mass of 1, 4-butanediol diglycidyl ether increases, the pores on the surface of the hyaluronic acid hydrogel are reduced, indicating that the degree of crosslinking of the hydrogel is increased.

The magnesium chondroitin sulfate hyaluronic acid hydrogels obtained in example 3 and example 4 were subjected to SEM morphology observation, and the results are shown in fig. 3 and fig. 4, respectively.

As can be seen from fig. 3 and 4, as the mass of chondroitin sulfate magnesium increases, the particles on the surface of hyaluronic acid increase, and the roughness of the surface increases.

(II)

The chondroitin sulfate magnesium hyaluronic acid hydrogels obtained in examples 1 and 2 and the hyaluronic acid hydrogel obtained in comparative example 1 were subjected to surface element spectrum analysis, and the results are shown in fig. 5 to 7.

As can be seen from FIGS. 5 to 7, the surface magnesium ion content of the chondroitin sulfate magnesium hyaluronic acid hydrogel gradually increased with the addition of the chondroitin sulfate magnesium.

(III)

(1) DMEM high-sugar medium containing the chondroitin sulfate magnesium hyaluronic acid hydrogel obtained in example 1-4 and the hyaluronic acid hydrogel obtained in comparative example 1-4 at a concentration of 100 mg/L was prepared, and OA chondrocytes were treated for 3 days while the proliferation of OA chondrocytes in each treatment group was tested using a simple DMEM high-sugar medium as a control, and the results are shown in FIG. 8.

As can be seen from fig. 8, OA chondrocytes significantly promoted cell proliferation after the treatment with the chondroitin sulfate magnesium hyaluronic acid hydrogel, wherein the promotion effect of example 1 was the best. Comparative example 3 had no significant cell proliferation effect, and comparative example 4 had an effect of inhibiting cell proliferation.

(2) DMEM high-sugar medium containing chondroitin sulfate magnesium hyaluronic acid hydrogel obtained in example 1 at concentrations of 50 mg/L, 100 mg/L, 500 mg/L and 1000m L was prepared, and OA chondrocytes were treated for 3 days, while proliferation of OA chondrocytes in each treatment group was tested using a simple DMEM high-sugar medium as a control, and the results are shown in FIG. 9.

As can be seen from FIG. 9, the OA chondrocyte cell-promoting effect was the best when the concentration of the chondroitin sulfate magnesium hyaluronic acid hydrogel obtained in example 1 was 100 mg/L.

(IV)

A DMEM high-glucose medium containing only 1.44 mg/L of magnesium chondroitin sulfate was prepared, a DMEM high-glucose medium containing the hydrogel of magnesium chondroitin sulfate hyaluronic acid obtained in example 1 at a concentration of 100 mg/L was prepared, OA chondrocytes were treated for 3 days, and inhibition of apoptosis of OA chondrocytes by each treatment group was tested using a simple DMEM high-glucose medium as a control, and the results are shown in FIGS. 10 to 12.

As can be seen from fig. 10 to 12, the magnesium chondroitin sulfate hyaluronic acid hydrogel obtained in example 1 was able to inhibit OA chondrocyte apoptosis, compared to the control and magnesium chondroitin sulfate alone.

(V)

DMEM high-glucose medium containing only 1.44 mg/L mg of chondroitin sulfate, DMEM high-glucose medium containing 100 mg/L of chondroitin sulfate magnesium hyaluronic acid hydrogel obtained in example 1 were prepared, and OA chondrocytes were treated for 3 days, while the apoptosis rate of OA chondrocytes in each treatment group was measured using a simple DMEM high-glucose medium as a control, and the results are shown in fig. 13.

As can be seen from fig. 13, the magnesium chondroitin sulfate hyaluronic acid hydrogel obtained in example 1 can significantly inhibit OA chondrocyte apoptosis compared to the control and magnesium chondroitin sulfate alone.

(VI)

DMEM high-glucose medium containing only 1.44 mg/L mg of chondroitin sulfate magnesium was prepared, DMEM high-glucose medium containing 100 mg/L of chondroitin sulfate magnesium hyaluronic acid hydrogel obtained in example 1 was prepared, OA chondrocytes were treated for 3 days, and OA chondrocyte inflammation and senescence-associated gene expression were measured for each treatment group using the simple DMEM high-glucose medium as a control, and the results are shown in fig. 14 and 15.

As can be seen from fig. 14, the magnesium chondroitin sulfate hyaluronic acid hydrogel obtained in example 1 can better inhibit the expression of the OA chondrocyte inflammatory gene interleukin 1 β, compared to the control and magnesium chondroitin sulfate alone.

As can be seen from fig. 15, the chondroitin sulfate magnesium hyaluronic acid hydrogel obtained in example 1 can inhibit OA chondrocyte apoptosis and expression of senescence gene p 53.

In summary, the preparation method of the chondroitin sulfate magnesium hyaluronic acid hydrogel provided by the application is simple and convenient to operate, and is suitable for industrial production. The prepared chondroitin sulfate magnesium hyaluronic acid hydrogel has good biocompatibility, good slow release effect and long action time. It can be used for preparing gel product with effects of inhibiting inflammation, filling articular cartilage defect, delaying cartilage aging and promoting articular cartilage repair. The gel product containing the chondroitin sulfate magnesium hyaluronic acid hydrogel is beneficial to the repair of articular cartilage of osteoarthritis patients.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. The preparation method of the chondroitin sulfate magnesium hyaluronic acid hydrogel is characterized by comprising the following steps of:

mixing a first mixed solution containing hyaluronic acid and magnesium chondroitin sulfate with a crosslinking agent to load the magnesium chondroitin sulfate on the crosslinked hyaluronic acid gel.

2. The preparation method of claim 1, wherein the first mixed solution and the cross-linking agent are subjected to heat preservation reaction at 30-50 ℃ for 2-6 h;

preferably, the first mixed solution and the cross-linking agent are subjected to incubation reaction for 6 hours at the temperature of 40 ℃.

3. The production method according to claim 1, wherein the first mixed solution is mixed with the crosslinking agent under an alkaline condition;

preferably, the first mixed solution is mixed with the cross-linking agent in a NaOH solution;

preferably, the NaOH concentration in the NaOH solution is 1-1.5 wt%;

more preferably, the concentration of NaOH in the NaOH solution is 1 wt%;

preferably, the volume ratio of the first mixed solution to the NaOH solution is 1: 0.7-0.9;

preferably, the crosslinking agent comprises 1, 4-butanediol diglycidyl ether or divinyl sulfone, more preferably 1, 4-butanediol diglycidyl ether;

preferably, the content of the cross-linking agent in the chondroitin sulfate magnesium hyaluronic acid hydrogel is less than 2 ppm.

4. The production method according to any one of claims 1 to 3, wherein the production of the first mixed solution includes: mixing the hyaluronic acid solution with chondroitin sulfate magnesium;

preferably, the chondroitin sulfate magnesium powder is added to the hyaluronic acid solution;

preferably, the hyaluronic acid solution and the chondroitin sulfate magnesium are mixed at 30-50 ℃, preferably at 50 ℃ with stirring.

5. The method according to claim 4, wherein the concentration of hyaluronic acid in the hyaluronic acid solution is 15 to 20 wt%;

preferably, the chondroitin sulfate magnesium is used in an amount of 1-3g per liter of the hyaluronic acid solution;

more preferably, the concentration of hyaluronic acid in the hyaluronic acid solution is 17 wt%, and the chondroitin sulfate magnesium is 1.44g per liter of the hyaluronic acid solution.

6. The method according to claim 1, further comprising crushing the hyaluronic acid gel loaded with the chondroitin sulfate magnesium, adjusting the pH to neutral or weakly alkaline, and filtering to obtain undersize;

preferably, the pH is adjusted with an acid, more preferably, HCl;

preferably, the filtration is through a 150-250 mesh screen, more preferably a 200 mesh screen.

7. The method of claim 6, further comprising drying the undersize;

preferably, freeze-drying is employed;

preferably, drying is carried out in a freeze dryer for 12-24h, more preferably for 12 h.

8. A magnesium chondroitin sulfate-hyaluronic acid hydrogel prepared by the preparation method according to any one of claims 1 to 7.

9. Use of the chondroitin sulfate magnesium hyaluronate hydrogel according to claim 8 for the preparation of a gel product for filling articular cartilage defects;

preferably, the chondroitin sulfate magnesium hyaluronic acid hydrogel is used for preparing a gel product for inhibiting chondrocyte apoptosis or senescence;

preferably, the chondroitin sulfate magnesium hyaluronic acid hydrogel is used for preparing a gel product for promoting articular cartilage repair, and more preferably, a gel product for promoting collagen II generation;

preferably, the chondroitin sulfate magnesium hyaluronic acid hydrogel is used for preparing an inflammation-inhibiting gel product.

10. A gel product comprising the magnesium chondroitin sulfate hyaluronic acid hydrogel of claim 8.