CN111499889B

CN111499889B - Chondroitin magnesium sulfate hyaluronic acid hydrogel, preparation method and application thereof, and gel product

Info

Publication number: CN111499889B
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: 2023-04-28
Anticipated expiration: 2040-05-07
Also published as: CN111499889A

Abstract

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

Description

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

Technical Field

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

Background

Chondroitin magnesium sulfate (magnesium chondroitin sulfate, mgCS) is a novel chondroitin sulfate composition which has the effects of anti-inflammatory, inhibiting chondrocyte apoptosis and aging, and promoting collagen synthesis.

The effect of directly treating the arthritis cartilage injury is short and the effect is still to be improved.

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

Disclosure of Invention

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

The second aim of the invention is to provide the chondroitin magnesium sulfate hyaluronic acid hydrogel prepared by the preparation method, which has good biocompatibility, good slow release effect and long acting time.

The third object of the present invention is to provide an application of the above chondroitin sulfate magnesium hyaluronate hydrogel, for example, the gel product with the effects of inhibiting inflammation, resisting apoptosis, delaying cartilage aging and promoting articular cartilage repair can be prepared.

A fourth object of the present invention consists in providing a gel product with the above-mentioned magnesium hyaluronate hydrogel of chondroitin sulfate, useful for the repair of articular cartilage in osteoarthritis patients.

The invention solves the technical problems by adopting the following technical scheme:

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

In some embodiments, the first mixed solution is reacted with the crosslinking agent at 30-50 ℃ for 2-6 hours.

In some embodiments, the first mixed solution is reacted 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 concentration in the NaOH solution is 1-1.5 wt.%, e.g., the NaOH concentration in the NaOH solution is 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 crosslinker comprises 1, 4-butanediol diglycidyl ether or divinyl sulfone, preferably 1, 4-butanediol diglycidyl ether.

In some embodiments, the crosslinker is present in the magnesium chondroitin sulfate hyaluronate hydrogel in an amount of < 2ppm.

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

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

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

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

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

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

Further comprises breaking hyaluronic acid gel loaded with chondroitin sulfate magnesium, adjusting pH to neutral or weak alkaline, and filtering to obtain undersize.

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, lyophilization is employed.

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

In addition, the invention also provides the chondroitin sulfate magnesium hyaluronate hydrogel which is prepared by the preparation method.

In addition, the invention also provides application of the chondroitin sulfate magnesium hyaluronate hydrogel in preparing gel products for filling articular cartilage defects.

In some embodiments, the magnesium hyaluronate hydrogel is used to prepare a gel product that inhibits chondrocyte apoptosis or aging.

In some embodiments, the magnesium chondroitin sulfate hyaluronate hydrogel is used to prepare a gel product that promotes repair of articular cartilage, and more preferably, to prepare a gel product that promotes collagen production of type ii.

In some embodiments, the magnesium chondroitin sulfate 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 hyaluronate hydrogel.

The chondroitin magnesium sulfate hyaluronic acid hydrogel provided by the application, the preparation method and the application thereof, and the gel product have the beneficial effects that:

the first mixed solution containing hyaluronic acid and chondroitin sulfate magnesium is crosslinked with the crosslinking agent, so that hyaluronic acid gel with higher molecular weight than uncrosslinked hyaluronic acid gel is obtained, and the sustained-release effect is better, 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 arthritis cartilage injury and the like than the pure chondroitin sulfate magnesium. The obtained chondroitin magnesium sulfate 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 hyaluronate hydrogel is beneficial to repairing 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 needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some examples of the present invention and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 is a SEM image of an aqueous hyaluronic acid gel of comparative example 1 of test example (I) of the present application;

FIG. 2 is a SEM image of the aqueous hyaluronic acid gel of comparative example 2 of test example (I) of the present application;

FIG. 3 is a SEM topography of the magnesium chondroitin sulfate hyaluronic acid hydrogel of example 3 in test example (one) of the present application;

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

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

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

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

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

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

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

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

FIG. 14 is a graph showing the results of expression of inflammatory genes of OA chondrocytes corresponding to each treatment group in the test example (sixth) of the present application;

FIG. 15 is a graph showing the results of apoptosis and senescence gene expression in OA chondrocytes corresponding to each treatment group in the test example (six) of the present application.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

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

The preparation method of the chondroitin sulfate magnesium hyaluronate hydrogel provided by the application comprises the following steps: the first mixed solution containing hyaluronic acid and chondroitin sulfate magnesium is mixed with a crosslinking agent to make chondroitin sulfate magnesium be loaded 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 insoluble in water. Hydrogels are more hydrophilic and have a lower surface tension, which reduces their adsorption of proteins in body fluids. In addition, the hydrogel also has better water vapor permeability and proper gas passing rate.

Common hydrogels are bulk, film, and liquid. Hydrogels can be classified into natural hydrogels and synthetic hydrogels according to sources. Chemical hydrogels and physical hydrogels can be classified according to the manner of crosslinking.

Hyaluronic Acid (HA) is a chain-like high molecular glycosaminoglycan formed by repeatedly and alternately linking D-glucuronic acid and N-acetamido glucose with beta-1, 3 and beta-1, 4 glycosidic bonds, is one of glycosaminoglycans, belongs to acidic glycosaminoglycans, is widely distributed on various parts of the human body, and HAs the following structure:

the gel prepared by combining the chondroitin sulfate magnesium and the hyaluronic acid can have better effect on arthritis cartilage injury and the like than pure chondroitin sulfate magnesium.

The inventors have found that the gel obtained by compounding the magnesium chondroitin sulfate and the hyaluronic acid is a non-crosslinked hyaluronic acid gel, and has the defects of high degradation speed and short filling effect maintaining time, so that the first mixed solution containing the hyaluronic acid solution and the magnesium chondroitin sulfate is crosslinked with the crosslinking agent, and the hyaluronic acid gel with higher molecular weight than the uncrosslinked hyaluronic acid gel can be obtained, so that the hyaluronic acid gel has better slow release effect and longer action time.

By way of reference, the first mixed solution and the crosslinking agent may be reacted under the condition of 30-50 ℃ (e.g., 30 ℃, 35 ℃, 40 ℃, 45 ℃, 50 ℃, etc.) for 2-6 hours (e.g., 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 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 subjected to heat preservation reaction under the preferable conditions, so that the first mixed solution and the cross-linking agent can be effectively cross-linked in a short time.

In this application, the first mixed solution and the crosslinking agent may be mixed under alkaline conditions, for example, in NaOH solution. The first mixed solution is mixed with the crosslinking agent under alkaline conditions for reasons including: the viscosity of hyaluronic acid in alkaline solution, especially NaOH solution, is reduced, and the solubility is increased, so that the hyaluronic acid is favorable for mixing and compatibility with chondroitin sulfate magnesium, and the chondroitin sulfate magnesium can be fully loaded on the crosslinked hyaluronic acid gel in the crosslinking process of the hyaluronic acid and the crosslinking agent.

Alternatively, the NaOH concentration in the NaOH solution may be 1-1.5wt% (e.g., 1wt%, 1.2wt%, 1.5wt%, etc.), preferably the NaOH concentration in the NaOH solution is 1wt%. 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 cross-linking agent may comprise 1, 4-butanediol diglycidyl ether or divinyl sulfone. Since the crosslinking agent is generally highly biotoxic, it is preferable to include 1, 4-butanediol diglycidyl ether having lower toxicity in order to secure biosafety of the gel. It is worth noting that the content of the cross-linking agent in the chondroitin sulfate magnesium hyaluronate hydrogel is less than 2ppm.

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

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

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

Further, the preparation method also comprises the steps of crushing the hyaluronic acid gel loaded with the chondroitin sulfate magnesium, adjusting the pH to be neutral or weak alkaline (pH is 7-7.35), and filtering to obtain undersize. It should be noted that, the hyaluronic acid gel loaded with the chondroitin sulfate magnesium is jelly-shaped, which is not beneficial to injection, and the crushed material can be granular by crushing the hyaluronic acid gel, so that an injectable mode can be realized. The pH is regulated to make the gel acidic or slightly alkaline, so that the gel is more suitable for the internal environment of a human body, and symptoms such as inflammation caused by acidity are avoided.

In some embodiments, when the pH is adjusted with an acid, it is preferable to adjust the pH with HCl.

In some embodiments, the filtration may be through a 150-250 mesh screen, for example a 200 mesh screen (75 μm pore size) to enhance injectability of the gel.

Further, drying the undersize product may also be included.

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

By way of reference, freeze-drying may be drying in a freeze dryer for 12-24 hours, preferably 12 hours.

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

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

In addition, the invention also provides a gel product which contains the chondroitin magnesium sulfate hyaluronic acid hydrogel, and the gel product can also contain other medical acceptable chemical components (refer to the prior art), and the description is omitted herein.

The features and capabilities of the present invention are described in further detail below in connection with the examples.

Example 1

The preparation steps of the chondroitin magnesium sulfate 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 17wt%, followed by adding 1.44g/L of chondroitin magnesium sulfate, and stirring uniformly to obtain a first mixed solution.

(2) Mixing the first mixed solution with NaOH solution with the concentration of 1.0wt% according to the volume ratio of 1:0.8, then adding 36uL of 1, 4-butanediol diglycidyl ether, and reacting at 50 ℃ for 6 hours at a constant temperature to obtain the hyaluronic acid gel loaded with chondroitin sulfate magnesium. The gel is broken, HCl is regulated to be neutral in pH value, and the gel is filtered by a 200-mesh filter screen, so that injectable chondroitin sulfate magnesium hyaluronate hydrogel is obtained.

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 17wt%, followed by adding 2.88g/L of chondroitin magnesium sulfate, and stirring uniformly to obtain a first mixed solution.

Example 3

(2) Mixing the first mixed solution with NaOH solution with the concentration of 1.0wt% according to the volume ratio of 1:0.8, then adding 72uL of 1, 4-butanediol diglycidyl ether, and reacting at 50 ℃ for 6 hours at a constant temperature to obtain the hyaluronic acid gel loaded with chondroitin sulfate magnesium. The gel is broken, HCl is regulated to be neutral in pH value, and the gel is filtered by a 200-mesh filter screen, so that injectable chondroitin sulfate magnesium hyaluronate hydrogel is obtained.

Example 4

Comparative example 1

This comparative example provides an aqueous hyaluronic acid hydrogel differing from example 1 only in that the raw material does not contain chondroitin magnesium sulfate.

Comparative example 2

This comparative example provides an aqueous hyaluronic acid hydrogel differing from example 3 only in that the raw material does not contain chondroitin magnesium sulfate.

Comparative example 3

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

Comparative example 4

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

Test examples

(one)

The results of SEM morphological observation of the hyaluronic acid hydrogels obtained in comparative examples 1 and 2 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 shrink, indicating that the degree of crosslinking of the hydrogel increases.

The results of SEM morphological observation of the chondroitin sulfate magnesium hyaluronate aqueous gels obtained in example 3 and example 4 are shown in fig. 3 and 4, respectively.

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

(II)

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

It can be seen from FIGS. 5 to 7 that the surface magnesium ion content of the hyaluronic acid hydrogel of chondroitin magnesium sulfate gradually increases as the chondroitin magnesium sulfate is added.

(III)

(1) The proliferation of OA chondrocytes was measured in each treatment group using a pure DMEM high-sugar medium as a control while preparing DMEM high-sugar media containing 100mg/L of the chondroitin magnesium sulfate hyaluronic acid hydrogels obtained in examples 1 to 4 and the hyaluronic acid hydrogels obtained in comparative examples 1 to 4, respectively, and the results are shown in FIG. 8.

As can be seen from fig. 8, OA chondrocytes were significantly promoted after the treatment with the acid soft ossein magnesium hyaluronate hydrogel, with the promoting effect of example 1 being 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 culture media containing chondroitin sulfate magnesium hyaluronate hydrogel obtained in example 1 at concentrations of 50mg/L, 100mg/L, 500mg/L and 1000mL, respectively, were prepared, OA chondrocytes were treated for 3 days, and the proliferation of OA chondrocytes was examined in each treatment group using the pure DMEM high-sugar culture medium as a control, and the results are shown in FIG. 9.

As can be seen from FIG. 9, the effect of promoting cell proliferation by OA chondrocytes was optimal when the concentration of chondroitin sulfate magnesium hyaluronate hydrogel obtained in example 1 was 100 mg/L.

(IV)

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

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

(V)

DMEM high sugar medium containing only 1.44mg/L of chondroitin magnesium sulfate was prepared, DMEM high sugar medium containing 100mg/L of chondroitin magnesium sulfate hyaluronic acid hydrogel obtained in example 1 was treated for 3 days, respectively, OA chondrocytes were tested for apoptosis rate of OA chondrocytes corresponding to each treatment group using pure DMEM high sugar 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 was able to significantly inhibit OA chondrocyte apoptosis compared to the control and the magnesium chondroitin sulfate alone.

(six)

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

As can be seen from fig. 14, the chondroitin magnesium sulfate hyaluronic acid hydrogel obtained in example 1 was able to better inhibit the expression of interleukin 1 beta, an inflammatory gene of OA chondrocytes, compared to the control and the pure chondroitin magnesium sulfate.

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

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

The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, and various modifications and variations may be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. 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 magnesium sulfate hyaluronic acid hydrogel is characterized by comprising the following steps of:

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

the first mixed solution and the cross-linking agent are mixed in NaOH solution; the concentration of NaOH in the NaOH solution is 1-1.5wt%; the volume ratio of the first mixed solution to the NaOH solution is 1:0.7-0.9;

the cross-linking agent comprises 1, 4-butanediol diglycidyl ether or divinyl sulfone; the content of the cross-linking agent in the chondroitin sulfate magnesium hyaluronate hydrogel is less than 2ppm; the dosage of the hyaluronic acid is 0.5g, and the dosage of the cross-linking agent is 36uL or 72 uL;

the concentration of the hyaluronic acid in the hyaluronic acid solution is 15-20wt%; the dosage of the chondroitin sulfate magnesium is 1-3g of the chondroitin sulfate magnesium in each liter of the hyaluronic acid solution;

the preparation of the first mixed solution comprises the following steps: adding the powder of the chondroitin sulfate magnesium into the hyaluronic acid solution; the first mixed solution and the cross-linking agent react for 2-6 hours at the temperature of 30-50 ℃;

the preparation method of the chondroitin magnesium sulfate hyaluronic acid hydrogel further comprises the following steps: crushing the hyaluronic acid gel loaded with the chondroitin sulfate magnesium, regulating the pH to be neutral or alkalescent, and filtering to obtain undersize;

the preparation method of the chondroitin magnesium sulfate hyaluronic acid hydrogel further comprises the following steps: drying the undersize; the drying mode is freeze drying.

2. The method according to claim 1, wherein the first mixed solution and the crosslinking agent are reacted at 40 ℃ for 6 hours.

3. The method of claim 1, wherein the NaOH concentration in the NaOH solution is 1wt%.

4. A method according to any one of claims 1 to 3, wherein the hyaluronic acid solution is mixed with the chondroitin magnesium sulphate at a temperature of 30 to 50 ℃.

5. The method according to claim 4, wherein the hyaluronic acid solution and the chondroitin sulfate magnesium are mixed with stirring at 50 ℃.

6. The method according to claim 1, wherein the concentration of hyaluronic acid in the hyaluronic acid solution is 17wt%, and the amount of the chondroitin sulfate magnesium is 1.44g of the chondroitin sulfate magnesium per liter of the hyaluronic acid solution.

7. The method of claim 1, wherein the pH is adjusted with an acid.

8. The process of claim 7, wherein the pH is adjusted with HCl.

9. The method of claim 1, wherein the filtration is performed through a 150-250 mesh screen.

10. The method of claim 9, wherein the filtration is through a 200 mesh screen.

11. The process according to claim 1, wherein the mixture is dried in a freeze dryer for 12 to 24 hours.

12. The method of claim 11, wherein the drying is performed for 12 hours.

13. A magnesium chondroitin sulfate hyaluronate hydrogel prepared by the method of any one of claims 1-12.

14. Use of the magnesium hyaluronate of chondroitin sulfate according to claim 13 for the preparation of a gel product for filling a articular cartilage defect.

15. The method of claim 14, wherein the magnesium hyaluronate hydrogel of chondroitin sulfate is used to prepare a gel product for inhibiting chondrocyte apoptosis or aging.

16. The method of claim 15, wherein the magnesium hyaluronate hydrogel of chondroitin sulfate is used to prepare a gel product that promotes repair of articular cartilage.

17. The method of claim 16, wherein the gel product is used to promote collagen production of type ii.

18. The method of claim 14, wherein the magnesium chondroitin sulfate hyaluronate hydrogel is used to prepare an anti-inflammatory gel product.

19. A gel product comprising the magnesium chondroitin sulfate hyaluronate hydrogel of claim 13.