CN113952506B - Preparation method of bionic cartilage surface layer repair hydrogel - Google Patents

Abstract

The invention belongs to the technical field of cartilage repair materials, and discloses a preparation method of a bionic cartilage surface repair hydrogel. Methods: 1) The biomimetic lubricant HPX was made into a solution with water to obtain the HPX solution; 2) The polyvinyl alcohol was mixed with the HPX solution to remove air bubbles, and then subjected to freezing and thawing cycles to obtain the biomimetic cartilage surface repair hydrogel; The biomimetic lubricant HPX is a mixture of anionic polymer HPA and zwitterionic polymer brush HPM; the concentration of HPA in the HPX solution is 1-10 mg/mL, and the concentration of HPM is 0.2-2 mg/mL. The process of the invention is simple and stable, and the prepared bionic cartilage surface repair hydrogel simulates the articular cartilage lubricating layer, and has good lubricating performance and cell compatibility.

Description

Preparation method of a biomimetic cartilage surface repair hydrogel

technical field

The invention belongs to the technical field of biomedical materials, and in particular relates to a preparation method of a bionic cartilage surface repair hydrogel with good lubricating performance and good biocompatibility.

Background technique

The surface of articular cartilage combined with synovial fluid has a lubricating effect, providing a low-friction and low-wear contact surface for articular cartilage to ensure that the joint can move freely. Therefore, the destruction of the surface layer of cartilage has a great impact on the lubrication performance of joints. For example, the destruction of composition and structure leads to the loss of proteoglycans such as lubricating factors and the phenotypic variation of surface chondrocytes, which reduces the lubrication performance of cartilage and further leads to other deep layers. The water and small molecule protein in the structure are lost. Therefore, the integrity of the cartilage surface structure is particularly important for maintaining the integrity of the articular cartilage structure and realizing its function. The progressive exacerbation of osteoarthritis often begins with the decline of lubrication function. At present, the lubrication timeliness and clinical operability of articular cartilage surface repair materials need to be further studied. For example, the strength does not match the mechanical properties of cartilage, and the surrounding cartilage and bone The tissue is not well combined and loosens and falls off, and there is a certain gap between the friction and lubrication performance of natural cartilage, etc. Then, it is of great significance to repair and restore the lubrication effect for the treatment of inhibiting the progressive exacerbation of osteoarthritis, and it provides a way of thinking for the treatment of joint diseases.

Polyvinyl alcohol (PVA) hydrogel is considered to be the most potential cartilage substitute and the material closest to the physical and chemical properties and mechanical behavior of natural cartilage. PVA hydrogel has good properties such as good biocompatibility, strong elasticity, good lubricity, and high water content. Physically cross-linked PVA hydrogel made by repeated freezing and thawing has good mechanical properties. However, when polyvinyl alcohol hydrogel is used as a cartilage repair prosthesis, it has poor integration with the surrounding cartilage and bone tissue, and is easy to wear and tear, resulting in a limited service life.

The invention simulates the lubricating layer based on the structure and function bionic of the natural articular cartilage surface, and prepares a bionic cartilage surface repair hydrogel with lubricating function.

Contents of the invention

Aiming at the shortcomings and deficiencies of the prior art, the purpose of the present invention is to provide a method for preparing a biomimetic cartilage surface repair hydrogel. The invention blends the brush-type bionic lubricant HPX and polyvinyl alcohol, and prepares the bionic cartilage surface repair hydrogel with lubricating function by freezing and thawing cycle method. The method of the invention is simple and stable, and the prepared biomimetic cartilage surface repair hydrogel with lubricating function is conducive to simulating the lubricating layer, has good lubricating effect, and simultaneously overcomes the defects of the polyvinyl alcohol hydrogel as a cartilage repair prosthesis.

The object of the present invention is achieved through the following technical solutions:

A preparation method for bionic cartilage surface repair hydrogel, comprising the following steps:

1) Use water to make the biomimetic lubricant HPX into a solution to obtain the HPX solution;

2) Mix polyvinyl alcohol and HPX solution to remove air bubbles, and then perform freeze-thaw cycle treatment to obtain bionic cartilage surface repair hydrogel;

The biomimetic lubricant HPX is a mixture of anionic polymer HPA and zwitterionic polymer brush HPM; the concentration of HPA in the HPX solution is 1-10 mg/mL, and the concentration of HPM is 0.2-2 mg/mL;

The structure of the anionic polymer HPA is

Wherein, n is an integer of 200-300, x is an integer of 280-427, and y is 90-150.

The structure of the zwitterionic polymer brush HPM is

Wherein n is an integer of 60-150, x is an integer of 589-686, and y is an integer of 125-230.

The freezing time is 6-8 hours, the thawing time is 12-16 hours, and the number of freezing-thawing cycles is ≥ 5 times, generally 5-10 times, preferably 7-10 times. The freezing temperature is -10°C to -25°C, and the thawing temperature is 0 to 25°C.

When the concentration of HPA is 1 mg/mL and the concentration of HPM is 0.2 mg/mL, the number of freezing and thawing cycles is more preferably 8 to 10 times.

When the HPA concentration in the HPX solution is 1-10 mg/mL and not 1 mg/mL, and the HPM concentration is 0.2-2 mg/mL and not 0.2 mg/mL, the number of freeze-thaw cycles is preferably 7-10.

The HPA concentration in the HPX solution is preferably 2-10 mg/mL, and the HPM concentration is preferably 0.3-2 mg/mL.

The molecular weight Mw of described polyvinyl alcohol is 89000-98000.

The concentration of the polyvinyl alcohol in the mixture of polyvinyl alcohol and HPX solution is 10-20 wt%.

The mixing in step 2) is heating and stirring, specifically: the heating temperature is 90-110° C., and the stirring time is 120-180 min.

In step 2), the removal of air bubbles is a standing treatment, and the standing time is ≥1h.

Before freezing and thawing cycle treatment, put the mixed solution for removing air bubbles into the mold, pressurize and flatten.

In the present invention, HPA and HPM are selected to modify PVA together, and the obtained hydrogel has the lowest friction coefficient, and the effect of using HAP or HPM alone is not as good as that of combining HPA and HPM. The invention generates hydrogen bonds through a freeze-thaw cycle method, and makes HPX form a hydration layer on the surface of the hydrogel through friction and sliding to reduce the friction coefficient.

The invention blends the bionic joint lubricant with the hydrogel to prepare the cartilage repair gel with the cartilage surface structure, which enhances the ability of the hydrogel to form a hydration film during friction and improves its lubricating performance.

Compared with the prior art, the present invention has the following advantages and beneficial effects:

(1) The structure and function of the biomimetic cartilage surface repair hydrogel prepared by the present invention simulates the lubricating layer of the bionic natural articular cartilage surface, which provides a way of thinking for the treatment and repair lubrication effect of inhibiting the progressive aggravation of osteoarthritis disease.

(2) The preparation method of the biomimetic cartilage surface repair hydrogel of the present invention has a simple and stable process, and the hydrogel prepared by freezing-thawing physical cross-linking has good lubricating function.

(3) The cell compatibility of the biomimetic cartilage surface repair hydrogel obtained in the present invention is good.

Description of drawings

Fig. 1 is the friction coefficient result diagram of the bionic cartilage surface repair hydrogel prepared in Example 2; A5M1 corresponds to the bionic cartilage surface repair hydrogel prepared in Example 2, and PVA corresponds to the hydrogel prepared by polyvinyl alcohol;

Fig. 2 is the water content result graph of the bionic cartilage surface repair hydrogel prepared in Example 2; A5M1 corresponds to the bionic cartilage surface repair hydrogel prepared in Example 2, and PVA corresponds to the hydrogel prepared by polyvinyl alcohol;

Fig. 3 is a diagram showing the cytotoxicity results of the biomimetic cartilage surface repair hydrogel prepared in Example 1; A1M0.5 corresponds to the biomimetic cartilage surface repair hydrogel prepared in Example 1, and PVA corresponds to the hydrogel prepared from polyvinyl alcohol.

Detailed ways

The present invention will be further described in detail below in conjunction with the embodiments and the accompanying drawings, but the embodiments of the present invention are not limited thereto. In the examples, HPA is prepared according to the method in patent CN201510548198.1; HPM is prepared according to the method in patent CN201810687134.3.

The molecular weight of HPA is 2.8-3*10 ⁷ , and that of HPM is 9-9.5*10 ⁷ .

In the present invention, the concentration of HPA in the HPX solution is 1～10mg/mL, and the concentration of HPM is 0.2～2mg/mL. The dosage relationship between HPA and HPM can also be other proportional relationships except satisfying the mass ratio of 5:1, such as: ( 1～20):1, as long as it is within the concentration range provided.

Example 1

The preparation method of the bionic cartilage surface repair hydrogel of the present embodiment comprises the following steps:

(1) Prepare a mixture of 1mg/mL HPA and 0.2mg/mL HPM, and the solvent is deionized water;

(2) Weigh 6g of polyvinyl alcohol (molecular weight 89000-98000) and blend it with 34mL of the solution in step (1), dissolve it completely by mechanical stirring at 90°C for 120min, and let it stand for 1h to remove air bubbles to obtain a mixed solution;

(3) Inject the mixed solution of step (2) into the mold, put it in the freezer for 8 hours at -20°C after pressurization, and then thaw at 4°C for 16 hours. Cross-linked to obtain bionic cartilage surface repair hydrogel.

The coefficient of friction of the hydrogel prepared in this example is 0.034; the water content is 81%.

Example 2

A preparation method for bionic cartilage surface repair hydrogel, comprising the following steps:

(1) Prepare a mixture of 5mg/mL HPA and 1mg/mL HPM, and the solvent is deionized water;

(2) Weigh 6g of polyvinyl alcohol (molecular weight 89000-98000) and add 34mL of the solution in step (1) for blending, stir it mechanically at 90°C for 120min to completely dissolve it, let stand for 1h to remove air bubbles, and obtain a mixed solution;

(3) Inject the solution of step (2) into the mold, put it in the freezer for 8 hours at -20°C after being pressurized and flattened, and then thaw at 4°C for 16 hours. Combined, the bionic cartilage surface repair hydrogel was obtained.

The coefficient of friction of the hydrogel prepared in this example is 0.025; the water content is 84%.

Example 3

A preparation method for bionic cartilage surface repair hydrogel, comprising the following steps:

(1) Prepare a mixture of 10mg/mL HPA and 2mg/mL HPM, the solvent is deionized water;

(2) Weigh 6g of polyvinyl alcohol (molecular weight 89000-98000) and add 34mL of the solution in step (1) for blending, mechanically stir at 90°C for 120min to dissolve completely, let stand for 2h to remove air bubbles, and obtain a mixed solution;

(3) Inject the solution of step (2) into the mold, put it in the freezer for 8 hours at -20°C after being pressurized and flattened, and then thaw at 4°C for 16 hours. Combined, the bionic cartilage surface repair hydrogel was obtained.

The coefficient of friction of the hydrogel prepared in this example is 0.025; the water content is 86%.

Example 4

A preparation method for bionic cartilage surface repair hydrogel, comprising the following steps:

(1) Prepare a mixture of 1mg/mL HPA and 0.2mg/mL HPM, and the solvent is deionized water;

(2) Weigh 6g of polyvinyl alcohol (molecular weight 89000-98000) and add 34mL of the solution in step (1) for blending, stir it mechanically at 90°C for 120min to completely dissolve it, let stand for 1h to remove air bubbles, and obtain a mixed solution;

(3) Inject the solution of step (2) into the mold, put it in the freezer for 8 hours at -20°C after pressurization and flattening, and then thaw at 4°C for 16 hours. Combined to obtain bionic cartilage surface repair hydrogel;

The coefficient of friction of the hydrogel prepared in this example is 0.045; the water content is 83%.

Example 5

A preparation method for bionic cartilage surface repair hydrogel, comprising the following steps:

(1) Prepare a mixture of 1mg/mL HPA and 0.2mg/mL HPM, and the solvent is deionized water;

(2) Weigh 6g of polyvinyl alcohol (molecular weight 89000-98000) and add 34mL of the solution in step (1) for blending, mechanically stir at 90°C for 180min to dissolve completely, let stand for 2h to remove air bubbles, and obtain a mixed solution;

(3) Inject the solution of step (2) into the mold, put it in the freezer for 8 hours at -20°C after being pressurized and flattened, and then thaw at 4°C for 16 hours. Combined, the bionic cartilage surface repair hydrogel was obtained.

The coefficient of friction of the hydrogel prepared in this example is 0.028; the water content is 81%.

Example 6

A preparation method for bionic cartilage surface repair hydrogel, comprising the following steps:

(1) Prepare a mixture of 5mg/mL HPA and 1mg/mL HPM, and the solvent is deionized water;

(2) Weigh 6g of polyvinyl alcohol and add 34mL of the solution in step (1) for blending, mechanically stir at 90°C for 180min to dissolve completely, let stand for 2h to remove air bubbles, and obtain a mixed solution;

(3) Inject the solution of step (2) into the mold, put it in the freezer for 8 hours at -20°C after being pressurized and flattened, and then thaw at 4°C for 16 hours. Combined to obtain bionic cartilage surface repair hydrogel;

The coefficient of friction of the hydrogel prepared in this example is 0.024; the water content is 84%.

Example 7

A preparation method for bionic cartilage surface repair hydrogel, comprising the following steps:

(1) Prepare a mixture of 10mg/mL HPA and 2mg/mL HPM, the solvent is deionized water;

(2) Weigh 6g of polyvinyl alcohol and add 34mL of the solution in step (1) for blending, mechanically stir at 90°C for 180min to dissolve completely, let stand for 2h to remove air bubbles, and obtain a mixed solution;

(3) Inject the solution of step (2) into the mold, put it in the freezer for 8 hours at -20°C after being pressurized and flattened, and then thaw at 4°C for 16 hours. Combined, the bionic cartilage surface repair hydrogel was obtained;

The coefficient of friction of the hydrogel prepared in this example is 0.02; the water content is 86%.

Comparative example 1

A preparation method for bionic cartilage surface repair hydrogel, comprising the following steps:

(1) Prepare 1mg/mL HPA solution, the solvent is deionized water;

(2) Weigh 6g of polyvinyl alcohol and add 34mL of the solution in step (1) for blending, mechanically stir at 90°C for 180min to dissolve completely, let stand for 2h to remove air bubbles, and obtain a mixed solution;

(3) Inject the mixed solution of step (2) into the mold, put it in the freezer for 8 hours at -20°C after pressurization, and then thaw at 4°C for 16 hours. Cross-linking to obtain bionic cartilage surface repair hydrogel;

The coefficient of friction of the hydrogel prepared in this comparative example is 0.030; the water content is 84%.

Comparative example 2

A preparation method for bionic cartilage surface repair hydrogel, comprising the following steps:

(1) Prepare a mixture of 0.2mg/mL HPM, the solvent is deionized water;

(2) Weigh 6g of polyvinyl alcohol and add 34mL of the solution in step (1) for blending, mechanically stir at 90°C for 180min to dissolve completely, let stand for 2h to remove air bubbles, and obtain a mixed solution;

(3) Inject the mixed solution of step (2) into the mold, put it in the freezer for 8 hours at -20°C after pressurization, and then thaw at 4°C for 16 hours. Cross-linking to obtain bionic cartilage surface repair hydrogel;

The coefficient of friction of the hydrogel prepared in this comparative example is 0.031; the water content is 83%.

The friction properties of the biomimetic cartilage surface repair hydrogel prepared in Example 2 were characterized. Blot the sample with filter paper to dry the water on the surface of the sample, fix the sample on a petri dish, add an appropriate amount of PBS, and then fix it on the sample platform of the testing machine and set the test parameters to test the reciprocating friction performance. Take 3 parallel samples for each group of tests , the friction coefficient results of the biomimetic cartilage surface repair hydrogel are shown in Figure 1. Fig. 1 is a result diagram of the friction coefficient of the biomimetic cartilage surface repair hydrogel prepared in Example 2; A5M1 corresponds to the biomimetic cartilage surface repair hydrogel prepared in Example 2, and PVA corresponds to the hydrogel prepared by polyvinyl alcohol. It can be seen from Figure 1 that the biomimetic cartilage surface repair hydrogel has good lubricating properties.

The water content of the biomimetic cartilage surface repair hydrogel prepared in Example 2 was characterized. Soak the sample in deionized water to reach swelling equilibrium, then take it out and dry the surface moisture with filter paper and weigh it, then put the sample into a freeze dryer to dry to constant weight, calculate the moisture content, and take 3 parallel samples for each group, The water content results of the biomimetic cartilage surface repair hydrogel are shown in Figure 2. Fig. 2 is a diagram showing the water content results of the biomimetic cartilage surface repair hydrogel prepared in Example 2; A5M1 corresponds to the biomimetic cartilage surface repair hydrogel prepared in Example 2, and PVA corresponds to the hydrogel prepared from polyvinyl alcohol. It can be seen from Figure 2 that the water content range of the biomimetic cartilage surface repair hydrogel is close to the water content requirement of natural cartilage.

The biomimetic cartilage surface repair hydrogel prepared in Example 1 was characterized for cytotoxicity. Rabbit chondrocytes were selected for the experiment. Rabbit chondrocytes were seeded in a 96-well plate at a density of 3×10 ³ /100 μL, cultured at 5% CO ₂ and 37°C for 24 hours, the medium was discarded, and the medium solution containing the extract of Example 1 was added Continue to culture for 1, 3, 5, and 7 days, remove the medium, add 100μLCCK-8 working solution to each well, and further culture the cells for 4 hours, use a microplate reader to measure the absorbance of each well at 450nm, and repair the hydrocoagulation of the bionic cartilage surface. The cytotoxicity results of the glue are shown in Figure 3. Fig. 3 is a diagram showing the cytotoxicity results of the biomimetic cartilage surface repair hydrogel prepared in Example 1; A1M0.5 corresponds to the biomimetic cartilage surface repair hydrogel prepared in Example 1, and PVA corresponds to the hydrogel prepared from polyvinyl alcohol. The results indicated that the prepared biomimetic cartilage surface repair hydrogel had good biocompatibility.

The above-mentioned embodiment is a preferred embodiment of the present invention, but the embodiment of the present invention is not limited by the embodiment, and any other changes, modifications, substitutions and combinations made without departing from the spirit and principle of the present invention , simplification, all should be equivalent replacement methods, and are all included in the protection scope of the present invention.

Claims (6)

1. A preparation method for bionic cartilage surface repair hydrogel, characterized in that: comprising the following steps: 1) Use water to make the biomimetic lubricant HPX into a solution to obtain the HPX solution; 2) Mix polyvinyl alcohol and HPX solution to remove air bubbles, and then perform freeze-thaw cycle treatment to obtain bionic cartilage surface repair hydrogel; The biomimetic lubricant HPX is a mixture of anionic polymer HPA and zwitterionic polymer brush HPM; the concentration of HPA in the HPX solution is 1-10 mg/mL, and the concentration of HPM is 0.2-2 mg/mL; The structure of the anionic polymer HPA is

In the HPA structure, n is an integer from 200 to 300, x is an integer from 280 to 427, and y is an integer from 90 to 150; The structure of the zwitterionic polymer brush HPM is

In the HPM structure, n is an integer of 60-150, x is an integer of 589-686, and y is an integer of 125-230; The freezing time is 6 to 8 hours, the thawing time is 12 to 16 hours, the freezing temperature is -10°C to -25°C, and the thawing temperature is 0 to 25°C; When the concentration of HPA is 1 mg/mL and the concentration of HPM is 0.2 mg/mL, the number of freeze-thaw cycles is 8 to 10; When the HPA concentration in the HPX solution is 1-10 mg/mL and not 1 mg/mL, and the HPM concentration is 0.2-2 mg/mL and not 0.2 mg/mL, the number of freeze-thaw cycles is 7-10; The molecular weight Mw of described polyvinyl alcohol is 89000-98000; The concentration of the polyvinyl alcohol in the mixture of polyvinyl alcohol and HPX solution is 10-20 wt%. 2. The preparation method of the biomimetic cartilage surface repair hydrogel according to claim 1, characterized in that: the concentration of HPA in the HPX solution is 2-10 mg/mL, and the concentration of HPM is 0.3-2 mg/mL. 3. The preparation method of the biomimetic cartilage surface repair hydrogel according to claim 1, characterized in that: the mixing in step 2) is heating and stirring, the heating temperature is 90-110°C, and the stirring time is 120-120°C. 180min; In step 2), the removal of air bubbles is a standing treatment, and the standing time is ≥1h. 4. A biomimetic cartilage surface repair hydrogel obtained by the preparation method described in any one of claims 1-3. 5. The application of the biomimetic cartilage surface repair hydrogel according to claim 4, characterized in that: the biomimetic cartilage surface repair hydrogel is used to prepare a medicine for the treatment of osteoarthritis and a lubricant for improving the lubricating friction performance of a prosthesis agent. 6. The application of the bionic cartilage surface repair hydrogel according to claim 4, characterized in that: the bionic cartilage surface repair hydrogel is used to prepare cartilage repair materials.

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