A photocurable hyaluronic acid-based composite hydrogel, its preparation and application

Abstract

This invention belongs to the technical field of biomedical materials and discloses a photocurable hyaluronic acid-based composite hydrogel and its preparation and application. Method: Modified hyaluronic acid, galactose polymer, and a photoinitiator are mixed in a buffer solution and photocured to obtain the hyaluronic acid-based composite hydrogel. The modified hyaluronic acid has the structural formula I, and the galactose polymer has the structural formula II. The composite hydrogel is used in the field of cartilage tissue engineering, including three-dimensional culture of chondrocytes and repair of damaged cartilage. This invention prepares a hydrogel containing both static covalent crosslinking and dynamic crosslinking of phenylboronic acid esters through photoinitiated free radical polymerization. The reaction conditions are mild, and the resulting hydrogel exhibits good biocompatibility and stress relaxation properties. Furthermore, the microenvironment of the composite hydrogel, composed of multiple components, is conducive to maintaining the morphology and function of chondrocytes.

Description

Technical Field

[0001] This invention belongs to the technical field of biomedical materials, specifically relating to a photocurable hyaluronic acid-based composite hydrogel and its preparation and application. Background Technology

[0002] Articular cartilage is an avascular, lymphatic-free connective tissue composed of chondrocytes, extracellular matrix, and a large amount of water, exhibiting very weak self-repair capabilities. Therefore, cartilage damage repair has always been a challenge in the field of regenerative medicine. Cartilage tissue engineering, which combines scaffold materials with cells and growth factors, is considered one of the most promising research directions for cartilage tissue repair. Currently, cartilage tissue engineering has been applied clinically in autologous chondrocyte transplantation (ACI), but it suffers from problems such as functional loss during chondrocyte proliferation, fibrosis of regenerated cartilage, and high surgical failure rates, limiting its widespread clinical application.

[0003] Hydrogels are water-rich three-dimensional networks formed through physical or chemical cross-linking. They possess structures and properties highly similar to the extracellular matrix, thus finding widespread research and application in the biomedical field. Three-dimensional encapsulation of chondrocytes with hydrogels facilitates the exchange and transport of nutrients and metabolic waste, provides space for proliferation and growth, and offers a suitable growth microenvironment for chondrocytes. The mechanical properties of biomaterials in direct contact with cells can influence cell behavior through mechanical stress transduction. Studies have shown that a stress-relaxed environment similar to the extracellular matrix is ​​more conducive to cell adhesion, migration, and proliferation.

[0004] Hyaluronic acid is an important component of the extracellular matrix of chondrocytes, and it can bind to CD44 receptors on the cell surface, influencing cell behavior. Hyaluronic acid stimulates the metabolic activity of chondrocytes and significantly promotes the secretion of proteoglycans and type II collagen. Chondrocytes can synthesize glycosaminoglycans from galactose. Galactose can promote the secretion of matrix by chondrocytes. Galactose polymers have a longer retention time in hydrogels than galactose molecules, and can provide the nutrients needed for matrix secretion by chondrocytes loaded in the gel.

[0005] The hydrogel prepared in this invention uses hyaluronic acid as the main material. Through the formation of dynamic phenylboronic acid ester bonds between the grafted phenylboronic acid functional groups and the galactose polymer, it not only creates a stress-relaxation microenvironment for chondrocytes, but also provides a large amount of nutrients required for their secretory matrix. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings and deficiencies of existing technologies and provide a photocurable hyaluronic acid-based composite hydrogel for cartilage tissue engineering and its preparation method. This invention utilizes photoinitiated free radical polymerization and the formation of phenylboronic acid ester bonds between phenylboronic acid groups and 1,2- or 1,3-diols to obtain a hydrogel with good biocompatibility and stress relaxation properties. It can encapsulate cells, be injected in situ, and photocured into a gel. The gelation process is mild and rapid, and it has broad application prospects in the field of cartilage tissue engineering. The composite hydrogel of this invention is used for cartilage tissue engineering.

[0007] To achieve the above objectives, the technical solution provided by this invention is as follows:

[0008] A method for preparing a photocurable hyaluronic acid-based composite hydrogel includes the following steps:

[0009] In a buffer solution, modified hyaluronic acid, galactose polymer, and photoinitiator are mixed and photocured to obtain a hyaluronic acid-based composite hydrogel.

[0010] The modified hyaluronic acid has the structural formula I:

[0011]

[0012] Where n > 0 and n is an integer.

[0013] The structural formula of the galactose polymer is Formula II:

[0014]

[0015] Where n is an integer between 25 and 100.

[0016] The modified hyaluronic acid has a mass concentration of 1-10% in the buffer solution, and the galactose polymer has a mass concentration of 0.1-5% in the buffer solution.

[0017] The buffer solution is a phosphate buffer solution.

[0018] The photoinitiator is one or more of photoinitiator I2959, photoinitiator LAP, and photoinitiator VA-086.

[0019] The photoinitiator has a mass concentration of 0.1–0.5% in the buffer solution. The density of the buffer solution is calculated in units of 1 g / mL.

[0020] The light curing is ultraviolet light curing, and the light exposure time is 1 to 5 minutes.

[0021] The preparation of the modified hyaluronic acid includes the following steps:

[0022] 1) Hyaluronic acid was activated in morpholine ethanesulfonic acid buffer using an activating agent; then 3-aminophenylboronic acid monohydrate was added, reacted, dialyzed and lyophilized to obtain phenylboronic acid modified hyaluronic acid;

[0023] 2) Sodium periodate was reacted with phenylboronic acid-modified hyaluronic acid in water, and then dialyzed and freeze-dried to obtain oxidized phenylboronic acid-modified hyaluronic acid.

[0024] 3) At low temperature, methacrylic anhydride was mixed with an aqueous solution of oxidized phenylboronic acid modified hyaluronic acid. The pH was adjusted to alkaline and the reaction was carried out. The supernatant was centrifuged and dialyzed to obtain oxidized phenylboronic acid and double bond modified hyaluronic acid.

[0025] In step 1), the mass ratio of hyaluronic acid to 3-aminophenylboronic acid monohydrate is 5:1 to 10:1.

[0026] In step 1), the activator is 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride or an EDC / NHS system.

[0027] In step 1), the reaction time is 12–36 hours. The reaction is carried out at room temperature in the dark.

[0028] In step 1), the dialysis refers to dialysis using a dialysis bag with a molecular weight cutoff of 8,000 to 14,000, and the dialysis time is 3 to 5 days.

[0029] In step 2), the mass ratio of the phenylboronic acid-modified hyaluronic acid to sodium periodate is 1:1 to 5:1.

[0030] In step 2), the reaction time is 0.5 to 6 hours.

[0031] In step 2), the dialysis refers to dialysis using a dialysis bag with a molecular weight cutoff of 8,000 to 14,000, and the dialysis time is 3 to 5 days.

[0032] In step 3), the low temperature is 1-4°C, the pH value is 8-10, and the reaction time is 12-24 hours.

[0033] In step 3), the mass ratio of the oxidized phenylboronic acid-modified hyaluronic acid to methacrylic anhydride is 1:2 to 1:7.

[0034] The methacrylic anhydride is added dropwise.

[0035] The pH adjustment to alkaline means adjusting the pH using an alkali, such as NaOH.

[0036] In step 3), the dialysis refers to dialysis using a dialysis bag with a molecular weight cutoff of 8,000 to 14,000, and the dialysis time is 3 to 5 days.

[0037] The preparation of the galactose polymer includes the following steps:

[0038] S1) 6-O-methacryloyl-diacetone galactose was dissolved in an organic solvent with an initiator and a chain transfer agent and subjected to polymerization; quenching reaction and subsequent processing were performed to obtain poly(6-O-methacryloyl-diacetone galactose).

[0039] S2) The poly(6-O-methacryloyl-diacetone galactose) obtained in step S1) is de-protected by the isopropylidene hydroxyl protecting group on the pyranose ring with trifluoroacetic acid solution, and then lyophilized by dialyzing to obtain poly(6-O-methacryloyl-D-galactose).

[0040] In step S1), the initiator is one or more of azobisisobutyronitrile, azobisisoheptanenitrile, dimethyl azobisisobutyrate, and benzoyl peroxide.

[0041] In step S1), the chain transfer agent is 4-cyano-4-(phenylthiocarbamoylthio)valerate.

[0042] In step S1), the organic solvent is one or more of 1,4-dioxane, tetrahydrofuran, and N,N-dimethylformamide.

[0043] In step S1), the molar ratio of 6-O-methacryloyl-diacetone galactose to chain transfer agent is 50:1 to 120:1, and the molar ratio of initiator to chain transfer agent is 1:4 to 1:6.

[0044] In step S1), the polymerization reaction is carried out under anaerobic conditions at 65–80°C for 12–48 hours.

[0045] In step S1), the quenching reaction refers to low-temperature quenching, where low temperature refers to -20℃ to -80℃.

[0046] In step S1), the subsequent treatment is to precipitate with methanol followed by vacuum drying.

[0047] In step S2), the volume ratio of trifluoroacetic acid to water in the trifluoroacetic acid solution is 5:1 to 3:1.

[0048] In step S2), the dialysis refers to dialysis using a dialysis bag with a molecular weight cutoff of 3000, and the dialysis time is 3 to 5 days.

[0049] The photocurable hyaluronic acid-based composite hydrogel was obtained by the above preparation method.

[0050] The photocurable hyaluronic acid-based composite hydrogel is used in the field of tissue engineering, particularly in cartilage tissue engineering, for three-dimensional culture of chondrocytes and / or cartilage repair.

[0051] The photocurable hyaluronic acid-based composite hydrogel is used for three-dimensional culture of chondrocytes, including the following steps:

[0052] (1) Chondrocytes were mixed with hydrogel prepolymer to obtain a complex; the cell density in the complex was 1×10⁻⁶. 5 ~1×10 7 The prepolymer solution is a mixture of 1 / mL of the hyaluronic acid-based composite hydrogel before photocuring.

[0053] (2) The complex was injected into the cell culture device and irradiated under 365nm ultraviolet light for 1-3 minutes to achieve three-dimensional encapsulation of chondrocytes.

[0054] The hydrogel of this invention is used in the field of cartilage tissue engineering, including three-dimensional culture of chondrocytes and repair of damaged cartilage. This invention prepares a hydrogel containing both static covalent crosslinks and dynamic crosslinks of phenylboronic acid esters through photo-initiated free radical polymerization. The reaction conditions are mild, and the resulting hydrogel exhibits good biocompatibility and stress relaxation properties. Furthermore, the microenvironment of the composite hydrogel, composed of multiple components, is conducive to maintaining the morphology and function of chondrocytes.

[0055] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0056] (1) The raw material hyaluronic acid used in this invention is a natural extract with a wide range of sources. It is an important component of the extracellular matrix of chondrocytes and can provide a more similar growth microenvironment for chondrocytes.

[0057] (2) The hydrogel preparation process of the present invention is simple and easy to operate, and the preparation conditions are mild, which is conducive to the three-dimensional encapsulation of chondrocytes.

[0058] (3) The hydrogel prepared by the present invention has stress relaxation properties, which is more conducive to the growth, proliferation and characteristic matrix secretion of chondrocytes.

[0059] (4) The galactose polymer in this invention can be used as a nutrient for chondrocytes. Attached Figure Description

[0060] Figure 1 The 1H NMR spectrum of the modified hyaluronic acid prepared in Example 1;

[0061] Figure 2 The 1H NMR spectrum of the galactose polymer prepared in Example 1;

[0062] Figure 3The stress relaxation curves of the composite hydrogels prepared in Examples 4-7 are shown.

[0063] Figure 4 Image showing the live / dead staining results of the hydrogel prepared in Example 8 used for in vitro three-dimensional culture of chondrocytes;

[0064] Figure 5 The hydrogel prepared in Example 8 was used for in vitro three-dimensional culture of chondrocytes, and the expression of the Aggrecan gene was observed after 7 days of culture. Detailed Implementation

[0065] The present invention will be further described below with reference to specific embodiments, but the implementation of the present invention is not limited thereto.

[0066] Example 1

[0067] (1) Dissolve 1g of hyaluronic acid in 100mM morpholine ethanesulfonic acid buffer. After complete dissolution, add 0.692g of carboxyl activator 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride and stir for 30min to activate the carboxyl group. Weigh 0.129g of 3-aminophenylboronic acid monohydrate and add it to the solution. React at room temperature in the dark for 24h. Place the reaction solution in a dialysis bag with a molecular weight cutoff of 8000-14000 and dialyze for three days. After dialysis, filter and freeze dry to obtain phenylboronic acid modified hyaluronic acid.

[0068] (2) Add 1g of the product from step (1) to 150mL of deionized water, stir at room temperature until it is completely dissolved, then add 5mL of 0.5M sodium periodate solution, react in the dark for 2h, add 1mL of ethylene glycol to quench the unreacted periodate, continue stirring for 1h, place in a dialysis bag with a molecular weight cutoff of 8000-14000 and dialyze for three days, freeze-dry after dialysis to obtain oxidized phenylboronic acid modified hyaluronic acid;

[0069] (3) Add 2g of the product from step (2) to 200mL of deionized water and stir in a low-temperature reactor at 4℃ until it is completely dissolved. Add 7.5mL of methacrylic anhydride dropwise using a constant pressure funnel. Add 5M sodium hydroxide solution dropwise until the pH of the reaction solution is stable between 8 and 9. Continue the reaction for 12h. After centrifuging the reaction solution at 10000rpm for 15min, take the supernatant and place it in a dialysis bag with a molecular weight cutoff of 8000-14000 for three days. After dialysis, freeze dry to obtain oxidized phenylboronic acid and double bond modified hyaluronic acid.

[0070] (4) Mix 6.56 g of 6-O-methacryloyl-diacetone galactose, 6.6 mg of initiator azobisisobutyronitrile and 55.8 mg of chain transfer agent 4-cyano-4-(phenylthiocarbamoylthio)valerate, dissolve in 20 mL of 1,4-dioxane, then deoxygenate the reaction solution by purging for 40 min, then place it in an oil bath preheated to 75 °C and react for 16 h, then transfer it to a -80 °C freezer to quench the reaction; after thawing, precipitate with 20 times the volume of methanol, centrifuge at 10000 rpm for 15 min and discard the solution, leaving the bottom precipitate to dry in a vacuum oven to obtain poly(6-O-methacryloyl-diacetone galactose);

[0071] (5) Dissolve the product of step (4) in trifluoroacetic acid solution (TFA / H2O=4 / 1), stir at room temperature for 4 hours, dialyze for three days with a dialysis bag with a molecular weight cutoff of 3000, and freeze dry to obtain poly(6-O-methacryloyl-D-galactose);

[0072] (6) Weigh 80mg of the product from step (3), 5mg of the product from step (5) and 2mg of photoinitiator I2959 and dissolve them in 1mL of phosphate buffer. Stir at room temperature in the dark until completely dissolved. Pour the prepolymer into a mold with a diameter of 10mm and a height of 5mm and cure it under a 365nm UV lamp for 2min to obtain a transparent composite gel.

[0073] The modified hyaluronic acid and galactose polymer prepared in this embodiment were subjected to NMR spectroscopy, and the results are shown in the figures below. Figure 1 , 2 .

[0074] Example 2

[0075] (1) Dissolve 1g of hyaluronic acid in 100mM morpholine ethanesulfonic acid buffer. After complete dissolution, add 0.692g of carboxyl activator 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride and stir for 30min to activate the carboxyl group. Weigh 0.158g of 3-aminophenylboronic acid monohydrate and add it to the solution. React at room temperature in the dark for 18h. Place the solution in a dialysis bag with a molecular weight cutoff of 8000-14000 and dialyze for three days. After dialysis, filter and freeze dry to obtain phenylboronic acid modified hyaluronic acid.

[0076] (2) Add 1g of the product from step (1) to 150mL of deionized water, stir at room temperature until it is completely dissolved, then add 8mL of 0.5M sodium periodate solution, react in the dark for 1h, add 2mL of ethylene glycol to quench the unreacted periodate, and continue stirring for 1h; place it in a dialysis bag with a molecular weight cutoff of 8000-14000 and dialyze for three days. After dialysis, freeze dry to obtain oxidized phenylboronic acid modified hyaluronic acid;

[0077] (3) Add 2g of the product from step (2) to 200mL of deionized water and stir in a low-temperature reactor at 2℃ until it is completely dissolved. Add 10.5mL of methacrylic anhydride dropwise using a constant pressure funnel. Add 5M sodium hydroxide solution dropwise until the pH of the reaction solution is stable between 8 and 9. Continue the reaction for 20h. After centrifuging the reaction solution at 10000rpm for 15min, take the supernatant and place it in a dialysis bag with a molecular weight cutoff of 8000-14000 for dialysis for three days. After dialysis, freeze dry to obtain oxidized phenylboronic acid and double bond modified hyaluronic acid.

[0078] (4) Mix 6.56 g of 6-O-methacryloyl-diacetone galactose, 8.2 mg of initiator azobisisobutyronitrile and 69.8 mg of chain transfer agent 4-cyano-4-(phenylthiocarbamoylthio)valerate, dissolve in 20 mL of 1,4-dioxane, then deoxygenate the reaction solution by purging for 60 min, then place it in an oil bath preheated to 70 °C and react for 24 h, then transfer it to a -80 °C freezer to quench the reaction; after thawing, precipitate with 20 times the volume of methanol, centrifuge at 10000 rpm for 15 min and discard the solution, leaving the bottom precipitate to dry in a vacuum oven to obtain poly(6-O-methacryloyl-diacetone galactose);

[0079] (5) Dissolve the product of step (4) in trifluoroacetic acid solution (TFA / H2O=5 / 1), stir at room temperature for 3 hours, dialyze for three days with a dialysis bag with a molecular weight cutoff of 3000, and freeze dry to obtain poly(6-O-methacryloyl-D-galactose);

[0080] (6) Weigh 60 mg of the product from step (3), 20 mg of the product from step (5), and 2 mg of photoinitiator I2959 and dissolve them in 1 mL of phosphate buffer solution. Stir at room temperature in the dark until completely dissolved. Pour the prepolymer solution into a mold with a diameter of 10 mm and a height of 5 mm and cure it under a 365 nm UV lamp for 2 min to obtain a transparent composite gel.

[0081] Example 3

[0082] (1) Dissolve 1g of hyaluronic acid in 100mM morpholine ethanesulfonic acid buffer. After complete dissolution, add 0.692g of carboxyl activator 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride and stir for 45min to activate the carboxyl group. Weigh 0.186g of 3-aminophenylboronic acid monohydrate and add it to the solution. React at room temperature in the dark for 30h. Place the solution in a dialysis bag with a molecular weight cutoff of 8000-14000 and dialyze for three days. After dialysis, filter and freeze dry to obtain phenylboronic acid modified hyaluronic acid.

[0083] (2) Add 1g of the product from step (1) to 150mL of deionized water, stir at room temperature until it is completely dissolved, then add 5mL of 0.2M sodium periodate solution, react in the dark for 4h, add 1mL of ethylene glycol to quench the unreacted periodate, and continue stirring for 1h; place it in a dialysis bag with a molecular weight cutoff of 8000-14000 and dialyze for three days. After dialysis, freeze dry to obtain oxidized phenylboronic acid modified hyaluronic acid;

[0084] (3) Add 2g of the product from step (2) to 200mL of deionized water and stir in a low-temperature reactor at 4℃ until it is completely dissolved. Add 7mL of methacrylic anhydride dropwise using a constant pressure funnel. Add 5M sodium hydroxide solution dropwise until the pH of the reaction solution is stable between 8 and 9. Continue the reaction for 16h. After centrifuging the reaction solution at 10000rpm for 15min, take the supernatant and place it in a dialysis bag with a molecular weight cutoff of 8000-14000 for dialysis for three days. After dialysis, freeze dry to obtain oxidized phenylboronic acid and double bond modified hyaluronic acid.

[0085] (4) 6.56 g of 6-O-methacryloyl-diacetone galactose, 13.7 mg of initiator azobisisobutyronitrile and 93.1 mg of chain transfer agent 4-cyano-4-(phenylthiocarbamoylthio)valerate were mixed and dissolved in 20 mL of 1,4-dioxane. The reaction solution was then deoxygenated by purging for 60 min and then placed in an oil bath preheated to 80 °C for 14 h. The reaction was then quenched in a -80 °C freezer. After thawing, the product was precipitated with 20 times the volume of methanol. After centrifugation at 10,000 rpm for 15 min, the solution was discarded, and the bottom precipitate was dried in a vacuum oven to obtain poly(6-O-methacryloyl-diacetone galactose).

[0086] (5) Dissolve the product of step (4) in trifluoroacetic acid solution (TFA / H2O=3 / 1), stir at room temperature for 5 h, dialyze for three days with a dialysis bag with a molecular weight cutoff of 3000, and freeze dry to obtain poly(6-O-methacryloyl-D-galactose);

[0087] (6) Weigh 20 mg of the product from step (3), 40 mg of the product from step (5) and 2 mg of photoinitiator I2959 and dissolve them in 1 mL of phosphate buffer. Stir at room temperature in the dark until completely dissolved. Inject the prepolymer into a mold with a diameter of 10 mm and a height of 5 mm and cure it under a 365 nm UV lamp for 2 min to obtain a transparent composite gel.

[0088] Example 4

[0089] Steps (1)-(3) are the same as in Example 1;

[0090] (4) Weigh 40 mg of the product from step (3) and 2 mg of photoinitiator I2959 and dissolve them in 1 mL of phosphate buffer. Stir at room temperature in the dark until completely dissolved. Inject the prepolymer into a mold with a diameter of 10 mm and a height of 5 mm and cure it under a 365 nm UV lamp for 2 min to obtain a transparent modified hyaluronic acid gel, named A4P0.

[0091] Example 5

[0092] Steps (1)-(5) are the same as in Example 1;

[0093] (6) Weigh 40 mg of the product from step (3), 5 mg of the product from step (5) and 2 mg of photoinitiator I2959 and dissolve them in 1 mL of phosphate buffer. Stir at room temperature in the dark until completely dissolved. Inject the prepolymer into a mold with a diameter of 10 mm and a height of 5 mm and cure it under a 365 nm UV lamp for 2 min to obtain a transparent composite gel, named A4P0.5.

[0094] Example 6

[0095] Steps (1)-(5) are the same as in Example 1;

[0096] (6) Weigh 40 mg of the product from step (3), 10 mg of the product from step (5) and 2 mg of photoinitiator I2959 and dissolve them in 1 mL of phosphate buffer. Stir at room temperature in the dark until completely dissolved. Inject the prepolymer into a mold with a diameter of 10 mm and a height of 5 mm and cure it under a 365 nm UV lamp for 2 min to obtain a transparent composite gel, named A4P1.

[0097] Example 7

[0098] Steps (1)-(5) are the same as in Example 1;

[0099] (6) Weigh 40 mg of the product from step (3), 20 mg of the product from step (5) and 2 mg of photoinitiator I2959 and dissolve them in 1 mL of phosphate buffer. Stir at room temperature in the dark until completely dissolved. Inject the prepolymer into a mold with a diameter of 10 mm and a height of 5 mm and cure it under a 365 nm UV lamp for 2 min to obtain a transparent composite gel, named A4P2.

[0100] Example 8

[0101] The prepolymerized hydrogel solutions prepared in Examples 4-7 were filtered through a 0.22 μm needle filter to remove bacteria. Chondrocytes were then resuspended in the prepolymerized solution to obtain a chondrocyte suspension. The cell suspension was injected into 48-well plates at a rate of 0.2 mL per well and irradiated under UV light for 2 min to form a gel. Chondrocyte-specific culture medium was added, and the plates were incubated in a cell culture incubator for 4 h. After replacing the culture medium, the cells were cultured again to observe chondrocyte behavior.

[0102] Performance testing:

[0103] The compressive stress relaxation of the composite hydrogels prepared in Examples 4-7 was tested using a dynamic thermomechanical analyzer (DMA, Q800), and the obtained stress relaxation curves are shown below. Figure 3 As shown in the figure, the results indicate that the stress relaxation rate of the composite gel increases with increasing galactose polymer content. This is because more phenylboronic acid ester bonds are formed with increasing galactose polymer content.

[0104] The chondrocyte encapsulation density in Example 8 was 5 × 10⁻⁶. 5 Gel materials at concentrations of 1 / mL were incubated with Calcein-AM and PI live / dead staining working solutions at 37°C for 15 min on days 1, 4, and 7 of in vitro culture. Observations and photographs were taken using a laser confocal microscope. The photographs are shown below. Figure 4 As shown. Figure 4 This image shows the live / dead staining results of the hydrogel used for in vitro three-dimensional culture of chondrocytes in Example 8. The results indicate that the chondrocytes maintained good activity throughout the three-dimensional culture process, demonstrating the good biocompatibility of the hydrogel.

[0105] The chondrocyte encapsulation density in Example 8 was 5 × 10⁻⁶. 6 After culturing gel material at concentrations of 1 / mL in vitro for 7 days, the expression of the cartilage-related characteristic gene Aggrecan was detected by polymerase chain reaction (PCR). The results are as follows: Figure 5 As shown. Figure 5 This image shows the expression of the Aggrecan gene after 7 days of in vitro three-dimensional culture of chondrocytes using the hydrogel in Example 8. The results indicate that the A4P1 group showed the best effect in promoting Aggrecan expression, demonstrating that the composite hydrogel has a matrix secretion-promoting effect on the cells it encapsulates.

[0106] The examples described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Any modifications or equivalent substitutions to the technical solutions of the present invention that do not depart from the spirit and scope of the present invention should be included therein.

Claims

1. A method for preparing a photocurable hyaluronic acid-based composite hydrogel, characterized in that: Includes the following steps: In a buffer solution, modified hyaluronic acid, galactose polymer, and photoinitiator are mixed and photocured to obtain a hyaluronic acid-based composite hydrogel. The modified hyaluronic acid has the structural formula I: , where n > 0 and n is an integer; The structural formula of the galactose polymer is Formula II: , where n is an integer from 25 to 100.

2. The method for preparing the photocurable hyaluronic acid-based composite hydrogel according to claim 1, characterized in that: The modified hyaluronic acid has a mass concentration of 1-10% in the buffer solution, and the galactose polymer has a mass concentration of 0.1-5% in the buffer solution. The buffer solution is a phosphate buffer solution; The photoinitiator is one or more of photoinitiator I2959, photoinitiator LAP, and photoinitiator VA-086; The photoinitiator has a mass concentration of 0.1% to 0.5% in the buffer solution; The light curing is ultraviolet light curing, and the light exposure time is 1 to 5 minutes.

3. The method for preparing the photocurable hyaluronic acid-based composite hydrogel according to claim 1, characterized in that: The preparation of the modified hyaluronic acid includes the following steps: 1) Hyaluronic acid was activated in morpholine ethanesulfonic acid buffer using an activating agent; then 3-aminophenylboronic acid monohydrate was added, reacted, dialyzed and lyophilized to obtain phenylboronic acid modified hyaluronic acid; 2) Sodium periodate was reacted with phenylboronic acid-modified hyaluronic acid in water, and then dialyzed and freeze-dried to obtain oxidized phenylboronic acid-modified hyaluronic acid. 3) At low temperature, methacrylic anhydride is mixed with an aqueous solution of oxidized phenylboronic acid modified hyaluronic acid. The pH value is adjusted to alkaline and the reaction is carried out. The supernatant is centrifuged and dialyzed to obtain oxidized phenylboronic acid and double bond modified hyaluronic acid.

4. The method for preparing the photocurable hyaluronic acid-based composite hydrogel according to claim 3, characterized in that: In step 1), the mass ratio of hyaluronic acid to 3-aminophenylboronic acid monohydrate is 5:1 to 10:1; In step 1), the activator is 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride or an EDC / NHS system; In step 1), the reaction time is 12–36 hours; the reaction is carried out at room temperature in the dark. In step 2), the mass ratio of the phenylboronic acid-modified hyaluronic acid to sodium periodate is 1:1 to 5:1; In step 2), the reaction time is 0.5–6 hours. In step 3), the low temperature is 1-4°C, the pH value is 8-10, and the reaction time is 12-24 hours. In step 3), the mass ratio of the oxidized phenylboronic acid-modified hyaluronic acid to methacrylic anhydride is 1:2 to 1:

7.

5. The method for preparing the photocurable hyaluronic acid-based composite hydrogel according to claim 3, characterized in that: In step 1), the dialysis refers to dialysis using a dialysis bag with a molecular weight cutoff of 8,000 to 14,000, and the dialysis time is 3 to 5 days. In step 2), the dialysis refers to dialysis using a dialysis bag with a molecular weight cutoff of 8,000 to 14,000, and the dialysis time is 3 to 5 days. In step 3), the dialysis refers to dialysis using a dialysis bag with a molecular weight cutoff of 8,000 to 14,000, and the dialysis time is 3 to 5 days.

6. The method for preparing the photocurable hyaluronic acid-based composite hydrogel according to claim 1, characterized in that: The preparation of the galactose polymer includes the following steps: S1) 6-O-methacryloyl-diacetone galactose was dissolved in an organic solvent with an initiator and a chain transfer agent and subjected to polymerization; quenching reaction and subsequent processing were performed to obtain poly(6-O-methacryloyl-diacetone galactose). S2) The poly(6-O-methacryloyl-diacetone galactose) obtained in step S1) is de-protected by the isopropylidene hydroxyl protecting group on the pyranose ring with trifluoroacetic acid solution, and then lyophilized by dialyzing to obtain poly(6-O-methacryloyl-D-galactose).

7. The method for preparing the photocurable hyaluronic acid-based composite hydrogel according to claim 6, characterized in that: In step S1), the initiator is one or more of azobisisobutyronitrile, azobisisoheptanenitrile, dimethyl azobisisobutyrate, and benzoyl peroxide; In step S1), the chain transfer agent is 4-cyano-4-(phenylthiocarbamoylthio)pentanoic acid; In step S1), the organic solvent is one or more of 1,4-dioxane, tetrahydrofuran, and N,N-dimethylformamide; In step S1), the molar ratio of 6-O-methacryloyl-diacetone galactose to chain transfer agent is 50:1 to 120:1, and the molar ratio of initiator to chain transfer agent is 1:4 to 1:

6. In step S1), the polymerization reaction is carried out under anaerobic conditions at 65–80°C for 12–48 hours. In step S1), the quenching reaction refers to low-temperature quenching, where low temperature refers to -20℃ to -80℃; In step S1), the subsequent treatment is to precipitate with methanol followed by vacuum drying; In step S2), the volume ratio of trifluoroacetic acid to water in the trifluoroacetic acid solution is 5:1 to 3:1; In step S2), the dialysis refers to dialysis using a dialysis bag with a molecular weight cutoff of 3000, and the dialysis time is 3 to 5 days.

8. A photocurable hyaluronic acid-based composite hydrogel obtained by the preparation method according to any one of claims 1 to 7.

9. The application of the photocurable hyaluronic acid-based composite hydrogel according to claim 8, characterized in that: The hyaluronic acid-based composite hydrogel is used as a repair material for three-dimensional culture of chondrocytes and / or repair of damaged cartilage.

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