A hydrogel for cartilage repair and a method for preparing the same

Abstract

The application belongs to the field of biomedical materials, and particularly relates to a hydrogel for cartilage repair and a preparation method thereof. The preparation method of the hydrogel comprises the following steps: (1) adding cross-linking modified chitosan and 8-O-acetylmagnol methyl ester into a collagen solution, adjusting pH to prepare a collagen polysaccharide solution; (2) adding metal ion microspheres into the collagen polysaccharide solution in step (1), stirring and reacting, then adding microbial transglutaminase for heating reaction, adjusting pH to prepare a mixed solution; and (3) adding 1,4-butanediol diglycidyl ether and growth factors into the mixed solution in step (2), adjusting pH, water-bathing and dialysis to prepare the hydrogel. The preparation method is simple in process, and the obtained hydrogel has excellent mechanical strength and adhesion.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials, and particularly relates to a hydrogel for cartilage repair and its preparation method. Background Technology

[0002] Hydrogels play a crucial role in cartilage repair. They are three-dimensional network structures composed of hydrophilic polymer chains, containing 90-99% water, which can mimic the natural tissue environment, providing an excellent matrix for cell growth and tissue regeneration, while also possessing drug-carrying capacity. Chitosan hydrogels, in particular, are widely used for loading mesenchymal stem cells due to their excellent properties. However, chitosan hydrogels also have some drawbacks, such as insufficient mechanical strength, low viscosity, and unsatisfactory drug loading ratios and drug release stability, which may hinder the formation of hyaline cartilage.

[0003] To overcome these drawbacks, researchers have attempted to hybridize chitosan with other materials, such as gellan gum, polyglutamic acid, or silk fibroin, to prepare hydrogels. These hybrid hydrogels not only improve mechanical properties and cell compatibility but also promote chondrocyte gene expression, making the repaired cartilage tissue more similar to hyaline cartilage. However, to date, how to construct polymeric hydrogels that simultaneously possess high mechanical strength and adhesion, environmental responsiveness and stability, and targeted therapeutic capabilities remains a pressing challenge in current research. Summary of the Invention

[0004] In order to overcome the shortcomings of the prior art, one of the objectives of this invention is to provide a method for preparing hydrogels for cartilage repair, which has a simple process.

[0005] The second objective of this invention is to provide a hydrogel for cartilage repair, which has excellent mechanical strength and adhesion properties.

[0006] One of the objectives of this invention is achieved through the following technical solution:

[0007] A method for preparing a hydrogel for cartilage repair includes the following steps:

[0008] (1) Add cross-linked modified chitosan and 8-O-acetylganoside methyl ester to collagen solution, adjust pH, and prepare collagen polysaccharide solution;

[0009] (2) Add the metal ion microspheres to the collagen polysaccharide solution in step (1), stir and react, then add microbial transglutaminase and heat to react, adjust the pH, and obtain a mixed solution.

[0010] (3) Add 1,4-butanediol diglycidyl ether and growth factor to the mixed solution in step (2), adjust the pH, and perform water bath and dialysis to obtain the hydrogel.

[0011] This invention employs a chemical cross-linking method to combine metal ion microspheres with collagen and cross-linked modified chitosan, thereby constructing a dual-carrier hydrogel loaded with 8-O-acetylganoside methyl ester and growth factors.

[0012] Preferably, the preparation method of the cross-linked modified chitosan in step (1) is as follows:

[0013] The crosslinked modified chitosan was prepared by adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide to an ethanol aqueous solution of the crosslinking agent for activation, followed by adding chitosan solution and reacting under light-protected conditions with an inert gas atmosphere.

[0014] The structural formula of the crosslinking agent is:

[0015] .

[0016] This invention utilizes a crosslinking agent to covalently crosslink chitosan to obtain crosslinked modified chitosan.

[0017] Preferably, the molar ratio of the crosslinking agent, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and N-hydroxysuccinimide is 1:(3-6):(3-6); the mass ratio of chitosan to crosslinking agent is 1:(0.25-0.29); the solvent of the chitosan solution is an aqueous acetic acid solution, and the concentration of chitosan is 1-3 wt%; the concentration of the aqueous ethanol solution of the crosslinking agent is 3-5 wt%; and the activation time is 1-3 h.

[0018] Preferably, the crosslinking agent is prepared by the following method:

[0019] S1. Under inert gas protection, mercaptoethylamine, magnolol and benzophenone were added to tetrahydrofuran and irradiated to prepare intermediate 1.

[0020] The structural formula of intermediate 1 is:

[0021]

[0022] S2. Add the intermediate 1 to dichloromethane, then add triethylamine and 3-(2-pyridinedithio)propionic acid N-hydroxysuccinimide ester, and react to obtain intermediate 2;

[0023] The structural formula of intermediate 2 is as follows:

[0024]

[0025] S3. Add a methanol solution of 3-mercaptopropionic acid to the N,N-dimethylformamide solution of the intermediate 2, and react to obtain the crosslinking agent.

[0026] The present invention obtains intermediate 1 by reacting magnolol and mercaptoethylamine, then reacts it with N-hydroxysuccinimide ester of 3-(2-pyridyldithio)propionic acid to obtain intermediate 2, and finally reacts it with 3-mercaptopropionic acid to obtain a crosslinking agent.

[0027] Preferably, in step S1, the molar ratio of mercaptoethylamine, magnolol, and benzophenone is 1:(2.2-2.4):(0.01-0.012); the irradiation reaction time is 1-2 h; in step S2, the molar ratio of intermediate 1,3-(2-pyridinedithio)propionic acid N-hydroxysuccinimide ester and triethylamine is 1:(2.3-2.7):(5-5.4); the reaction time is 16-18 h.

[0028] Preferably, in step S3, the molar ratio of intermediate 2 and 3-mercaptopropionic acid is 1:(2-2.2); the concentration of the N,N-dimethylformamide solution of intermediate 2 is 0.03-0.04 mol / L, and the concentration of the methanol solution of 3-mercaptopropionic acid is 0.3-0.4 mol / L; the reaction time is 12-16 h.

[0029] Preferably, in step (1), the mass ratio of collagen, cross-linked modified chitosan, and 8-O-acetylganoside methyl ester is 1:1:(0.05-0.2); the concentration of the collagen solution is 0.5-1 g / mL; the collagen is selected from one of bone collagen, fish bone collagen, silk protein, and collagen fermented by genetically engineered bacteria; and the pH is adjusted to 5.4-5.9.

[0030] Preferably, based on the mass of collagen in the collagen polysaccharide solution, the mass ratio of the metal ion microspheres, microbial transglutaminase, and collagen in step (2) is 1:1:(4-5); the stirring reaction temperature is 25-27℃ and the time is 3-4h; the heating reaction temperature is 37-40℃ and the time is 0.5-1h; the pH is adjusted to 6.4-6.9; the microbial transglutaminase is glutamine transferase; the metal ion microspheres are selected from one of calcium carbonate-coated iron-selenopeptide microspheres, calcium carbonate-coated copper-selenopeptide microspheres, and calcium carbonate-coated iron / copper-selenopeptide microspheres.

[0031] Preferably, based on the mass of the metal ion microspheres, the mass ratio of 1,4-butanediol diglycidyl ether, growth factor and metal ion microspheres in step (3) is 1:1:1; the growth factor is transforming growth factor; the pH is adjusted to 7.4-7.9; the temperature of the water bath is 60-65℃ and the time is 5-8h.

[0032] The second objective of this invention is achieved by the following technical solution:

[0033] A hydrogel for cartilage repair was prepared using the above-described preparation method.

[0034] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0035] 1. This invention provides a method for preparing a hydrogel for cartilage repair, which uses a chemical cross-linking method to combine metal ion microspheres with collagen and cross-linked modified chitosan to construct a dual-carrier hydrogel loaded with 8-O-acetylganoside methyl ester and growth factors.

[0036] 2. The cross-linked modified chitosan of this invention is prepared by covalent cross-linking chitosan with a self-made cross-linking agent, possessing a stable three-dimensional network structure. The introduction of the cross-linking structure and benzene rings can significantly improve the mechanical strength of the hydrogel. The magnolol-biphenyl bisphenol structure can form multiple hydrogen bonds with the tissue surface, which helps to improve adhesion performance. It can also synergistically work with amide bonds and thioether groups to enhance the interfacial compatibility, tissue affinity, and loading and stabilization capacity of growth factors and metal ion microspheres of the hydrogel. In addition, the disulfide bonds introduced by the cross-linking agent molecules can undergo reversible "breakage-recombination" under physiological conditions to form a dynamic covalent bond network, giving the hydrogel a certain degree of self-repair capability and enabling it to better withstand repeated mechanical stimulation from joint activity. At the same time, the disulfide bonds are sensitive to ROS and can be specifically reduced and broken, allowing the hydrogel to intelligently and rapidly degrade at cartilage injury / inflammation sites, effectively promoting cartilage repair.

[0037] 3. The metal ion microspheres of this invention are obtained by encapsulating selenium peptides and ferrous or copper ions in calcium carbonate microspheres, and modifying the surface of the microspheres with folic acid and cysteine. These metal ion microspheres not only provide sufficient calcium for the cartilage repair process, but also significantly improve the stability and activity of growth factors. Furthermore, the 8-O-acetylganoside methyl ester loaded in the hydrogel works synergistically with the metal ion microspheres, enhancing the promoting effect of transforming growth factor on the cartilage differentiation of bone marrow mesenchymal stem cells (BMSCs), thereby improving the quality of newly formed chondrocytes, ensuring the stability of the articular cartilage repair process, and ultimately achieving efficient in vivo repair of hyaline cartilage in the knee joint. It also effectively inhibits inflammatory responses.

[0038] 4. The hydrogel prepared by this invention possesses excellent mechanical strength and adhesion properties. It can be customized and shaped according to the specific condition of the patient. It can closely adhere to the cartilage injury site and combine with surrounding tissues through physical and chemical actions to form a stable repair interface. This not only improves the repair effect but also reduces the occurrence of postoperative complications. In addition, this hydrogel has the advantages of good stability, high biological efficacy, and no toxic side effects. It can also be made into injectable small particles. Attached Figure Description

[0039] Figure 1 This is a SEM image of the calcium carbonate-loaded iron-selenium peptide microspheres of the present invention.

[0040] Figure 2 The 1H NMR spectrum of intermediate 1;

[0041] Figure 3 The 1H NMR spectrum of intermediate 2;

[0042] Figure 4 The image shows the 1H NMR spectrum of the crosslinking agent.

[0043] Figure 5 The infrared spectrum of the cross-linked modified chitosan prepared in Example 4 is shown in Figure 4, where curve a is the infrared spectrum of chitosan and curve b is the infrared spectrum of cross-linked modified chitosan.

[0044] Figure 6 The figures show the in vitro cytotoxicity results of the hydrogel prepared in Example 1, where a is the in vitro cytotoxicity result of the blank control group, b is the in vitro cytotoxicity result of the hydrogel prepared in Example 1, and c is the in vitro cytotoxicity result of the positive control group. Detailed Implementation

[0045] The present invention will now be further described with reference to the accompanying drawings and specific embodiments. It should be noted that, without conflict, the various embodiments or technical features described below can be arbitrarily combined to form new embodiments. Specific conditions not specified in the embodiments are performed according to conventional conditions or conditions recommended by the manufacturer. Unless otherwise specified, all reagents or instruments used are conventional products obtained through commercial channels.

[0046] In this invention, the metal ion microspheres are exemplified by calcium carbonate-coated iron-selenopeptide microspheres. The preparation method is as follows: 0.15 g of cysteine ​​is added to 250 mL of saturated calcium hydroxide solution to adjust the pH to 8. Then, 0.8 g of selenopeptide is added and fully dissolved. Carbon dioxide gas is then introduced, and the reaction proceeds for 15 min. Next, 0.03 g of ferrous chloride is added, and the pH is adjusted to 7. After reacting for 5 min, 0.2 g of folic acid is added, and the pH is adjusted to 6.5. The reaction proceeds for 10 min. After filtration and drying, calcium carbonate-coated iron-selenopeptide microspheres are obtained. See [Image of SEM image of calcium carbonate-coated iron-selenopeptide microspheres]. Figure 1 .

[0047] Preparation Example 1

[0048] A crosslinking agent is prepared by the following steps:

[0049]

[0050] S1. Under argon protection, with the amounts of magnolol, mercaptoethylamine, benzophenone, and tetrahydrofuran in the ratio of 1 mmol:2.3 mmol:0.011 mmol:7 mL, mercaptoethylamine, magnolol, and benzophenone were added to tetrahydrofuran. The reaction was carried out under UV light (wavelength 366 nm, 18 × 4 W) for 1.5 h. After the reaction was completed, the reaction solution was concentrated by rotary evaporation, and the residue was purified by column chromatography (V... 正庚烷 V 丙酮 =1:2), yielding intermediate 1 (yield 84.7%). The 1H NMR spectrum of intermediate 1 is shown below. Figure 2 The NMR and mass spectrometry data are shown below:

[0051] 1 HNMR: (C 22 H 32 S2O2N2, 400MHz, DMSO-d6) δ: 1.50 (s, 4H), 1.97-2.01 (m, 4H), 2.40-2.44 (m, 4H), 2.61-2.72 (m, 8H), 3.00-3.04 (m, 4H), 6.98-7.02 (d, 2H), 7.04-7.08 (d, 2H), 7.65 (s, 2H), 8.98 (s, 2H). MS (ESI) m / z=420.19 [M].

[0052] S2. Using intermediate 1, SPDP, triethylamine, and dichloromethane in a ratio of 1 mmol: 2.5 mmol: 5.2 mmol: 8 mL, intermediate 1 was dissolved in dichloromethane and cooled to 4 °C. Then, triethylamine and N-hydroxysuccinimide 3-(2-pyridinedithio)propionic acid (SPDP, CAS: 68181-17-9) were added, and the mixture was reacted at room temperature for 17 h. The reaction solution was diluted with dichloromethane, and then washed successively with saturated sodium bicarbonate solution, distilled water, and saturated brine. The mixture was dried over anhydrous magnesium sulfate, concentrated under reduced pressure, and the residue was purified by column chromatography (V... 三氯甲烷 V 异丙醇 =100:0~100:7), yielding intermediate 2 (yield 76.4%). The 1H NMR spectrum of intermediate 2 is shown below. Figure 3 The NMR and mass spectrometry data are shown below:

[0053] 1 HNMR: (C 38 H 46S6O4N4, 400MHz, DMSO-d6) δ: 1.97-2.01 (m, 4H), 2.40-2.50 (m, 8H), 2.61-2.72 (m, 8H), 2.82-2.86 (m, 4H), 3.55-3.59 (m, 4H), 6.98-7. 02 (d, 2H), 7.04-7.08 (d, 2H), 7.19-7.23 (m, 2H), 7.37-7.41 (d, 2H), 7.61-7.67 (m, 4H), 8.01 (s, 2H), 8.24-8.28 (d, 2H), 8.98 (s, 2H). MS (ESI) m / z=814.18 [M].

[0054] S3. With an intermediate 2 and 3-mercaptopropionic acid in a molar ratio of 1:2.1, a 0.35 mol / L methanol solution of 3-mercaptopropionic acid was added dropwise to a 0.035 mol / L N,N-dimethylformamide solution of intermediate 2. The reaction was allowed to proceed at room temperature for 14 h, followed by quenching with water. The mixture was then extracted with ethyl acetate, and the organic phase was washed successively with distilled water and saturated brine. After drying with anhydrous magnesium sulfate, the mixture was concentrated under reduced pressure, and the residue was purified by column chromatography (V... 二氯甲烷 :V 甲醇 :V 甲酸 =80:15:1), yielding a crosslinking agent (yield 63.7%). The 1H NMR spectrum of the crosslinking agent is shown below. Figure 4 The NMR and mass spectrometry data are shown below:

[0055] 1 HNMR: (C 34 H 48 S6O8N2, 400MHz, DMSO-d6) δ: 1.97-2.01 (m, 4H), 2.40-2.51 (m, 12H), 2.61-2.72 (m, 8H), 2.81-2.86 (m, 8H), 3. 55-3.59 (m, 4H), 6.98-7.02 (d, 2H), 7.04-7.08 (d, 2H), 7.65 (s, 2H), 8.01 (s, 2H), 8.98 (s, 2H), 12.17 (s, 2H). MS (ESI) m / z=804.17 [M].

[0056] Preparation Example 2

[0057] A crosslinking agent is prepared by the following steps:

[0058] S1. Under argon protection, with the amounts of magnolol, mercaptoethylamine, benzophenone, and tetrahydrofuran in the ratio of 1 mmol:2.2 mmol:0.01 mmol:5 mL, mercaptoethylamine, magnolol, and benzophenone were added to tetrahydrofuran. The reaction was carried out under UV light (wavelength 366 nm, 18 × 4 W) for 1 h. After the reaction was completed, the reaction solution was concentrated by rotary evaporation, and the residue was purified by column chromatography (V... 正庚烷 V 丙酮 =1:2), to obtain intermediate 1 (yield 81.9%), the NMR and mass spectrometry results of intermediate 1 are the same as those of preparation example 1.

[0059] S2. Using intermediate 1, SPDP, triethylamine, and dichloromethane in a ratio of 1 mmol: 2.3 mmol: 5 mmol: 7.5 mL, intermediate 1 was dissolved in dichloromethane and cooled to 0 °C. Then, triethylamine and SPDP were added, and the mixture was reacted at room temperature for 16 h. The reaction solution was diluted with dichloromethane, and then washed successively with saturated sodium bicarbonate solution, distilled water, and saturated brine. The solution was dried over anhydrous magnesium sulfate, concentrated under reduced pressure, and the residue was purified by column chromatography (V... 三氯甲烷 V 异丙醇 =100:0~100:7), intermediate 2 was obtained (yield 74.1%), and the NMR and mass spectrometry results of intermediate 2 were the same as those of preparation example 1.

[0060] S3. With an intermediate 2 and 3-mercaptopropionic acid in a molar ratio of 1:2, a 0.3 mol / L methanol solution of 3-mercaptopropionic acid was added dropwise to a 0.03 mol / L N,N-dimethylformamide solution of intermediate 2. The reaction was allowed to proceed for 12 h at room temperature, followed by quenching with water. The mixture was then extracted with ethyl acetate, and the organic phase was washed successively with distilled water and saturated brine. After drying with anhydrous magnesium sulfate, the mixture was concentrated under reduced pressure, and the residue was purified by column chromatography (V... 二氯甲烷 :V 甲醇 :V 甲酸 =80:15:1), to obtain a crosslinking agent (yield 61.5%). The NMR and mass spectrometry results of the crosslinking agent were the same as those in Preparation Example 1.

[0061] Preparation Example 3

[0062] A crosslinking agent is prepared by the following steps:

[0063] S1. Under argon protection, with the amounts of magnolol, mercaptoethylamine, benzophenone, and tetrahydrofuran in the ratio of 1 mmol:2.4 mmol:0.012 mmol:8 mL, mercaptoethylamine, magnolol, and benzophenone were added to tetrahydrofuran. The reaction was carried out under UV light (wavelength 366 nm, 18 × 4 W) for 2 h. After the reaction was completed, the reaction solution was concentrated by rotary evaporation, and the residue was purified by column chromatography (V... 正庚烷 V 丙酮=1:2), to obtain intermediate 1 (yield 82.4%), the NMR and mass spectrometry results of intermediate 1 are the same as those of preparation example 1.

[0064] S2. Using intermediate 1, SPDP, triethylamine, and dichloromethane in a ratio of 1 mmol:2.7 mmol:5.4 mmol:8.5 mL, intermediate 1 was dissolved in dichloromethane and cooled to 5°C. Then, triethylamine and SPDP were added, and the mixture was reacted at room temperature for 18 h. The reaction solution was diluted with dichloromethane, then washed successively with saturated sodium bicarbonate solution, distilled water, and saturated brine. The solution was dried over anhydrous magnesium sulfate, concentrated under reduced pressure, and the residue was purified by column chromatography (V... 三氯甲烷 V 异丙醇 =100:0~100:7), intermediate 2 was obtained (yield 73.8%), and the NMR and mass spectrometry results of intermediate 2 were the same as those of preparation example 1.

[0065] S3. With a molar ratio of intermediate 2 to 3-mercaptopropionic acid of 1:2.2, a methanol solution of 3-mercaptopropionic acid of 0.4 mol / L was added dropwise to a 0.04 mol / L N,N-dimethylformamide solution of intermediate 2. The reaction was allowed to proceed at room temperature for 16 h, followed by quenching with water. The mixture was then extracted with ethyl acetate, and the organic phase was washed successively with distilled water and saturated brine. After drying with anhydrous magnesium sulfate, the mixture was concentrated under reduced pressure, and the residue was purified by column chromatography (V... 二氯甲烷 :V 甲醇 :V 甲酸 =80:15:1), and a crosslinking agent was obtained (yield 62.3%). The NMR and mass spectrometry results of the crosslinking agent were the same as those in Preparation Example 1.

[0066] Preparation Example 4

[0067] A cross-linked modified chitosan is prepared using the following steps:

[0068] The crosslinking agent of Preparation Example 1 was dissolved in a 60% (v / v) aqueous ethanol solution to obtain an ethanol aqueous solution of crosslinking agent with a concentration of 4 wt%. 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) were added to the ethanol aqueous solution of the crosslinking agent with a molar ratio of 1:5:5 and activated for 2 h to obtain a mixed solution.

[0069] Chitosan was dissolved in 0.12 mol / L acetic acid solution to obtain a chitosan solution with a concentration of 2 wt%. The chitosan solution was added to the mixture at a mass ratio of chitosan to crosslinking agent of 1:0.27. The mixture was reacted overnight in the dark under nitrogen protection. After dialyzing with deionized water and freeze-drying, crosslinked modified chitosan was obtained.

[0070] Preparation Example 5

[0071] A cross-linked modified chitosan is prepared using the following steps:

[0072] The crosslinking agent of Preparation Example 2 was dissolved in an ethanol aqueous solution with a volume fraction of 60% to obtain an ethanol aqueous solution with a concentration of 3wt% of the crosslinking agent; EDC and NHS were added to the ethanol aqueous solution of the crosslinking agent with a molar ratio of 1:3:3, and the mixture was activated for 1 hour to obtain a mixed solution.

[0073] Chitosan was dissolved in 0.1 mol / L acetic acid solution to obtain a chitosan solution with a concentration of 1 wt%. The chitosan solution was added to the mixture at a mass ratio of chitosan to crosslinking agent of 1:0.25. The mixture was reacted overnight in the dark under nitrogen protection. After dialyzing with deionized water, the mixture was freeze-dried to obtain crosslinked modified chitosan.

[0074] Preparation Example 6

[0075] A cross-linked modified chitosan is prepared using the following steps:

[0076] The crosslinking agent of Preparation Example 3 was dissolved in an ethanol aqueous solution with a volume fraction of 60% to obtain an ethanol aqueous solution with a concentration of 5 wt% of the crosslinking agent; EDC and NHS were added to the ethanol aqueous solution of the crosslinking agent with a molar ratio of 1:6:6 and activated for 3 h to obtain a mixed solution.

[0077] Chitosan was dissolved in 0.15 mol / L acetic acid solution to obtain a chitosan solution with a concentration of 3 wt%. The chitosan solution was added to the mixture at a mass ratio of chitosan to crosslinking agent of 1:0.29. The mixture was reacted overnight in the dark under nitrogen protection. After dialyzing with deionized water and freeze-drying, crosslinked modified chitosan was obtained.

[0078] Example 1

[0079] A method for preparing a hydrogel for cartilage repair includes the following steps:

[0080] (1) With a mass ratio of 1:1:0.1 for collagen, cross-linked modified chitosan and 8-O-acetylganoside methyl ester, cross-linked modified chitosan and 8-O-acetylganoside methyl ester were added to a collagen solution with a concentration of 0.7 g / mL. The pH was adjusted to 5.6 using hydrochloric acid solution to obtain a collagen polysaccharide solution.

[0081] (2) Based on the mass of collagen in the collagen polysaccharide solution, according to the mass ratio of calcium carbonate-coated iron-selenopeptide microspheres, glutamine transferase and collagen of 1:1:4.5, add calcium carbonate-coated iron-selenopeptide microspheres to the collagen polysaccharide solution in step (1), stir at 26°C for 3.5h, let stand for 2.5h, then add glutamine transferase, stir at 38°C for 0.6h, adjust the pH to 6.8 with sodium hydroxide solution, let stand for 2.5h, and obtain a mixed solution;

[0082] (3) According to the mass ratio of 1,4-butanediol diglycidyl ether, growth factor and calcium carbonate-loaded iron-selenopeptide microspheres in step (2) of 1:1:1, 1,4-butanediol diglycidyl ether and transforming growth factor were added to the mixed solution in step (2), the pH was adjusted to 7.5 using phosphate buffer, and the reaction was carried out in a constant temperature water bath at 62℃ for 7h. Then, the mixture was dialyzed in distilled water without a pyrogen source for 4d to obtain the hydrogel.

[0083] Example 1 also provides a hydrogel for cartilage repair, which is prepared using the above-described preparation method.

[0084] Example 2

[0085] A method for preparing a hydrogel for cartilage repair includes the following steps:

[0086] (1) With the mass ratio of silk protein, cross-linked modified chitosan and 8-O-acetylganoside methyl ester being 1:1:0.05, cross-linked modified chitosan and 8-O-acetylganoside methyl ester of Preparation Example 5 were added to a silk protein solution with a concentration of 0.5 g / mL. The pH was adjusted to 5.4 using hydrochloric acid solution to obtain a collagen polysaccharide solution.

[0087] (2) Based on the mass of collagen in the collagen polysaccharide solution, according to the mass ratio of calcium carbonate-coated iron-selenopeptide microspheres, glutamine transferase and collagen of 1:1:4, add calcium carbonate-coated iron-selenopeptide microspheres to the collagen polysaccharide solution in step (1), stir at 25°C for 4 hours, let stand for 3 hours, then add glutamine transferase, stir at 37°C for 1 hour, adjust the pH to 6.9 with sodium hydroxide solution, let stand for 3 hours, and obtain a mixed solution;

[0088] (3) According to the mass ratio of 1,4-butanediol diglycidyl ether, growth factor and calcium carbonate-loaded iron-selenopeptide microspheres in step (2) of 1:1:1, 1,4-butanediol diglycidyl ether and transforming growth factor were added to the mixed solution in step (2), the pH was adjusted to 7.4 using phosphate buffer, and the reaction was carried out in a constant temperature water bath at 60℃ for 8 hours. Then, the mixture was dialyzed in distilled water without a pyrogen source for 2 days to obtain the hydrogel.

[0089] Example 2 also provides a hydrogel for cartilage repair, which is prepared using the above preparation method.

[0090] Example 3

[0091] A method for preparing a hydrogel for cartilage repair includes the following steps:

[0092] (1) With a mass ratio of fish bone collagen, cross-linked modified chitosan and 8-O-acetylganoside methyl ester of 1:1:0.2, cross-linked modified chitosan and 8-O-acetylganoside methyl ester of 1 g / mL fish bone collagen solution were added to the solution and the pH was adjusted to 5.9 with hydrochloric acid solution to obtain collagen polysaccharide solution;

[0093] (2) Based on the mass of collagen in the collagen polysaccharide solution, according to the mass ratio of calcium carbonate-coated iron-selenopeptide microspheres, glutamine transferase and collagen of 1:1:5, add calcium carbonate-coated iron-selenopeptide microspheres to the collagen polysaccharide solution in step (1), stir at 27°C for 3 hours, let stand for 2 hours, then add glutamine transferase, stir at 40°C for 0.5 hours, adjust the pH to 6.4 with sodium hydroxide solution, let stand for 2 hours, and obtain a mixed solution;

[0094] (3) According to the mass ratio of 1,4-butanediol diglycidyl ether, growth factor and calcium carbonate-loaded iron-selenopeptide microspheres in step (2) of 1:1:1, 1,4-butanediol diglycidyl ether and transforming growth factor were added to the mixed solution in step (2), the pH was adjusted to 7.9 using phosphate buffer, and the reaction was carried out in a constant temperature water bath at 65°C for 5 hours. Then, the mixture was dialyzed in distilled water without a pyrogen source for 5 days to obtain the hydrogel.

[0095] Example 3 also provides a hydrogel for cartilage repair, which is prepared using the above preparation method.

[0096] Comparative Example 1

[0097] The difference between Comparative Example 1 and Example 1 is that chitosan was used in step (1) to replace the cross-linked modified chitosan in Preparation Example 4.

[0098] Comparative Example 2

[0099] The difference between Comparative Example 2 and Example 1 is that 8-O-acetylganoside methyl ester is omitted in step (1).

[0100] Comparative Example 3

[0101] The difference between Comparative Example 3 and Example 1 is that step (2) omits the calcium carbonate-loaded iron-selenium peptide microspheres.

[0102] Experimental Example 1

[0103] The cross-linked modified chitosan obtained in Preparation Example 4 was analyzed by Fourier transform infrared spectroscopy (FT-IR), and the results are as follows: Figure 5 As shown.

[0104] Figure 5 This is the infrared spectrum of the cross-linked modified chitosan obtained in Preparation Example 4 of this invention, where curve a is the infrared spectrum of chitosan and curve b is the infrared spectrum of cross-linked modified chitosan. Figure 5 It is known that cross-linked modified chitosan has a cross-linking effect at 1706 cm⁻¹. -1 and 1621cm -1 The characteristic peaks at these locations correspond to the absorption peaks of the amide I band and the NH absorption peak of the amide II band of secondary amides, respectively, while the characteristic peak at 1600 cm⁻¹ on protochitosan is also present. -1 The significant decrease in the bending vibration peak of the primary amine (-NH2) at the site indicates that the crosslinking agent crosslinks with the -NH2 on chitosan through two -COOH groups to form amide bonds (-CONH-), indicating that the crosslinking modification of chitosan was successful.

[0105] Experiment Example 2

[0106] The mechanical and adhesive properties of the hydrogels prepared in the examples and comparative examples were tested, as follows:

[0107] The hydrogels prepared in Examples 1-3 and Comparative Examples 1-3 were used to prepare dumbbell-shaped test samples with dimensions of 20 mm × 10 mm × 100 μm (length × width × thickness).

[0108] Tensile properties: Tensile tests were conducted at a tensile rate of 1 mm / min and a tensile force of 50 N.

[0109] Compression performance: The sample was compressed along its long axis at a speed of 0.5 mm / min until the deformation reached 10%, and the compressive strength and compressive modulus were calculated.

[0110] Adhesion performance: The adhesion ability of the test sample to pigskin was determined by referring to the ASTM F2255 lap shear test and the ASTM F2256 180-degree peel test. The area was 10mm × 10mm, and the average value was used to calculate the viscosity strength. The results are shown in Table 1.

[0111] Table 1

[0112]

[0113] As can be seen from Table 1, the hydrogel obtained by this invention has excellent mechanical and adhesive properties.

[0114] Compared to Example 1, Comparative Example 1, which replaced the cross-linked modified chitosan of this invention with chitosan, showed a significant decrease in both the mechanical and adhesive properties of the resulting hydrogel. These experimental results demonstrate that the cross-linked modified chitosan of this invention can improve the mechanical and adhesive properties of the hydrogel. Specifically, the cross-linked modified chitosan of this invention possesses a stable three-dimensional network structure. The introduction of the cross-linked structure and benzene rings significantly enhances the mechanical strength of the hydrogel. The magnolol-biphenyl bisphenol structure can form multiple hydrogen bonds with the tissue surface, which helps improve adhesion. Furthermore, it can synergistically work with amide bonds and thioether groups to enhance the interfacial compatibility, tissue affinity, and loading and stabilization capacity of growth factors and metal ion microspheres in the hydrogel.

[0115] Experimental Example 3

[0116] The repair effects of the hydrogels prepared in the examples and comparative examples were tested, as follows:

[0117] laboratory animals

[0118] A total of 70 SD rats weighing 200-220g were used, and 10 rats were randomly selected as the control group.

[0119] Constructing a cartilage defect model

[0120] All rats were anesthetized by intraperitoneal injection of 800 μL of 10% chloroacetaldehyde hydrate solution. After the right knee joint of each rat was completely shaved, a cylindrical full-thickness cartilage defect with a diameter of 2 mm and a depth of 3 mm was made at the right knee joint using sterile surgical scissors, reaching the subchondral bone. Blood clots within the defect were removed to obtain the rat model.

[0121] Grouping and Dosing

[0122] The model rats were randomly divided into Example 1-3 groups and Comparative Example 1-3 groups, with 10 rats in each group. The corresponding hydrogels were then implanted in each group. The control group received no treatment and was directly sutured. The wounds were disinfected with povidone-iodine solution and injected with penicillin G sodium antibiotic solution at a dose of 120 mg / rat. The rats were housed separately and injected with the same dose of penicillin G sodium antibiotic solution for 2 consecutive days. After 12 weeks, CT scans were performed to observe the repair of osteochondral bone. The International Cartilage Repair Society (ICRS) scoring criteria were used to score the cartilage and the average value was calculated. The specific scoring criteria are shown in Table 2. The results of the cartilage repair experiment are shown in Table 3.

[0123] Table 2

[0124]

[0125] Table 3

[0126]

[0127] As can be seen from Table 3, the hydrogel obtained by this invention repairs the defective tissue in the same way as the surrounding normal cartilage, with no obvious boundary integration and a generally smooth surface.

[0128] Compared to Example 1, the repair capacity of the hydrogel cartilage obtained in Comparative Examples 1-3 was reduced, with Comparative Example 1 showing the worst effect, followed by Comparative Example 3. Specific analysis is as follows:

[0129] In Comparative Example 1, the use of chitosan instead of cross-linked modified chitosan resulted in a particularly significant decrease in cartilage repair capacity. This is because the disulfide bonds introduced by the cross-linking agent molecules can undergo reversible "breakage-recombination" under physiological conditions, forming a dynamic covalent bond network. This gives the hydrogel a certain degree of self-repair capability, allowing it to better withstand repeated mechanical stimulation from joint activity. Simultaneously, the disulfide bonds are sensitive to ROS and can be specifically reduced and broken, enabling the hydrogel to intelligently and rapidly degrade at cartilage injury / inflammation sites, effectively promoting cartilage repair.

[0130] Comparative Example 2, omitting 8-O-acetylganoside methyl ester, showed a slight decrease in cartilage repair capacity. Comparative Example 3, omitting calcium carbonate-encapsulated iron-selenopeptide microspheres, showed the next lowest cartilage repair capacity. This is because the 8-O-acetylganoside methyl ester loaded in the hydrogel and the calcium carbonate-encapsulated iron-selenopeptide microspheres work synergistically, not only enhancing the promoting effect of transforming growth factor on bone marrow mesenchymal stem cell (BMSC) cartilage differentiation, thereby improving the quality of newly formed chondrocytes, ensuring the stability of the articular cartilage repair process, and ultimately achieving efficient in vivo repair of hyaline cartilage in the knee joint, but also effectively inhibiting the inflammatory response. In addition, the calcium carbonate-encapsulated iron-selenopeptide microspheres can provide sufficient calcium for the cartilage repair process and can also significantly improve the stability and activity of growth factors.

[0131] Experiment Example 4

[0132] The in vitro cytotoxicity of the hydrogel prepared in Example 1 was tested as follows:

[0133] L929 mouse fibroblasts in logarithmic growth phase were digested with trypsin to adjust the cell density to 1×10⁻⁶. 5 Cells were seeded at a density of 100 μL / mL into 96-well cell culture plates. After incubation at 37°C and 5% CO2 for 24 hours, the culture medium was discarded, and 100 μL of 50 μg / mL hydrogel from Example 1 was added. Cells were then cultured for another 24 hours. After the culture period, cell morphology was observed under a microscope. Results are shown in [link to results]. Figure 6 In addition, a blank control group was set up using DMEM serum-free medium and a positive control group was set up using DMEM complete medium supplemented with 5% phenol.

[0134] Figure 6 The figures show the in vitro cytotoxicity results of the hydrogel prepared in Example 1, where a represents the in vitro cytotoxicity results of the blank control group, b represents the in vitro cytotoxicity results of the hydrogel prepared in Example 1, and c represents the in vitro cytotoxicity results of the positive control group. Observation Figure 6 It can be seen that there was no significant change in cell morphology, indicating that the hydrogel prepared in Example 1 had no significant cytotoxicity.

[0135] The above embodiments are merely preferred embodiments of the present invention and should not be construed as limiting the scope of protection of the present invention. Any non-substantial changes and substitutions made by those skilled in the art based on the present invention shall fall within the scope of protection claimed by the present invention.

Claims

1. A method for preparing a hydrogel for cartilage repair, characterized in that, Includes the following steps: (1) Add cross-linked modified chitosan and 8-O-acetylganoside methyl ester to collagen solution, adjust pH, and prepare collagen polysaccharide solution; (2) Add the metal ion microspheres to the collagen polysaccharide solution in step (1), stir and react, then add microbial transglutaminase and heat to react, adjust the pH, and obtain a mixed solution. (3) Add 1,4-butanediol diglycidyl ether and growth factor to the mixed solution in step (2), adjust the pH, and perform water bath and dialysis to obtain the hydrogel; The preparation method of the cross-linked modified chitosan in step (1) is as follows: 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide were added to an ethanol-water solution of the crosslinking agent for activation. Then, a chitosan solution was added, and the reaction was carried out in the dark under inert gas protection to obtain the crosslinked modified chitosan. The mass ratio of chitosan to crosslinking agent was 1:(0.25-0.29). The structural formula of the crosslinking agent is: In step (1), the mass ratio of collagen, cross-linked modified chitosan, and methyl 8-O-acetylganoside is 1:1:(0.05-0.2); the concentration of the collagen solution is 0.5-1 g / mL. Based on the mass of collagen in the collagen polysaccharide solution, the mass ratio of the metal ion microspheres, microbial transglutaminase and collagen in step (2) is 1:1:(4-5); the metal ion microspheres are selected from one of the following: calcium carbonate-loaded iron-selenium peptide microspheres, calcium carbonate-loaded copper-selenium peptide microspheres, and calcium carbonate-loaded iron / copper-selenium peptide microspheres. Based on the mass of the metal ion microspheres, the mass ratio of 1,4-butanediol diglycidyl ether, growth factor and metal ion microspheres in step (3) is 1:1:1; the growth factor is transforming growth factor.

2. The method for preparing the hydrogel for cartilage repair according to claim 1, characterized in that, The molar ratio of the crosslinking agent, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and N-hydroxysuccinimide is 1:(3-6):(3-6); the solvent of the chitosan solution is an aqueous acetic acid solution, and the concentration of chitosan is 1-3 wt%; the concentration of the aqueous ethanol solution of the crosslinking agent is 3-5 wt%; and the activation time is 1-3 h.

3. The method for preparing the hydrogel for cartilage repair according to claim 2, characterized in that, The crosslinking agent is prepared as follows: S1. Under inert gas protection, mercaptoethylamine, magnolol and benzophenone were added to tetrahydrofuran and irradiated to prepare intermediate 1. The structural formula of intermediate 1 is: S2. Add the intermediate 1 to dichloromethane, then add triethylamine and 3-(2-pyridinedithio)propionic acid N-hydroxysuccinimide ester, and react to obtain intermediate 2; The structural formula of intermediate 2 is as follows: S3. Add a methanol solution of 3-mercaptopropionic acid to the N,N-dimethylformamide solution of the intermediate 2, and react to obtain the crosslinking agent.

4. The method for preparing the hydrogel for cartilage repair according to claim 3, characterized in that, In step S1, the molar ratio of mercaptoethylamine, magnolol, and benzophenone is 1:(2.2-2.4):(0.01-0.012); the irradiation reaction time is 1-2 h. In step S2, the molar ratio of intermediate 1,3-(2-pyridinedithio)propionic acid N-hydroxysuccinimide ester and triethylamine is 1:(2.3-2.7):(5-5.4); the reaction time is 16-18 h.

5. The method for preparing the hydrogel for cartilage repair according to claim 3, characterized in that, In step S3, the molar ratio of intermediate 2 and 3-mercaptopropionic acid is 1:(2-2.2); the concentration of the N,N-dimethylformamide solution of intermediate 2 is 0.03-0.04 mol / L, and the concentration of the methanol solution of 3-mercaptopropionic acid is 0.3-0.4 mol / L; the reaction time is 12-16 h.

6. The method for preparing the hydrogel for cartilage repair according to claim 1, characterized in that, The collagen mentioned in step (1) is selected from one of bone collagen, fish bone collagen, silk protein, and collagen fermented by genetically engineered bacteria; the pH is adjusted to 5.4-5.

9.

7. The method for preparing the hydrogel for cartilage repair according to claim 1, characterized in that, The stirring reaction in step (2) is carried out at a temperature of 25-27°C for 3-4 hours; the heating reaction is carried out at a temperature of 37-40°C for 0.5-1 hours; the pH is adjusted to 6.4-6.9; and the microbial transglutaminase is a glutamin transferase.

8. The method for preparing the hydrogel for cartilage repair according to claim 1, characterized in that, In step (3), the pH is adjusted to 7.4-7.9; the temperature of the water bath is 60-65℃ and the time is 5-8h.

9. A hydrogel for cartilage repair, characterized in that, It is prepared by the preparation method according to any one of claims 1-8.

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