A recombinant collagen gel and a method for preparing the same

Abstract

This invention relates to the field of biomedical materials technology and discloses a recombinant collagen gel and its preparation method. The preparation method is as follows: recombinant collagen is dissolved in glacial acetic acid solution and stirred until completely dissolved to obtain a recombinant collagen solution. The recombinant collagen solution is added to phosphate buffer, and then modified chitosan, phenylboronic acid-modified sodium alginate, and glycerol are added. The mixture is stirred for 30-40 minutes to obtain a gel precursor solution. An aldehyde-modified epoxy-thiol polymer is dissolved in dimethyl sulfoxide to prepare a stock solution. The stock solution is then added dropwise to the gel precursor solution, and the mixture is stirred for another 5-10 minutes. The mixture is injected into a sterile mold, allowed to stand for 12-24 hours, defoamed, sterilized, and then sealed and stored at 4°C to obtain the recombinant collagen gel. The recombinant collagen gel prepared by this invention has good mechanical properties, self-healing properties, is safe and non-toxic, and has high biocompatibility.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, specifically to a recombinant collagen gel and its preparation method. Background Technology

[0002] Collagen, with its excellent biocompatibility, cell adhesion, and biodegradability, has become a core raw material in the field of biomedical materials, widely used in soft tissue filling, wound repair, and other applications. With the development of biomanufacturing technology, recombinant collagen has achieved efficient in vitro expression through genetic engineering. Its amino acid sequence is highly homologous to natural human collagen, effectively avoiding the inherent defects of animal-derived collagen such as viral contamination and high immunogenicity. This significantly improves its safety and purity, making it a preferred raw material for medical materials.

[0003] However, existing recombinant collagen gels primarily improve performance through physical blending or chemical cross-linking, which still suffers from the following problems: physical blending relies solely on weak forces such as hydrogen bonds and electrostatics, resulting in a loose network structure with low mechanical strength and poor environmental adaptability; while chemical cross-linking can enhance gel rigidity, traditional small-molecule cross-linking agents are prone to residue, triggering cytotoxicity and skin sensitization reactions; even with single physical cross-linking or dynamic cross-linking, there are still issues with insufficient mechanical strength and poor structural stability, making it difficult to meet the support requirements for clinical use. Furthermore, existing gels have limited functions, lack self-repair properties, are prone to failure after mechanical damage, and have poor injection compatibility, further restricting their application scope.

[0004] Therefore, developing a recombinant collagen gel that combines excellent mechanical properties, self-healing function, high biocompatibility, and good injectability has become a key issue that urgently needs to be addressed in this field. Summary of the Invention

[0005] In order to overcome the shortcomings of the prior art, the present invention aims to provide a recombinant collagen gel and its preparation method. The recombinant collagen gel prepared by the present invention has good mechanical properties, self-repair properties, is safe and non-toxic, and has high biocompatibility.

[0006] To achieve the above objectives, the present invention provides the following technical solution: a recombinant collagen gel, comprising the following weight components: 2-4 parts by weight of recombinant collagen, 1-2 parts by weight of modified chitosan, 1-1.5 parts by weight of phenylboronic acid-modified sodium alginate, 0.5-1 parts by weight of aldehyde-modified epoxy-thiol polymer, 35-40 parts by weight of phosphate buffer, and 0.2-0.3 parts by weight of glycerol;

[0007] The phenylboronic acid-modified sodium alginate is obtained by reacting oxidized sodium alginate with 3-aminophenylboronic acid;

[0008] The aldehyde-based epoxy-thiol polymer is obtained by reacting ethylene glycol diglycidyl ether with 1,3-propanedithiol to obtain the epoxy-thiol polymer, and then reacting it with p-aldehyde benzoic acid.

[0009] Further, the modified chitosan is prepared as follows: 150-160 mL of a 5% (v / v) acetic acid solution and 180-190 mL of an ethanol solution are added to a reactor to prepare a solvent. Then, 1.5-1.6 g of chitosan is added and stirred to dissolve. Next, 2.25-2.4 g of succinic anhydride is added to 60-64 mL of acetone and stirred to dissolve. This solution is then added dropwise to the reactor over 25-30 minutes. After the addition is complete, the reactor is heated at 25-30°C. The reaction was carried out for 12-14 hours. After the reaction was completed, the precipitate was collected and dispersed in an ethanol solution. The pH was adjusted to 9-10 by adding sodium hydroxide solution with a molar concentration of 1.25 mol / L. The precipitate was collected again and dissolved in deionized water. The solution was then placed in a dialysis bag with a capacity of 8000-14000 Da and dialyzed with deionized water for 48-50 hours. After dialysis, the solution was freeze-dried at -50℃ to -60℃ for 24-48 hours to obtain modified chitosan.

[0010] In the above steps, the primary amino group at the C2 position of the chitosan molecular chain has strong nucleophilicity. In a weakly acidic to slightly alkaline environment, the lone pair electrons on the amino group attack the carbonyl carbon of succinic anhydride, causing the succinic anhydride to open its ring. This allows the succinic anhydride to be grafted onto the chitosan molecular chain through an amide reaction, introducing a hydrophilic free carboxyl group and improving the water solubility of chitosan.

[0011] Furthermore, the preparation method of the phenylboronic acid modified sodium alginate is as follows:

[0012] Step 1: Add 3-3.1g of sodium alginate to 150-160mL of deionized water, stir to dissolve, and obtain sodium alginate solution. Then, completely dissolve 1.5-1.6g of sodium periodate in 5-6mL of deionized water to obtain sodium periodate solution. Add the sodium periodate solution dropwise to the sodium alginate solution over 15-20 minutes. After the addition is complete, react at room temperature in the dark for 24-26 hours. After the reaction is complete, add 2-2.2mL of ethylene glycol to react for 2-3 hours. After the reaction is complete, add 1-1.1g of sodium chloride and mix well. Place the solution in a 14000Da dialysis bag and dialyze with deionized water in the dark for 72-76 hours, changing the water 3-5 times a day. After dialysis, purify and freeze-dry at -50℃ to -60℃ for 24-48 hours to obtain oxidized sodium alginate.

[0013] Step 2: Add sodium oxidized alginate to phosphate buffer and stir to dissolve. Then add 3-aminophenylboronic acid and mix well. Stir and react at room temperature for 20-24 hours. After the reaction is complete, place the reaction solution in a dialysis bag with a capacity of 8000-14000 Da and dialyze with deionized water for 72-76 hours, changing the water 3-4 times a day. After dialysis, freeze-dry at -50℃ to -60℃ for 24-48 hours to obtain phenylboronic acid modified sodium alginate.

[0014] In the above steps, sodium periodate, as a highly selective oxidant, specifically oxidizes the vicinal diols at the C2 and C3 positions on the sugar ring of sodium alginate. The oxidation leads to the breakage of the C2-C3 carbon-carbon bond, the opening of the sugar ring, and the original hydroxyl group is oxidized into two active aldehyde groups to generate oxidized sodium alginate. Subsequently, the primary amino group on 3-aminophenylboronic acid acts as a nucleophile to attack the aldehyde carbon on the oxidized sodium alginate, removing one molecule of water and forming a Schiff base bond to obtain phenylboronic acid-modified sodium alginate.

[0015] Furthermore, in step two, the ratio of phosphate buffer, sodium alginate, and 3-aminophenylboronic acid is 90-100 mL: 2-2.1 g: 0.15-0.18 g.

[0016] Furthermore, the preparation method of the aldehyde-modified epoxy-thiol polymer is as follows:

[0017] S1: Add 10-12 mL of tetrahydrofuran solvent to the reactor and pre-cool it in a cold trap at -8 to -12℃ for 20-25 min. Then add 1.61-1.65 g of ethylene glycol diglycidyl ether and 44.25-44.45 mg of lithium hydroxide. After stirring evenly, add 0.94-0.98 mL of 1,3-propanedithiol and stir the reaction in an ice-water bath at 0-5℃ for 5-8 h. After the reaction is completed, add 0.48-0.5 g of ethylene glycol diglycidyl ether at -10℃ to -15℃ to quench the reaction for 1-2 h. After the reaction is completed, precipitate with ether and dissolve in dichloromethane for repeated precipitation. Centrifuge to remove the supernatant. Add the crude product to 40 mL of anhydrous ethanol, stir, centrifuge, add an appropriate amount of dichloromethane to dissolve, and rotary evaporate to obtain epoxy-thiol polymer.

[0018] S2: Under nitrogen protection, add epoxy-thiol polymer and p-aldehyde benzoic acid to N,N-dimethylformamide solvent, stir and mix, then add 4-dimethylaminopyridine and N,N-dicyclohexylcarbodiimide, stir and react at room temperature for 24-26 h. After the reaction is complete, filter, precipitate with 150 mL of diethyl ether solution, dissolve in dichloromethane, repeat precipitation, centrifuge, wash with acetone 2-3 times, rotary evaporate, and dry to obtain aldehyde-modified epoxy-thiol polymer.

[0019] In the above steps, under the action of an alkaline catalyst, thiols are deprotonated to form highly nucleophilic sulfide anions. These sulfide anions efficiently and selectively attack the end epoxy rings of the epoxy resin, resulting in ring-opening polymerization to obtain an epoxy-thiol polymer. Subsequently, N,N-dicyclohexylcarbodiimide acts as a dehydrating agent to activate the carboxyl group of p-aldehyde benzoic acid, forming a highly active O-acyl isourea intermediate. 4-Dimethylaminopyridine acts as a catalyst to accelerate acyl transfer, causing the secondary hydroxyl groups on the main chain of the epoxy-thiol polymer to undergo esterification with the activated carboxyl groups, forming ester bonds. This efficiently grafts benzaldehyde groups onto the polymer side chains, resulting in an aldehyde-modified epoxy-thiol polymer.

[0020] Further, the ratio of N,N-dimethylformamide, epoxy-thiol polymer, p-aldehyde benzoic acid, 4-dimethylaminopyridine, and N,N-dicyclohexylcarbodiimide in S2 is 12-15 mL: 3.9-4 g: 1.03-1.05 g: 0.51-0.53 g: 0.86-0.88 g.

[0021] Furthermore, the preparation method of the recombinant collagen gel is as follows:

[0022] (1) Dissolve recombinant collagen in glacial acetic acid solution with a molar concentration of 0.1 mol / L and stir until completely dissolved to obtain recombinant collagen solution. Under light-protected conditions, add recombinant collagen solution to phosphate buffer and stir at 0-5℃ for 12-16 h. Then add modified chitosan, phenylboronic acid modified sodium alginate and glycerol and stir at 60-80 r / min for 30-40 min to obtain gel precursor solution.

[0023] (2) The aldehyde-modified epoxy-thiol polymer was dissolved in dimethyl sulfoxide to prepare a stock solution with a mass concentration of 10%. The stock solution was then added dropwise to the gel precursor solution over a period of 10-15 minutes. After the addition was complete, the mixture was stirred for 5-10 minutes. The mixture was then injected into a sterile mold and placed in a constant temperature incubator at 37°C for 12-24 hours. After defoaming and sterilization, the mixture was sealed and stored at 4°C to obtain recombinant collagen gel.

[0024] Furthermore, the concentration of the recombinant collagen solution is 1-2 wt%.

[0025] Compared with the prior art, the present invention has the following beneficial technical effects:

[0026] This invention constructs a dual network structure of Schiff base covalent crosslinking and borate ester dynamic crosslinking, using recombinant collagen as a bioactive framework to provide a natural biocompatibility basis for cell adhesion and tissue repair. Modified chitosan, after modification with succinic anhydride, exhibits significantly improved water solubility, avoiding the aggregation problem of unmodified chitosan. Simultaneously, it forms a dense crosslinked network with other components, enhancing the overall structural stability of the gel. Phenylboronic acid-modified sodium alginate introduces dynamic crosslinking sites and enhances network support through its own polysaccharide segments. The aldehyde-modified epoxy-thiol polymer uses flexible thioether segments as crosslinking bridges, balancing crosslinking strength and network stability. The complex's flexibility synergistically endows the gel with excellent mechanical properties and good injectability. The phenylboronic acid groups in the phenylboronic acid-modified sodium alginate can form dynamic and reversible borate ester bonds with the vicinal diols on the polysaccharide backbone. Combined with the dynamic characteristics of Schiff bases, this endows the gel with excellent self-healing ability, broadening the gel's application scenarios and stability while extending the material's service life. Using aldehyde-modified epoxy-thiol polymers as crosslinking agents, the large molecular weight makes it difficult for the polymers to migrate or remain in the gel, avoiding the cytotoxicity and sensitization risks of traditional small molecule crosslinking agents. This results in high biosafety and compliance with medical material safety standards. Detailed Implementation

[0027] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are described clearly and completely. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0028] The reagents used in the following specific embodiments are of analytical grade. Additionally:

[0029] Recombinant collagen: Product number ProtYouth humanized type III collagen stock solution T16, manufactured by Shanxi Jinbo Biopharmaceutical Co., Ltd.

[0030] Chitosan: Deacetylation degree of 95%, viscosity of 100 mPa·s, manufactured by Shanghai Maclean Biochemical Technology Co., Ltd.

[0031] Phosphate buffer: pH 7.4, manufactured by Gibco, USA.

[0032] Example 1

[0033] (1) Add 150 mL of acetic acid solution with a volume fraction of 5% and 180 mL of ethanol solution to the reactor, mix to prepare solvent, add 1.5 g of chitosan, stir to dissolve, then add 2.25 g of succinic anhydride to 60 mL of acetone, stir to dissolve, and then add it dropwise to the reactor for 25 min. After the addition is complete, react at 25 °C for 12 h. After the reaction is complete, collect the precipitate, disperse the precipitate in ethanol solution, add sodium hydroxide solution with a molar concentration of 1.25 mol / L to adjust the pH to 9, collect the precipitate again, dissolve the obtained precipitate in deionized water, then place the solution in an 8000 Da dialysis bag and dialyze with deionized water for 48 h. After the dialysis is complete, freeze dry at -60 °C for 24 h to obtain modified chitosan.

[0034] (2) Add 3g of sodium alginate to 150mL of deionized water, stir to dissolve, and obtain sodium alginate solution. Then completely dissolve 1.5g of sodium periodate in 5mL of deionized water to obtain sodium periodate solution. Add the sodium periodate solution dropwise to the sodium alginate solution for 15min. After the addition is complete, react for 24h at room temperature in the dark. After the reaction is complete, add 2mL of ethylene glycol to react for 2h. After the reaction is complete, add 1g of sodium chloride and mix well. Place the solution in a 14000Da dialysis bag and dialyze with deionized water in the dark for 72h. Change the water 3 times a day. After the dialysis is complete, purify and freeze dry at -60℃ for 24h to obtain oxidized sodium alginate.

[0035] (3) Add 2g of oxidized sodium alginate to 90mL of phosphate buffer, stir to dissolve, then add 0.15g of 3-aminophenylboronic acid, mix well, stir and react at room temperature for 20h. After the reaction is completed, place the reaction solution in an 8000Da dialysis bag and dialyze with deionized water for 72h, changing the water 3 times a day. After the dialysis is completed, freeze dry at -60℃ for 24h to obtain phenylboronic acid modified sodium alginate.

[0036] (4) Add 10 mL of tetrahydrofuran solvent to the reactor, pre-cool it in a cold trap at -12℃ for 20 min, then add 1.61 g of ethylene glycol diglycidyl ether and 44.25 mg of lithium hydroxide, stir evenly, then add 0.94 mL of 1,3-propanedithiol, stir and react for 5 h in an ice-water bath at 0℃. After the reaction is completed, add 0.48 g of ethylene glycol diglycidyl ether at -10℃ to quench the reaction for 1 h. After the reaction is completed, precipitate with ether, dissolve in dichloromethane and repeat precipitation, centrifuge to remove the supernatant, add the crude product to 40 mL of anhydrous ethanol, stir, centrifuge, add an appropriate amount of dichloromethane to dissolve and rotary evaporate to obtain epoxy-thiol polymer;

[0037] (5) Under nitrogen protection, 3.9 g of epoxy-thiol polymer and 1.03 g of p-aldehyde benzoic acid were added to 12 mL of N,N-dimethylformamide solvent and stirred. Then, 0.51 g of 4-dimethylaminopyridine and 0.86 g of N,N-dicyclohexylcarbodiimide were added and stirred at room temperature for 24 h. After the reaction was completed, the mixture was filtered and precipitated with 150 mL of diethyl ether solution. The precipitate was dissolved in dichloromethane and the precipitation was repeated. The mixture was centrifuged and then washed twice with acetone. The mixture was then rotary evaporated and dried to obtain aldehyde-modified epoxy-thiol polymer.

[0038] (6) Dissolve 2 parts by weight of recombinant collagen in glacial acetic acid solution with a molar concentration of 0.1 mol / L, stir until completely dissolved to obtain a recombinant collagen solution with a concentration of 1 wt%. Under light-protected conditions, add the recombinant collagen solution to 35 parts by weight of phosphate buffer, stir at 0°C for 12 h, then add 1 part by weight of modified chitosan, 1 part by weight of sodium alginate modified with phenylboronic acid, and 0.2 parts by weight of glycerol, stir at 60 r / min for 30 min to obtain a gel precursor solution;

[0039] (7) 0.5 parts by weight of aldehyde-modified epoxy-thiol polymer were added to dimethyl sulfoxide and dissolved to prepare a stock solution with a mass concentration of 10%. The stock solution was then added dropwise to the gel precursor solution over a period of 10 min. After the addition was complete, the mixture was stirred for another 5 min. The mixture was then injected into a sterile mold and placed in a constant temperature incubator at 37°C for 12 h. After defoaming and sterilization, the mixture was sealed and stored at 4°C to obtain recombinant collagen gel.

[0040] Example 2

[0041] (1) Add 160 mL of acetic acid solution with a volume fraction of 5% and 190 mL of ethanol solution to the reactor, mix to prepare solvent, add 1.6 g of chitosan, stir to dissolve, then add 2.4 g of succinic anhydride to 64 mL of acetone, stir to dissolve, and then add it dropwise to the reactor for 30 min. After the addition is complete, react at 30 °C for 14 h. After the reaction is complete, collect the precipitate, disperse the precipitate in ethanol solution, add sodium hydroxide solution with a molar concentration of 1.25 mol / L to adjust the pH to 10, collect the precipitate again, dissolve the obtained precipitate in deionized water, then place the solution in a 14000 Da dialysis bag and dialyze with deionized water for 50 h. After dialysis, freeze dry at -50 °C for 48 h to obtain modified chitosan.

[0042] (2) Add 3.1g of sodium alginate to 160mL of deionized water, stir and dissolve to obtain sodium alginate solution, then completely dissolve 1.6g of sodium periodate in 6mL of deionized water to obtain sodium periodate solution, add sodium periodate solution dropwise to sodium alginate solution for 20min, after the addition is complete, react for 26h at room temperature in the dark, after the reaction is complete, add 2.2mL of ethylene glycol to react for 3h, after the reaction is complete, add 1.1g of sodium chloride, mix well, place the solution in a 14000Da dialysis bag, dialyze with deionized water in the dark for 76h, change the water 5 times a day, after dialysis, purify, freeze dry at -50℃ for 48h to obtain oxidized sodium alginate;

[0043] (3) Add 2.1g of oxidized sodium alginate to 100mL of phosphate buffer, stir to dissolve, then add 0.18g of 3-aminophenylboronic acid, mix well, stir and react at room temperature for 24h. After the reaction is completed, place the reaction solution in a 14000Da dialysis bag and dialyze with deionized water for 76h, changing the water 4 times a day. After the dialysis is completed, freeze dry at -50℃ for 48h to obtain phenylboronic acid modified sodium alginate.

[0044] (4) Add 12 mL of tetrahydrofuran solvent to the reactor, pre-cool it in a cold trap at -8℃ for 25 min, then add 1.65 g of ethylene glycol diglycidyl ether and 44.45 mg of lithium hydroxide, stir evenly, then add 0.98 mL of 1,3-propanedithiol, stir and react for 8 h in an ice-water bath at 5℃. After the reaction is completed, add 0.5 g of ethylene glycol diglycidyl ether at -15℃ to quench the reaction for 2 h. After the reaction is completed, precipitate with ether, dissolve in dichloromethane and repeat precipitation, centrifuge to remove the supernatant, add the crude product to 40 mL of anhydrous ethanol, stir, centrifuge, add an appropriate amount of dichloromethane to dissolve and rotary evaporate to obtain epoxy-thiol polymer;

[0045] (5) Under nitrogen protection, 4 g of epoxy-thiol polymer and 1.05 g of p-aldehyde benzoic acid were added to 15 mL of N,N-dimethylformamide solvent and stirred. Then, 0.53 g of 4-dimethylaminopyridine and 0.88 g of N,N-dicyclohexylcarbodiimide were added and stirred at room temperature for 26 h. After the reaction was completed, the mixture was filtered and precipitated with 150 mL of diethyl ether solution. The precipitate was dissolved in dichloromethane and the precipitation was repeated. The mixture was centrifuged and then washed three times with acetone. The mixture was then rotary evaporated and dried to obtain aldehyde-modified epoxy-thiol polymer.

[0046] (6) Dissolve 4 parts by weight of recombinant collagen in glacial acetic acid solution with a molar concentration of 0.1 mol / L, and stir until completely dissolved to obtain a recombinant collagen solution with a concentration of 2 wt%. Under light-protected conditions, add the recombinant collagen solution to 40 parts by weight of phosphate buffer, stir at 5°C for 16 h, and then add 2 parts by weight of modified chitosan, 1.5 parts by weight of phenylboronic acid modified sodium alginate and 0.3 parts by weight of glycerol. Stir at 80 r / min for 40 min to obtain a gel precursor solution.

[0047] (7) Dissolve 1 part by weight of aldehyde-modified epoxy-thiol polymer in dimethyl sulfoxide to prepare a stock solution with a mass concentration of 10%. Then add the stock solution dropwise to the gel precursor solution for 15 min. After the addition is complete, continue stirring for 10 min. Pour the mixture into a sterile mold and place it in a constant temperature incubator at 37℃ for 24 h. After defoaming and sterilization, seal and store at 4℃ to obtain recombinant collagen gel.

[0048] Example 3

[0049] (1) Add 155 mL of acetic acid solution with a volume fraction of 5% and 185 mL of ethanol solution to the reactor, mix to prepare a solvent, add 1.55 g of chitosan, stir to dissolve, then add 2.33 g of succinic anhydride to 62 mL of acetone, stir to dissolve, and then add it dropwise to the reactor for 28 min. After the addition is complete, react at 28 °C for 13 h. After the reaction is complete, collect the precipitate, disperse the precipitate in ethanol solution, add sodium hydroxide solution with a molar concentration of 1.25 mol / L to adjust the pH to 9, collect the precipitate again, dissolve the obtained precipitate in deionized water, then place the solution in a 11000 Da dialysis bag and dialyze with deionized water for 49 h. After the dialysis is complete, freeze dry at -55 °C for 36 h to obtain modified chitosan.

[0050] (2) Add 3.05g of sodium alginate to 155mL of deionized water, stir and dissolve to obtain sodium alginate solution, then completely dissolve 1.55g of sodium periodate in 5.5mL of deionized water to obtain sodium periodate solution, add sodium periodate solution dropwise to sodium alginate solution for 18min, after the addition is complete, react for 25h at room temperature in the dark, after the reaction is complete, add 2.1mL of ethylene glycol to react for 2.5h, after the reaction is complete, add 1.05g of sodium chloride, mix well, place the solution in a 14000Da dialysis bag, dialyze with deionized water in the dark for 74h, change the water 4 times a day, after dialysis, purify, freeze dry at -55℃ for 36h to obtain oxidized sodium alginate;

[0051] (3) Add 2.05 g of oxidized sodium alginate to 95 mL of phosphate buffer, stir to dissolve, then add 0.16 g of 3-aminophenylboronic acid, mix well, stir and react at room temperature for 22 h. After the reaction is completed, place the reaction solution in a 11000 Da dialysis bag and dialyze with deionized water for 74 h, changing the water 3 times a day. After the dialysis is completed, freeze dry at -55℃ for 36 h to obtain phenylboronic acid modified sodium alginate.

[0052] (4) Add 11 mL of tetrahydrofuran solvent to the reactor, pre-cool it in a cold trap at -10℃ for 22 min, then add 1.63 g of ethylene glycol diglycidyl ether and 44.35 mg of lithium hydroxide, stir evenly, then add 0.96 mL of 1,3-propanedithiol, stir and react for 6 h in an ice-water bath at 2℃. After the reaction is completed, add 0.49 g of ethylene glycol diglycidyl ether at -12℃ to quench the reaction for 1.5 h. After the reaction is completed, precipitate with ether, dissolve in dichloromethane and repeat precipitation, centrifuge to remove the supernatant, add the crude product to 40 mL of anhydrous ethanol, stir, centrifuge, add an appropriate amount of dichloromethane to dissolve and rotary evaporate to obtain epoxy-thiol polymer;

[0053] (5) Under nitrogen protection, 3.95 g of epoxy-thiol polymer and 1.04 g of p-aldehyde benzoic acid were added to 13 mL of N,N-dimethylformamide solvent and stirred. Then, 0.52 g of 4-dimethylaminopyridine and 0.87 g of N,N-dicyclohexylcarbodiimide were added and stirred at room temperature for 25 h. After the reaction was completed, the mixture was filtered and precipitated with 150 mL of diethyl ether solution. The precipitate was dissolved in dichloromethane and the precipitation was repeated. The mixture was centrifuged and then washed twice with acetone. The mixture was then rotary evaporated and dried to obtain aldehyde-modified epoxy-thiol polymer.

[0054] (6) Dissolve 3 parts by weight of recombinant collagen in glacial acetic acid solution with a molar concentration of 0.1 mol / L, stir until completely dissolved to obtain a recombinant collagen solution with a concentration of 1 wt%. Under light-protected conditions, add the recombinant collagen solution to 38 parts by weight of phosphate buffer, stir at 2°C for 14 h, then add 1.5 parts by weight of modified chitosan, 1 part by weight of sodium alginate modified with phenylboronic acid, and 0.2 parts by weight of glycerol, stir at 70 r / min for 35 min to obtain a gel precursor solution;

[0055] (7) 0.5 parts by weight of aldehyde-modified epoxy-thiol polymer were added to dimethyl sulfoxide and dissolved to prepare a stock solution with a mass concentration of 10%. The stock solution was then added dropwise to the gel precursor solution over a period of 12 min. After the addition was complete, the mixture was stirred for another 8 min. The mixture was then injected into a sterile mold and placed in a constant temperature incubator at 37°C for 18 h. After defoaming and sterilization, the mixture was sealed and stored at 4°C to obtain recombinant collagen gel.

[0056] Comparative Example 1

[0057] The main difference between this comparative example and Example 3 is that glutaraldehyde is used instead of aldehyde-modified epoxy-thiol polymer.

[0058] Comparative Example 2

[0059] The main difference between this comparative example and Example 3 is that sodium alginate is used instead of phenylboronic acid-modified sodium alginate.

[0060] Comparative Example 3

[0061] The main difference between this comparative example and Example 3 is that chitosan is used instead of modified chitosan.

[0062] Comparative Example 4

[0063] The main difference between this comparative example and Example 3 is that no aldehyde-modified epoxy-thiol polymer is added.

[0064] Performance testing

[0065] The recombinant collagen gels prepared in Examples 1-3 and Comparative Examples 1-4 were subjected to performance tests.

[0066] (1) Mechanical property testing

[0067] Compression strength: The recombinant collagen gels prepared in Examples 1-3 and Comparative Examples 1-4 were cut into cylinders with a height of 10 mm and a diameter of 10 mm. They were equilibrated in PBS buffer at pH 7.4 at room temperature for 24 h, and then placed on a general-purpose compressor at a compression rate of 1 mm·min⁻¹. -1 The sample is compressed at a certain speed until it breaks or is compressed to 50% of its original height. The compression stress-strain curve is recorded, and the maximum compression stress is taken as the compression strength. Each group is tested at least 3 times, and the average value is taken.

[0068] Pushing force: A 2 mL syringe filled with the recombinant collagen gel prepared in Examples 1-3 and Comparative Examples 1-4 was fitted with a 27G needle, fixed to a general-purpose material compressor, and the pushing speed was set to 30 mm·min. -1 Record the pushing force curve and take the maximum pushing force as the test result.

[0069] The test results are shown in Table 1.

[0070] Table 1: Mechanical Performance Tests

[0071] project Compressive strength (kPa) Pushing force (N) Example 1 128.2 5.6 Example 2 136.5 5.9 Example 3 133.9 5.8 Comparative Example 1 86.3 7.7 Comparative Example 2 73.6 8.1 Comparative Example 3 69.4 9.0 Comparative Example 4 32.5 The gel is too weak to be tested.

[0072] As can be seen from Table 1, the recombinant collagen gels prepared in Examples 1-3 have good mechanical properties.

[0073] (2) Self-healing performance test: The recombinant collagen gels prepared in Examples 1-3 and Comparative Examples 1-4 were made into cylinders with a diameter of 20 mm and a height of 5 mm, and stained with Rhodamine B. Then, the stained hydrogel and the unstained hydrogel were cut in the middle, and the cut surfaces were tightly joined together without external pressure. They were left to stand at 25°C and observed every 1 hour. The time required for complete healing of the interface was recorded. The healing judgment criteria were: if the gel does not break from the interface when gently held or stretched with tweezers and the interface is not visible to the naked eye, it is considered to be completely healed. If it does not heal within 24 hours, it is recorded as "not healed". The test results are shown in Table 2.

[0074] Table 2: Self-healing performance test

[0075] project Healing time (h) Judgment Result Example 1 7.8 Complete healing Example 2 7.1 Complete healing Example 3 7.3 Complete healing Comparative Example 1 17.8 Complete healing Comparative Example 2 ＞24 Unhealed Comparative Example 3 ＞24 Unhealed Comparative Example 4 —— The gel is too weak to be tested.

[0076] As can be seen from Table 2, the recombinant collagen gels prepared in Examples 1-3 have good self-repair properties.

[0077] (3) Biological evaluation

[0078] Referring to GB / T16886.5-2017 "Biological Evaluation of Medical Devices Part 5: In Vitro Cytotoxicity Tests", cytotoxicity experiments were performed on the recombinant collagen gels prepared in Examples 1-3 and Comparative Examples 1-4: The gel samples were added to DMEM medium containing 10% fetal bovine serum at a ratio of 0.2 g / mL, and extracted at 37°C for 24 h. The extract was collected, and L929 cells were inoculated at a rate of 1×10⁻⁶ cells / mL. 4 Cells were seeded per well in a 96-well plate and cultured for 24 hours. The original culture medium was discarded, and the above-mentioned extract was added. The cells were cultured for another 24 hours. Cell viability was determined using the CCK-8 assay. DMEM containing 10% serum was used as a negative control, and DMEM containing 5% DMSO was used as a positive control. Each group had 6 replicates, and the average value of the results was taken.

[0079] According to GB / T16886.10-2024 "Biological Evaluation of Medical Devices Part 10: Skin Sensitization Tests", the recombinant collagen gels prepared in Examples 1-3 and Comparative Examples 1-4 were subjected to skin sensitization tests. The gel samples were added to physiological saline at a ratio of 0.2 g / mL and extracted at 37°C for 24 h. The extract was collected as the test sample. Five albino guinea pigs were selected for each group, and five negative controls (physiological saline) were set up. During the induction period, the test sample was applied to the back for 48 h. After 14 days, the test sample was applied to the ventral side for 24 h. The skin reaction was observed at 24 h and 48 h after the stimulation. The skin reaction was recorded according to the Magnusson and Kligman grading system: Grade 0 (no reaction), Grade 1 (scattered erythema, no redness and swelling), Grade 2 (obvious erythema, slight redness and swelling), Grade 3 (severe erythema, moderate edema), Grade 4 (severe erythema, severe edema / vesicles).

[0080] The test results are shown in Table 3.

[0081] Table 3: Biological Evaluation

[0082] project Cell viability (%) 24-hour excitation results 48h excitation results Example 1 96.8 Level 0 Level 0 Example 2 98.6 Level 0 Level 0 Example 3 97.3 Level 0 Level 0 Comparative Example 1 82.3 Level 1 Level 1 Comparative Example 2 94.2 Level 0 Level 0 Comparative Example 3 88.5 Level 0 Level 0 Comparative Example 4 95.1 Unable to form Unable to form

[0083] As can be seen from Table 3, the recombinant collagen gels prepared in Examples 1-3 have good biological properties.

[0084] The comparison shows that in Comparative Example 1, glutaraldehyde was used instead of aldehyde-modified epoxy-thiol polymer. The resulting crosslinked gel network was short and lacked elasticity. The aldehyde-modified epoxy-thiol polymer contains flexible thioether chains that can dissipate stress under pressure. After replacement, the strength decreased significantly. Glutaraldehyde forms irreversible covalent bonds with amino groups, and repair can only be achieved through weak molecular chain entanglement, leading to a significant reduction in healing efficiency. Furthermore, glutaraldehyde itself has well-known cytotoxicity and tissue irritation. Its unreacted residual monomers can significantly reduce cell survival and induce sensitization. In Comparative Example 2, sodium alginate was used instead of phenylboronic acid-modified sodium alginate. The absence of phenylboronic acid groups resulted in the loss of dynamically reversible borate ester bonds within the gel. Relying solely on the weak electrostatic interaction between carboxyl groups and cations, effective dynamic crosslinking could not be formed. In Comparative Example 3, the network was loose, and the overall double cross-linked network structure collapsed into a single network, resulting in a decrease in performance. In Comparative Example 4, chitosan was used instead of modified chitosan. Unmodified chitosan had poor solubility in phosphate buffer at pH 7.4, leading to severe macroscopic phase separation and precipitation. These precipitated insoluble particle clusters disrupted the uniformity of the gel network, resulting in a decrease in performance. In Comparative Example 4, no aldehyde-modified epoxy-thiol polymer was added. The recombinant collagen, modified chitosan, and sodium alginate in the system could only be barely maintained by electrostatic attraction and hydrogen bonds, failing to construct a stable three-dimensional network and making effective testing impossible. However, the recombinant collagen, sodium alginate, and other raw materials in the system were non-toxic. The gel was ineffective only due to the lack of a cross-linking agent, and the biological properties were not affected.

[0085] It should be noted that, in this document, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0086] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

[0087] Those skilled in the art should understand that the above descriptions are merely several specific embodiments of the present invention, and not all embodiments.

Claims

1. A recombinant collagen gel, characterized in that, It includes the following components by weight: 2-4 parts by weight of recombinant collagen, 1-2 parts by weight of modified chitosan, 1-1.5 parts by weight of sodium alginate modified with phenylboronic acid, 0.5-1 parts by weight of aldehyde-modified epoxy-thiol polymer, 35-40 parts by weight of phosphate buffer, and 0.2-0.3 parts by weight of glycerol. The phenylboronic acid-modified sodium alginate is obtained by reacting oxidized sodium alginate with 3-aminophenylboronic acid; The aldehyde-based epoxy-thiol polymer is obtained by reacting ethylene glycol diglycidyl ether with 1,3-propanedithiol to obtain the epoxy-thiol polymer, and then reacting it with p-aldehyde benzoic acid.

2. The recombinant collagen gel according to claim 1, characterized in that, The modified chitosan is prepared as follows: 150-160 mL of a 5% (v / v) acetic acid solution and 180-190 mL of an ethanol solution are added to a reactor to prepare a solvent. 1.5-1.6 g of chitosan is added and stirred to dissolve. Then, 2.25-2.4 g of succinic anhydride is added to 60-64 mL of acetone and stirred to dissolve. This solution is then added dropwise to the reactor over 25-30 minutes. After the addition is complete, the reactor is cooled at 25-30°C. After 12-14 hours of reaction, collect the precipitate and disperse it in an ethanol solution. Adjust the pH to 9-10 by adding a 1.25 mol / L sodium hydroxide solution. Collect the precipitate again and dissolve it in deionized water. Then, place the solution in a dialysis bag with a capacity of 8000-14000 Da and dialyze it with deionized water for 48-50 hours. After dialysis, freeze-dry the solution at -50℃ to -60℃ for 24-48 hours to obtain modified chitosan.

3. The recombinant collagen gel according to claim 1, characterized in that, The preparation method of the phenylboronic acid modified sodium alginate is as follows: Step 1: Add 3-3.1g of sodium alginate to 150-160mL of deionized water, stir to dissolve, and obtain sodium alginate solution. Then, completely dissolve 1.5-1.6g of sodium periodate in 5-6mL of deionized water to obtain sodium periodate solution. Add the sodium periodate solution dropwise to the sodium alginate solution over 15-20 minutes. After the addition is complete, react at room temperature in the dark for 24-26 hours. After the reaction is complete, add 2-2.2mL of ethylene glycol to react for 2-3 hours. After the reaction is complete, add 1-1.1g of sodium chloride and mix well. Place the solution in a 14000Da dialysis bag and dialyze with deionized water in the dark for 72-76 hours, changing the water 3-5 times a day. After dialysis, purify and freeze-dry at -50℃ to -60℃ for 24-48 hours to obtain oxidized sodium alginate. Step 2: Add sodium oxidized alginate to phosphate buffer and stir to dissolve. Then add 3-aminophenylboronic acid and mix well. Stir and react at room temperature for 20-24 hours. After the reaction is complete, place the reaction solution in a dialysis bag with a capacity of 8000-14000 Da and dialyze with deionized water for 72-76 hours, changing the water 3-4 times a day. After dialysis, freeze-dry at -50℃ to -60℃ for 24-48 hours to obtain phenylboronic acid modified sodium alginate.

4. The recombinant collagen gel according to claim 3, characterized in that, In step two, the ratio of phosphate buffer, sodium alginate, and 3-aminophenylboronic acid is 90-100 mL: 2-2.1 g: 0.15-0.18 g.

5. The recombinant collagen gel according to claim 1, characterized in that, The preparation method of the aldehyde-based epoxy-thiol polymer is as follows: S1: Add 10-12 mL of tetrahydrofuran solvent to the reactor and pre-cool it in a cold trap at -8 to -12℃ for 20-25 min. Then add 1.61-1.65 g of ethylene glycol diglycidyl ether and 44.25-44.45 mg of lithium hydroxide. After stirring evenly, add 0.94-0.98 mL of 1,3-propanedithiol and stir the reaction in an ice-water bath at 0-5℃ for 5-8 h. After the reaction is completed, add 0.48-0.5 g of ethylene glycol diglycidyl ether at -10℃ to -15℃ to quench the reaction for 1-2 h. After the reaction is completed, precipitate with ether and dissolve in dichloromethane for repeated precipitation. Centrifuge to remove the supernatant. Add the crude product to 40 mL of anhydrous ethanol, stir, centrifuge, add an appropriate amount of dichloromethane to dissolve, and rotary evaporate to obtain epoxy-thiol polymer. S2: Under nitrogen protection, add epoxy-thiol polymer and p-aldehyde benzoic acid to N,N-dimethylformamide solvent, stir and mix, then add 4-dimethylaminopyridine and N,N-dicyclohexylcarbodiimide, stir and react at room temperature for 24-26 h. After the reaction is complete, filter, precipitate with 150 mL of diethyl ether solution, dissolve in dichloromethane, repeat precipitation, centrifuge, wash with acetone 2-3 times, rotary evaporate, and dry to obtain aldehyde-modified epoxy-thiol polymer.

6. The recombinant collagen gel according to claim 5, characterized in that, The ratio of N,N-dimethylformamide, epoxy-thiol polymer, p-aldehyde benzoic acid, 4-dimethylaminopyridine, and N,N-dicyclohexylcarbodiimide in S2 is 12-15 mL: 3.9-4 g: 1.03-1.05 g: 0.51-0.53 g: 0.86-0.88 g.

7. A method for preparing recombinant collagen gel as described in any one of claims 1-6, characterized in that, The preparation method of the recombinant collagen gel is as follows: (1) Dissolve recombinant collagen in glacial acetic acid solution with a molar concentration of 0.1 mol / L and stir until completely dissolved to obtain recombinant collagen solution. Under light-protected conditions, add recombinant collagen solution to phosphate buffer and stir at 0-5℃ for 12-16 h. Then add modified chitosan, phenylboronic acid modified sodium alginate and glycerol and stir at 60-80 r / min for 30-40 min to obtain gel precursor solution. (2) The aldehyde-modified epoxy-thiol polymer was dissolved in dimethyl sulfoxide to prepare a stock solution with a mass concentration of 10%. The stock solution was then added dropwise to the gel precursor solution over a period of 10-15 minutes. After the addition was complete, the mixture was stirred for 5-10 minutes. The mixture was then injected into a sterile mold and placed in a constant temperature incubator at 37°C for 12-24 hours. After defoaming and sterilization, the mixture was sealed and stored at 4°C to obtain recombinant collagen gel.

8. The method for preparing recombinant collagen gel according to claim 7, characterized in that, The concentration of the recombinant collagen solution is 1-2 wt%.