Preparation method of modified collagen hydrogel
By combining collagen with oxidized methyl cellulose to form reversible imine bonds, the problems of low mechanical properties and biocompatibility of collagen hydrogels are solved, and higher mechanical properties and self-healing properties are achieved. It is suitable for Wound heals and prevents bacterial penetration.
Patent Information
- Application Number
- CN202411606197.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-12
- Publication Date
- 2025-02-07
AI Technical Summary
The existing collagen hydrogels have low mechanical properties in wound treatment, are prone to diffusion and inactivate, and cannot achieve ideal therapeutic effects. The crosslinking agents used in chemical crosslinking will cause tissue calcification and have low biocompatibility.
By combining collagen with oxidized methylcellulose (OMC), the aldehyde group of OMC forms a reversible imine bond with the amino group in collagen, dynamic crosslinking is achieved, and the mechanical properties and self-healing properties of the hydrogel are improved.
It improves the mechanical properties and self-healing properties of collagen hydrogels, avoids calcification problems caused by crosslinking agents, maintains good biocompatibility, and is suitable for wound healing and prevents bacterial penetration.
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Figure CN119391008A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of biomedicine, and in particular to a method for preparing a modified collagen hydrogel. Background Art
[0002] Bacteria can penetrate into the subcutaneous tissue after skin surgery or injury, which can not only cause skin infection, but also be life-threatening in severe cases. At present, the use of dressings with good antibacterial properties to cover the wound can not only prevent bacterial penetration and bacterial growth, but also effectively prevent cross-infection of the wound, providing an ideal environment for wound healing. Compared with traditional medical dressings, collagen hydrogel has high coverage and good absorbency, can insulate, maintain the moisture environment around the wound, and is suitable for postoperative wound hemostasis and promotion of wound healing. It is also light in dry state and easy to transport. However, the inherent low mechanical properties of existing commercial products during wound treatment make them rapidly diffuse and inactivate, and cannot achieve the ideal therapeutic effect. In order to improve the integrity and stability of the collagen structure, it is usually necessary to cross-link it to a certain extent, among which physical cross-linking can improve its mechanical properties to a certain extent, but the improvement effect is not significant; while the cross-linking agent used in chemical cross-linking will cause a certain degree of calcification of the tissue, resulting in low biocompatibility of the product; in addition, collagen hydrogel can be compounded with other materials to improve its mechanical properties. Methylcellulose (MC) is a water-soluble polymer derived from cellulose. It is made by introducing methyl groups into cellulose through an etherification reaction. It has good biocompatibility and is widely used in the field of pharmaceutical excipients, such as oral and local injection preparations. However, due to weak physical bonding, MC hydrogels have the disadvantage of low mechanical strength and are easily destroyed during application. Therefore, it is urgent to develop a preparation method for modified collagen hydrogels to solve the above technical problems.
[0003] In view of this, the present invention is proposed. Summary of the invention
[0004] The purpose of the present invention is to provide a method for preparing a modified collagen hydrogel. Collagen sponge obtained from animal tissue is dispersed in water for injection containing additives to obtain a collagen intermediate. The ortho-hydroxy structure of a cellulose derivative is introduced into an aldehyde group to obtain oxidized methyl cellulose (OMC). OMC can improve the mechanical properties of the filling material and establish a reversible intermolecular crosslink with collagen. The composite material composed of OMC and collagen is rapidly crosslinked through a dynamic Schiff base bond under physiological conditions, has excellent self-healing properties and a higher elastic modulus, is an ideal hydrogel material after surgery, has broad application prospects, and is conducive to popularization and application.
[0005] In order to achieve the above object, the present invention provides a method for preparing a modified collagen hydrogel, comprising the following steps: S1: Soak the bovine Achilles tendon in 1M sodium hydroxide solution for 60 minutes, wash it with purified water several times and then crush it, add pepsin and stir it for 72 hours to obtain a crude extract, salt it out with 5℃ sodium chloride solution overnight, remove the supernatant to obtain collagen precipitate, dialyze to remove impurities, and crush it after freeze-drying; S2: dispersing the crushed product in water for injection containing additives, and homogenizing under high pressure to obtain a collagen intermediate; S3: adding sodium periodate solution to the methyl cellulose solution at room temperature and in the dark, adding ethylene glycol to neutralize the excess sodium periodate after the reaction, dialyzing with purified water, and freeze-drying to obtain white sponge-like oxidized methyl cellulose; S4: Pipette the collagen intermediate and oxidized methyl cellulose into a beaker, add water for injection and stir at a rate of 20 rpm until cross-linked, pour into a mold and place at 4°C for 24 hours, and prepare the modified collagen hydrogel by irradiation sterilization.
[0006] Preferably, in S2, the mass ratio of the additive composition in the water for injection is carbomer: mannitol: alginate = 1:3:4, and the mass ratio of the additive is the mass percentage of the final collagen powder.
[0007] Preferably, in S3, the mass fraction of the methyl cellulose solution is 1%, the concentration of the sodium periodate solution is 1.5%, the reaction time is 5 hours, the product is placed in a dialysis bag MWCO 3500 and dialyzed for 3 days, and the aldehyde content of the finally obtained oxidized methyl cellulose is 17.5%±0.12%.
[0008] Preferably, in S4, the concentration ratio of the transferred collagen intermediate and oxidized methyl cellulose is 1:2-3, and as the amount of oxidized methyl cellulose added increases, the self-healing time of the modified collagen hydrogel gradually decreases.
[0009] Preferably, in S4, the modified collagen hydrogel is sterilized using cobalt 60 at a radiation dose of 25 KGy.
[0010] The present invention provides a method for preparing a modified collagen hydrogel, which has the following beneficial effects: 1. The present invention prepares a soluble collagen intermediate by processing animal tissue, and uses the soluble collagen intermediate and oxidized methyl cellulose as raw materials to prepare a modified collagen gel after a cross-linking reaction. The preparation method of the present invention is simple, the cross-linking reaction reduces the enzymatic hydrolysis rate and avoids the use of a cross-linking agent, and does not cause the cytotoxicity caused by the modification of a general chemical cross-linking agent, and maintains the good biocompatibility of the collagen hydrogel, which provides a basis for the application of modified collagen in the field of dressing materials.
[0011] 2. The present invention introduces the Schiff base reaction into the dynamic hydrogel, and it occurs in an aqueous solution at room temperature or physiological temperature. The dissolution process does not require adjustment of the acidity and alkalinity of the solution, thus avoiding toxic hazards during the operation.
[0012] 3. The reaction of the aldehyde group and the amino group to form a Schiff base (imine) bond in the present invention allows dynamic equilibrium, and the internal cross-linking of the hydrogel has dynamic equilibrium, which gives the hydrogel excellent self-healing properties and a higher elastic modulus.
[0013] 4. The oxidized methylcellulose of the present invention enhances the mechanical properties of the collagen hydrogel and can effectively prevent the structural collapse after water absorption. As the amount of oxidized methylcellulose added increases, the self-healing time of the modified collagen hydrogel gradually decreases. BRIEF DESCRIPTION OF THE DRAWINGS
[0014] Figure 1 is the infrared spectrum of the modified collagen hydrogel; Figure 2 Macroscopic characterization of the self-healing behavior of modified collagen hydrogels. DETAILED DESCRIPTION
[0015] The present invention is further described below in conjunction with specific embodiments and drawings to help understand the content of the present invention.
[0016] The present invention provides a method for preparing a modified collagen hydrogel, comprising the following steps: S1: Soak the bovine Achilles tendon in 1M sodium hydroxide solution for 60 minutes, wash it with purified water several times and then crush it, add pepsin and stir it for 72 hours to obtain a crude extract, salt it out with 5℃ sodium chloride solution overnight, remove the supernatant to obtain collagen precipitate, dialyze to remove impurities, and crush it after freeze-drying; S2: The crushed product is dispersed in water for injection containing additives, and a collagen intermediate is obtained after high-pressure homogenization; the mass ratio of the additive composition in the water for injection is carbomer: mannitol: alginate = 1:3:4, and the mass ratio of the additive is the mass percentage of the final collagen crushed product.
[0017] S3: At room temperature and in the dark, add a 1.5% sodium periodate solution to a 1% methyl cellulose solution. After reacting for 5 hours, add ethylene glycol to neutralize the excess sodium periodate. Place the product in a dialysis bag MWCO 3500 for dialyzation for 3 days. After freeze-drying, a white sponge-like oxidized methyl cellulose is obtained. The aldehyde content of the final oxidized methyl cellulose is 17.5%±0.12%.
[0018] S4: Pipette the collagen intermediate and oxidized methyl cellulose with a concentration ratio of 1:2-3 into a beaker, add water for injection and stir at a rate of 20rpm until cross-linking, pour into a mold and place at 4°C for 24 hours to reduce the interference of bubbles in the product and ensure the mechanical properties. The modified collagen hydrogel was prepared by irradiation sterilization. The modified collagen hydrogel was sterilized with cobalt 60 at a irradiation dose of 25KGy. With the increase of the amount of oxidized methyl cellulose added, the self-healing time of the modified collagen hydrogel gradually decreased. Example 1
[0019] The bovine Achilles tendon was soaked in 1 M sodium hydroxide solution for 60 min, washed several times with purified water and then crushed, enzymatically hydrolyzed by adding pepsin and stirring for 72 hours to obtain a crude extract, which was then salted out with 5°C low-temperature sodium chloride solution overnight, and the supernatant was removed to obtain collagen precipitate. After dialysis, impurities were removed to obtain a collagen intermediate, which was freeze-dried and crushed; The crushed product is dispersed in water for injection containing additives, wherein the mass ratio of the additives is carbomer: mannitol: alginate = 1:3:4, and the mass ratio of the additives is the mass percentage of the final collagen crushed product; A 1.5% concentration of sodium periodate was added to a 1% mass fraction methylcellulose solution at room temperature and in the dark. After 5 hours of reaction, a certain amount of ethylene glycol was added to neutralize the excess periodate. The product was dialyzed in purified water for 3 days and freeze-dried to obtain a white sponge-like oxidized methylcellulose, the aldehyde content of which was measured to be about 17.5%.
[0020] Collagen intermediate and oxidized methylcellulose with a concentration ratio of 1:2 were transferred into a beaker, purified water was added and stirred at a rate of 20 rpm until cross-linked, poured into a mold and placed at low temperature for 24 h, and sterilized with cobalt 60 at a radiation dose of 25 KGy to prepare a modified collagen hydrogel.
[0021] The chemical structure was analyzed by FTIR to confirm the formation of imine bonds. Oxidized methylcellulose (OMC) with aldehyde groups reacts with free amino groups on collagen chains through Schiff bases to induce the formation of reversible imine bonds, such as Figure 1 The following is the infrared spectrum of the modified collagen hydrogel. After adding collagen, the vicinal diol structure of MC is oxidized to two aldehyde groups to form OMC at 1730 cm -1 The aldehyde absorption peak at 1685 cm -1 The absorption peak at became stronger, confirming that a Schiff base reaction occurred between the aldehyde group on OMC and the amino group on collagen to form a Schiff base (imine) bond, and the modified collagen hydrogel was successfully prepared.
[0022] like Figure 2The figure shows the macroscopic characterization of the self-healing behavior of the modified collagen hydrogel. At 37 °C, two hydrogel fragments stained with rhodamine B and methylene blue were bonded together. After incubation for about 30 minutes, the colors of the two hydrogels diffused and they were successfully combined to form a whole. In addition, when the repaired hydrogel was manually stretched, it did not break from the incision, confirming the self-healing ability of the modified collagen hydrogel. Example 2
[0023] The collagen intermediate was prepared according to the method of Example 1.
[0024] Oxidized methyl cellulose was prepared according to the method of Example 1.
[0025] Collagen intermediate and oxidized methylcellulose with a concentration ratio of 1:2.5 were transferred into a beaker, purified water was added and stirred at a rate of 20 rpm until cross-linked, poured into a mold and placed at 4°C for 24 h, and irradiated with cobalt 60 at a dose of 25 KGy to prepare a modified collagen hydrogel. The same test method showed that the hydrogel with a damaged morphology could achieve self-healing in about 26 minutes. Example 3
[0026] The collagen intermediate was prepared according to the method of Example 1.
[0027] Oxidized methyl cellulose was prepared according to the method of Example 1.
[0028] Collagen intermediate and oxidized methylcellulose with a concentration ratio of 1:3 were transferred into a beaker, purified water was added and stirred at a rate of 20 rpm until cross-linked, poured into a mold and placed at 4°C for 24 h, and irradiated with cobalt 60 at a dose of 25 KGy to prepare a modified collagen hydrogel. The same test method showed that the hydrogel with a damaged morphology could achieve self-healing in about 20 minutes.
[0029] The self-healing abilities of the hydrogels with different ratios in Examples 1-3 are shown in Table 1.
[0030] Table 1 Self-healing ability of hydrogels with different ratios
[0031] It can be seen from Table 1 that with the increase of the amount of oxidized methylcellulose added, the self-healing time of the modified collagen hydrogel gradually decreases.
[0032] The invention concept is described in detail using specific examples herein, and the description of the above embodiments is only used to help understand the core idea of the invention. It should be pointed out that any obvious modification, equivalent substitution or other improvement made by a person of ordinary skill in the art without departing from the invention concept should be included in the protection scope of the present invention.
Claims
1. A method for preparing a modified collagen hydrogel, characterized in that: The steps include: S1: Soak the bovine Achilles tendon in 1M sodium hydroxide solution for 60 minutes, wash it with purified water several times and then crush it, add pepsin and stir it for 72 hours to obtain a crude extract, salt it out with 5℃ sodium chloride solution overnight, remove the supernatant to obtain collagen precipitate, dialyze to remove impurities, and crush it after freeze-drying; S2: dispersing the crushed product in water for injection containing additives, and homogenizing under high pressure to obtain a collagen intermediate; S3: adding sodium periodate solution to the methyl cellulose solution at room temperature and in the dark, adding ethylene glycol to neutralize the excess sodium periodate after the reaction, dialyzing with purified water, and freeze-drying to obtain white sponge-like oxidized methyl cellulose; S4: Pipette the collagen intermediate and oxidized methyl cellulose into a beaker, add water for injection and stir at a rate of 20 rpm until cross-linked, pour into a mold and place at 4°C for 24 hours, and prepare the modified collagen hydrogel by irradiation sterilization.
2. The method for preparing a modified collagen hydrogel according to claim 1, characterized in that: In S2, the mass ratio of the additive composition in the water for injection is carbomer: mannitol: alginate = 1:3:4, and the mass ratio of the additive is the mass percentage of the final collagen powder.
3. The method for preparing a modified collagen hydrogel according to claim 1, characterized in that: In the S3, the mass fraction of the methyl cellulose solution is 1%, the concentration of the sodium periodate solution is 1.5%, the reaction time is 5 hours, and the product is placed in a dialysis bag MWCO 3500 for dialysing for 3 days. The aldehyde content of the finally obtained oxidized methyl cellulose is 17.5%±0.12%.
4. The method for preparing a modified collagen hydrogel according to claim 1, characterized in that: In the S4, the concentration ratio of the transferred collagen intermediate and oxidized methyl cellulose is 1:2-3. As the amount of oxidized methyl cellulose added increases, the self-healing time of the modified collagen hydrogel gradually decreases.
5. The method for preparing a modified collagen hydrogel according to claim 1, characterized in that: In S4, the modified collagen hydrogel is sterilized by irradiation with cobalt 60 at a dose of 25 KGy.