A decellularized matrix composite hydrogel dressing for acute and chronic wound healing and a method of preparation thereof
Patent Information
- Application Number
- CN202611137826.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-07-29
- Publication Date
- 2026-08-28
AI Technical Summary
[0003]构建水凝胶敷料的材料有很多，包括聚乙烯醇，聚乙二醇，壳聚糖，透明质酸以及纤维素等，但这些材料分子结构单一，很难模拟天然皮肤组织的结构与功能；脱细胞基质材料（decellularized extracellular matrix, dECM）是指通过物理、化学或生物方法去除天然组织或器官中的细胞成分和免疫原性物质，同时完整保留其细胞外基质（ECM）的三维结构及关键生物活性成分（如胶原蛋白、弹性蛋白、蛋白聚糖、糖蛋白和多种生长因子）的一种天然生物材料，凭借其仿生的三维结构、优异的生物相容性和主动诱导组织再生的能力，在促进慢性伤口愈合方面具有巨大的优势与潜力；但脱细胞基质材料制备的凝胶材料，一般力学性能较弱，同时缺乏抗菌，抗炎等生物学功能，因此，将dECM材料和其他功能高分子材料以及纳米材料结合，各取所长，有望制备一种理想的凝胶敷料
本发明实施例提供的复合水凝胶敷料，以猪脾脏来源的脱细胞基质，透明质酸以及羧甲基壳聚糖为原料，通过化学修饰功能基团，并通过分子间的迈克尔加成与二硫键交联，构建凝胶本体，即将天然生物材料与高分子材料相结合，兼具二者优势，具有良好的粘弹性和快速松弛性能，有望成为理想的伤口敷料产品。
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Figure CN122643501A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of hydrogels, and more specifically, to a decellularized matrix composite hydrogel dressing for acute and chronic wound healing and its preparation method. Background Technology
[0002] Diabetic chronic wounds are one of the most common complications of diabetes, imposing a heavy economic burden on patients. Hydrogels are polymeric materials that are natural or synthetic polymers cross-linked by physical or chemical means and contain a certain amount of water. They have been extensively studied due to their similarity to the extracellular matrix, good biocompatibility, strong exudate absorption capacity, and ability to maintain a moist microenvironment that promotes healing. They can be used in tissue engineering, drug carriers, medical dressings, and cell culture.
[0003] Many materials can be used to construct hydrogel dressings, including polyvinyl alcohol, polyethylene glycol, chitosan, hyaluronic acid, and cellulose. However, these materials have simple molecular structures and are difficult to mimic the structure and function of natural skin tissue. Decellularized extracellular matrix (dECM) refers to a natural biomaterial that removes cellular components and immunogenic substances from natural tissues or organs through physical, chemical, or biological methods, while completely preserving the three-dimensional structure of the extracellular matrix (ECM) and key bioactive components (such as collagen, elastin, proteoglycans, glycoproteins, and various growth factors). With its biomimetic three-dimensional structure, excellent biocompatibility, and ability to actively induce tissue regeneration, it has great advantages and potential in promoting chronic wound healing. However, gel materials prepared from decellularized matrix materials generally have weak mechanical properties and lack antibacterial and anti-inflammatory biological functions. Therefore, combining dECM materials with other functional polymers and nanomaterials, leveraging their respective strengths, holds promise for preparing an ideal gel dressing. Summary of the Invention
[0004] The purpose of this invention is to provide a decellularized matrix composite hydrogel dressing for acute and chronic wound healing. The decellularized matrix composite hydrogel dressing is prepared by chemical modification and cross-linking methods. The hydrogel has good viscoelasticity and rapid relaxation properties.
[0005] Another objective of this invention is to provide a method for preparing a decellularized matrix composite hydrogel dressing for acute and chronic wound healing. The method uses a decellularized matrix derived from porcine spleen, hyaluronic acid, and carboxymethyl chitosan as raw materials. By chemically modifying functional groups and cross-linking with disulfide bonds between molecules, a gel matrix is constructed. This method combines natural biomaterials with polymer materials, possessing the advantages of both, and can serve as an ideal wound dressing product.
[0006] The technical problem solved by this invention is achieved by the following technical solution.
[0007] On one hand, embodiments of the present invention provide a method for preparing a decellularized matrix composite hydrogel dressing for acute and chronic wound healing, comprising the following steps: S1, decellularized matrix of pig spleen was prepared into a hydrogel solution, methacrylic anhydride was added dropwise at room temperature to adjust the pH to between 9 and 10, and the mixture was stirred overnight at 4°C. After the reaction was completed, the pH was adjusted to physiological pH, and the mixture was dialyzed at 4°C for 4 days using a dialysis bag. The solution was then freeze-dried to obtain PECM-MA.
[0008] S2, hyaluronic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and N-hydroxysuccinimide were dissolved in MES buffer solution and stirred to form solution one; L-cysteine was dissolved in MES buffer solution and stirred to form solution two, and activated for 20-60 min; solution two was added to solution one, and the reaction was carried out at room temperature for 12-24 h. The reaction solution was dialyzed for 3 days and lyophilized to obtain the product HA-SH; S3. Take PECM-MA, HA-SH, and carboxymethyl chitosan, add them to deionized water, dissolve and stir evenly to form a hydrogel.
[0009] In some embodiments of the present invention, in step S1, the volume ratio of methacrylic anhydride to decellularized matrix hydrogel solution is (5-15) μL:1mL.
[0010] In some embodiments of the present invention, in step S2, the mass ratio of hyaluronic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and N-hydroxysuccinimide is 10-15:8:5.
[0011] In some embodiments of the present invention, in step S2, in solution one, the concentrations of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide are both 1-10 mg / mL, and the concentration of hyaluronic acid is 0.001-0.02 g / mL.
[0012] In some embodiments of the present invention, in step S2, the concentration of L-cysteine in solution two is 1-100 mg / mL.
[0013] In some embodiments of the present invention, in step S2, the volume ratio of solution one to solution two is 10-20:1.
[0014] In some embodiments of the present invention, in step S2, the concentration of the MES buffer solution is 0.01-0.2 M; the pH is between 5 and 7.
[0015] In some embodiments of the present invention, in step S3, by mass percentage: PECM-MA 3-5%, HA-SH 2-8%, carboxymethyl chitosan 4-8%, and the balance being water.
[0016] In some embodiments of the present invention, in step S3, by mass percentage: PECM-MA 3%, HA-SH 4%, carboxymethyl chitosan 4%, and the balance being water.
[0017] On the other hand, embodiments of the present invention provide a decellularized matrix composite hydrogel dressing for acute and chronic wound healing, which is prepared by the above-described preparation method.
[0018] Compared with the prior art, the embodiments of the present invention have at least the following advantages or beneficial effects: The composite hydrogel dressing provided in this invention uses decellularized matrix derived from porcine spleen, hyaluronic acid, and carboxymethyl chitosan as raw materials. By chemically modifying functional groups and cross-linking with disulfide bonds between molecules, a gel matrix is constructed. This combines natural biomaterials with polymer materials, possessing the advantages of both. It exhibits good viscoelasticity and rapid relaxation properties, and is expected to become an ideal wound dressing product. Attached Figure Description
[0019] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.
[0020] Figure 1 This is a physical example of the hydrogel PHC-2 from an embodiment of the present invention. Figure 1 ; Figure 2 This is a physical example of the hydrogel PHC-2 from an embodiment of the present invention. Figure 2 ; Figure 3 This is a test of the stress relaxation performance of hydrogels in an embodiment of the present invention; Figure 4 Hydrogel degradation performance test of embodiments of the present invention; Figure 5 This is an example of a hydrogel cell live / dead staining experiment according to an embodiment of the present invention; Figure 6 This invention provides a rat skin wound repair model using hydrogel PHC-2 as an example. Figure 7 This is a rat skin wound repair model used as a control group in this embodiment of the invention. Detailed Implementation
[0021] 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 will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.
[0022] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other. The present invention will now be described in detail with reference to specific embodiments.
[0023] On one hand, embodiments of the present invention provide a method for preparing a decellularized matrix composite hydrogel dressing for acute and chronic wound healing, comprising the following steps: S1, take the decellularized matrix of pig spleen and prepare it into a hydrogel solution. Add methacrylic anhydride dropwise at room temperature to adjust the pH to between 9 and 10. Stir overnight at 4°C. After the reaction is complete, adjust the pH to physiological pH. Dialyze the solution in a dialysis bag at 4°C for 4 days. Freeze-dry the solution to obtain PECM-MA. S2, hyaluronic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and N-hydroxysuccinimide were dissolved in MES buffer solution and stirred to form solution one; L-cysteine was dissolved in MES buffer solution and stirred to form solution two, and the reaction was activated for 20-60 min to form hyaluronic acid as an intermediate for subsequent reaction with L-cysteine; solution two was added to solution one, and the reaction was carried out at room temperature for 12-24 h. The reaction solution was dialyzed for 3 days and lyophilized to obtain the product HA-SH; S3. Take PECM-MA, HA-SH, and carboxymethyl chitosan, add them to deionized water, dissolve and stir evenly to form a hydrogel.
[0024] When used, the decellularized matrix in the hydrogel provides cell adhesion sites, promotes cell migration and proliferation, and also promotes angiogenesis and reepithelialization; thiolated hyaluronic acid has moisturizing, anti-inflammatory and wound-healing effects; carboxymethyl chitosan has good antibacterial and adhesive properties, and also participates in regulating the mechanical properties of the gel.
[0025] In step S1, the volume ratio of methacrylic anhydride to decellularized matrix hydrogel is 5-15:1.
[0026] In step S2, the mass ratio of hyaluronic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and N-hydroxysuccinimide is 10-15:8:5.
[0027] In solution one, the concentrations of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide are both 1-10 mg / mL, and the concentration of hyaluronic acid is 0.001-0.02 g / mL. In step S2, the concentration of L-cysteine in solution two is 1-100 mg / mL; In step S2, the volume ratio of solution one to solution two is 10-20:1.
[0028] In step S2, the concentration of the MES buffer solution is 0.01-0.2 M; the pH is between 5 and 7.
[0029] In step S3, by mass percentage: PECM-MA 3-5%, HA-SH 2-8%, carboxymethyl chitosan 4-8%, and the balance is water.
[0030] The preferred composition, by mass percentage, is: PECM-MA 3%, HA-SH 4%, carboxymethyl chitosan 4%, with the remainder being water.
[0031] The features and performance of the present invention will be further described in detail below with reference to embodiments.
[0032] Example 1 Prepare the hydrogel using the following steps: S1. Porcine spleen decellularized matrix was prepared into a hydrogel solution (the concentration of porcine spleen decellularized matrix was 10 mg / mL). Methacrylic anhydride was slowly added dropwise at a volume ratio of 15 (µL):1 (mL) to the decellularized matrix hydrogel solution at room temperature. The pH was adjusted to 9-10 with sodium hydroxide solution. The mixture was stirred overnight (12 h) at 4 °C. After the reaction was completed, the mixture was neutralized with dilute hydrochloric acid to adjust the pH to physiological pH. The mixture was dialyzed at 4 °C for 4 days using a dialysis bag with a molecular weight cutoff of 8000 Da. The solution was then freeze-dried to obtain a white spongy product, PECM-MA.
[0033] S2, hyaluronic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and N-hydroxysuccinimide were dissolved in MES buffer solution (pH=5.5) and stirred to form solution one; L-cysteine was dissolved in MES buffer solution of the same concentration and stirred to form solution two, and activated for 20-60 min; solution two was added to solution one at a volume ratio of 15:1, and the reaction was carried out at room temperature for 24 h. The reaction solution was dialyzed for 3 days and lyophilized to obtain the product HA-SH; In solution one, the mass ratio of hyaluronic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and N-hydroxysuccinimide is 15:8:5.
[0034] In solution two, the concentration of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride was 4 mg / mL, the concentration of N-hydroxysuccinimide was 2.5 mg / mL, and the concentration of hyaluronic acid was 5 mg / mL.
[0035] In solution two, the concentration of L-cysteine is 50 mg / mL.
[0036] S3, according to the following mass fraction ratio, take PECM-MA, HA-SH, and carboxymethyl chitosan (CMC), add them to deionized water, dissolve and stir evenly to form hydrogels PHC-1 to PHC-5.
[0037] PHC-1: PECM-MA 3%, HA-SH 2%, CMC 4%; PHC-2: PECM-MA 3%, HA-SH 4%, CMC 4%; PHC-3: PECM-MA 3%, HA-SH 8%, CMC 4%; PHC-4: PECM-MA 5%, HA-SH 2%, CMC 4%; PHC-5: PECM-MA 3%, HA-SH 4%, CMC 8%.
[0038] The stress relaxation properties of the prepared hydrogels PHC-1 to PHC-5 were tested, and the results are as follows: Figure 3 As shown, from Figure 3 The results show that PHC-1, PHC-2, and PHC-5 gel samples exhibit good relaxation properties, with gel strength decreasing to less than 10% of the initial value within 20 seconds. This is mainly due to the low degree of crosslinking and dynamic bond crosslinking. Considering the initial strength of the gel, PHC-2 was selected as the gel for subsequent animal experiments. By controlling the component content, the degree of addition reaction and disulfide bond content within the material differ, resulting in different stress relaxation properties. Studies have shown that stress relaxation properties are beneficial for vascular and epithelial regeneration and reduce scar formation, thereby accelerating wound healing and improving healing quality.
[0039] A picture of the PHC-2 hydrogel is attached. Figure 1 and 2 As shown, from Figure 1 This indicates that hydrogels possess a certain degree of adhesiveness, allowing them to adhere to the skin without detaching. From... Figure 2 This suggests that the hydrogel has a certain degree of injectability, as it can be directly ejected from a syringe. Figure 1 and Figure 2 It can be explained that the hydrogel in the embodiments of the present invention can be used as a dressing to adhere to the wound and fill the irregular surface.
[0040] The degradation performance of hydrogels PHC-1, PHC-2, and PHC-3 was tested. The test method was as follows: the hydrogels were immersed in PBS, and the dissolution of the hydrogels was observed and recorded.
[0041] Test results are as follows Figure 4 As shown. From Figure 4 The results show that hydrogel PHC-1 completely degraded after being soaked in PBS for two weeks, leaving the sample in a solution state; PHC-1 and PHC-2 swelled but did not degrade. With increasing hyaluronic acid content, the degree of cross-linking increased, and the stability further improved.
[0042] Using PHC-1 / 2 / 3 as samples, cell viability / death staining experiments were performed: The prepared hydrogel material was placed in cell culture medium at a concentration of 10 mg / mL. After soaking for 24 hours, the extract was filtered and extracted. Cells were then cultured in this extract for 24 hours. Finally, viability / death staining was performed and photographs were taken. Specifically, the extract concentration was 10 mg / mL, and L929 cells were cultured for 24 hours before staining. The results are shown below. Figure 5 As shown, this demonstrates that the hydrogel has good biocompatibility.
[0043] Animal experiment: SD rats (approximately 250g in weight) were collected and acclimatized for three days. They were then anesthetized with isoflurane, and the hair on their backs was removed. A circular, full-thickness skin wound with a diameter of 1.0 cm was then created using a circular skin harvesting tool. The control group received PBS solution, while the sample group received PHC-2 hydrogel. The hydrogel was injected into the wound using a syringe, and after filling the wound, the gel sample was smoothed with a spatula. The medication was administered every two days, and the wound size was recorded by photograph.
[0044] The result is as follows Figure 6 (Sample group) and Figure 7 (Control group) As shown, from bottom to top, the first row is the image of the wound on day 0, the second row is day 2, and the subsequent rows are day 4, 7, 9, and 11 respectively. From Figure 6 It can be concluded that hydrogel has a certain effect on promoting wound healing (as shown in the pictures on day 7 and day 9), and the wound surface is smoother. From... Figure 7 It can be concluded that the surface of the wound is not smooth and has obvious scars.
[0045] The embodiments described above are some, but not all, embodiments of the present invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.
Claims
1. A method for preparing a decellularized matrix composite hydrogel dressing for acute and chronic wound healing, characterized in that, Includes the following steps: S1, take the decellularized matrix of pig spleen and prepare it into a hydrogel solution. Add methacrylic anhydride dropwise at room temperature to adjust the pH to between 9 and 10. Stir overnight at 4°C. After the reaction is complete, adjust the pH to physiological pH. Dialyze the solution in a dialysis bag at 4°C for 4 days. Freeze-dry the solution to obtain PECM-MA. S2, hyaluronic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and N-hydroxysuccinimide were dissolved in MES buffer solution and stirred to form solution one; L-cysteine was dissolved in MES buffer solution and stirred to form solution two, and activated for 20-60 min; solution two was added to solution one, and the reaction was carried out at room temperature for 12-24 h. The reaction solution was dialyzed for 3 days and lyophilized to obtain the product HA-SH; S3. Take PECM-MA, HA-SH, and carboxymethyl chitosan, add them to deionized water, dissolve and stir evenly to form a hydrogel.
2. The method for preparing the decellularized matrix composite hydrogel dressing for acute and chronic wound healing according to claim 1, characterized in that, In step S1, the volume ratio of methacrylic anhydride to decellularized matrix hydrogel solution is (5-15) μL:1mL.
3. The method for preparing the decellularized matrix composite hydrogel dressing for acute and chronic wound healing according to claim 1, characterized in that, In step S2, the mass ratio of hyaluronic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and N-hydroxysuccinimide is 10-15:8:
5.
4. The method for preparing the decellularized matrix composite hydrogel dressing for acute and chronic wound healing according to claim 1, characterized in that, In step S2, in solution one, the concentrations of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide are both 1-10 mg / mL, and the concentration of hyaluronic acid is 0.001-0.02 g / mL.
5. The method for preparing the decellularized matrix composite hydrogel dressing for acute and chronic wound healing according to claim 1, characterized in that, In step S2, the concentration of L-cysteine in solution two is 1-100 mg / mL.
6. The method for preparing the decellularized matrix composite hydrogel dressing for acute and chronic wound healing according to claim 1, characterized in that, In step S2, the volume ratio of solution one to solution two is 10-20:
1.
7. The method for preparing the decellularized matrix composite hydrogel dressing for acute and chronic wound healing according to claim 1, characterized in that, In step S2, the concentration of the MES buffer solution is 0.01-0.2 M; pH is between 5 and 7.
8. The method for preparing the decellularized matrix composite hydrogel dressing for acute and chronic wound healing according to claim 1, characterized in that, In step S3, by mass percentage: PECM-MA 3-5%, HA-SH 2-8%, carboxymethyl chitosan 4-8%, balance water.
9. The method for preparing the decellularized matrix composite hydrogel dressing for acute and chronic wound healing according to claim 1, characterized in that, In step S3, by mass percentage: PECM-MA 3%, HA-SH 4%, carboxymethyl chitosan 4%, balance water.
10. A decellularized matrix composite hydrogel dressing for acute and chronic wound healing, characterized in that, It is prepared by the preparation method according to any one of claims 1-9.