CN118217445A

CN118217445A - Composite hydrogel for repairing skin wound and preparation method and application thereof

Info

Publication number: CN118217445A
Application number: CN202410311190.2A
Authority: CN
Inventors: 王进; 陈振华; 钱宇; 韦宗佚
Original assignee: Jiangxi Science and Technology Normal University
Current assignee: Jiangxi Science and Technology Normal University
Priority date: 2024-03-19
Filing date: 2024-03-19
Publication date: 2024-06-21

Abstract

The invention belongs to the technical field of medical wound repair materials, and particularly relates to a composite hydrogel for repairing skin wounds, and a preparation method and application thereof. Dissolving methacrylic acid gelatin (GelMA) and sodium alginate (Alg) in water, uniformly mixing, adding phenyl-2, 4, 6-trimethylbenzoyl lithium phosphinate in a dark place, performing a crosslinking reaction under an ultraviolet lamp, and soaking in CaCl ₂ solution after the reaction is finished to obtain GelMA/Alg hydrogel; and then soaking the obtained GelMA/Alg hydrogel in a tannic acid solution to finally obtain the composite hydrogel for repairing skin wounds. The preparation method is simple, the material cost is low, and the prepared composite hydrogel has the characteristics of high biocompatibility, antioxidation and antibiosis, and can accelerate the repair of skin wounds.

Description

Composite hydrogel for repairing skin wound and preparation method and application thereof

Technical Field

The invention belongs to the technical field of medical wound repair materials, and particularly relates to a composite hydrogel for repairing skin wounds, and a preparation method and application thereof.

Background

The skin acts as a first barrier to the human body and it prevents microorganisms, chemicals and other harmful substances from entering the body. However, when the skin is damaged and there is no better way to treat, inflammation is induced, resulting in longer recovery cycles, suffering to the patient and costly economic loss. Therefore, the development of materials capable of enabling skin wounds to heal quickly is of great significance to clinical treatment.

There are many dressings for treating skin wounds, such as foams, rubbers, gauze, bandages, and the like. Traditional dressings require the incorporation of antibiotics to promote wound healing. In addition, over time they lose the ability to provide a moist environment for the wound and fail to provide adequate exudation. The high water content of the hydrogel can maintain a moist environment around the wound, which helps painlessly remove necrotic and infected tissue, and stimulates the growth of new tissue. Hydrogels can provide a protective barrier that minimizes the risk of infection while stimulating migration of epithelial and fibroblasts and promoting the wound healing process. Hydrogels exhibit swelling behaviour which enables them to absorb large amounts of exudates and oxygen and water vapour can easily pass through them which makes them more effective for wound healing. The hydrogel dressing may be loaded with therapeutic drugs and different bioactive molecules to accelerate wound closure and avoid infection.

However, conventional gelatin hydrogels have low mechanical properties and lack functionality. These drawbacks limit their application in the biomedical field. Therefore, finding out the combination of active substances and the improved gelatin hydrogel to construct hydrogel for accelerating the healing of skin wounds becomes a key for meeting the clinical application of the hydrogel to the repair of skin wounds.

Disclosure of Invention

The invention aims to solve the defects of the prior art, provides a composite hydrogel for repairing skin wounds, and a preparation method and application thereof, and specifically adopts the following technical scheme:

a method for preparing composite hydrogel for repairing skin wound, comprising the following steps:

Dissolving methacrylic acid gelatin and sodium alginate in water, uniformly mixing, adding phenyl-2, 4, 6-trimethylbenzoyl lithium phosphinate in a dark place, performing a crosslinking reaction under an ultraviolet lamp, and soaking in CaCl ₂ solution after the reaction is finished to obtain GelMA/Alg hydrogel;

Soaking the obtained GelMA/Alg hydrogel in tannic acid solution with the concentration of 0.01-0.05% w/v for 4 h%, and finally obtaining the composite hydrogel (GelMA/Alg-TA) for repairing skin wound.

The methacrylated gelatin (GelMA) employed in the present invention is an effective biopolymer that can be used to load and deliver bioactive compounds and other drugs to promote wound healing. Sodium alginate (Alg) is a linear polysaccharide that is hydrophilic, biocompatible, and cost-effective. The invention adopts Alg and GelMA to form double-network hydrogel, improves the mechanical property of the hydrogel, and overcomes the difficulty that GelMA is a good hydrogel dressing due to lower mechanical property. Meanwhile, tannic Acid (TA) is added into the hydrogel to stabilize the structure of the hydrogel, so that the mechanical property of the hydrogel is improved, and meanwhile, the functionality of the hydrogel is increased. The polyphenol structure of TA has free radical scavenging, antibacterial and anti-inflammatory activities, and due to the existence of phenolic hydroxyl in the polyphenol structure of TA, the TA forms hydrogen bonds with amino groups and carboxyl groups on polysaccharide and collagen, so that the macromolecular structure is stabilized. Therefore, the tannic acid composite hydrogel with high biocompatibility, antioxidation and antibacterial properties is prepared by a physical and chemical double-crosslinking preparation method.

As a further preferred embodiment, the process for preparing the methacrylated gelatin is as follows:

Dissolving gelatin and methacrylic anhydride in water, stirring at 50deg.C for 1.5h, dialyzing in dialysis bag for 72. 72 h, adjusting pH to 7.4, and lyophilizing for 48h to obtain methacrylic gelatin.

The invention adopts methacrylic acid gelatin (GelMA) to prepare an excellent wound dressing material by an ultraviolet crosslinking method, and has the characteristics of good cell adhesion, strong permeability to nutrient substances and oxygen, and the like. In addition, the GelMA prepared by the preparation method has low requirement on substitution degree compared with the GelMA purchased directly, and the synthesis condition is mild, so that the GelMA prepared by the preparation method has better effect.

As a further preferred embodiment, the ratio of gelatin to methacrylic anhydride is 10 g:6 mL. When the ratio of the amount of gelatin to methacrylic anhydride is too high, the substitution degree is not changed, and when the ratio of the amount is 10 g:4 mL, the substitution level is reduced, which affects the preparation of the subsequent gel, when the dosage ratio is 10 g:6 mL, more facilitating the subsequent preparation of the gel.

As a further preferred embodiment, the ratio of the amounts of the methacrylic acid gelatin, sodium alginate and the phenyl-2, 4, 6-trimethylbenzoyl lithium phosphinate is 1 g:0.2 g:20 mg.

As a further preferred embodiment, the CaCl ₂ solution has a concentration of 3% w/v and a soaking time of 2 h. The calcium chloride is soaked in CaCl ₂ solution to crosslink Ca ²⁺ ions with sodium alginate, the concentration of the calcium chloride is 3% w/v, which is favorable for the increase of the toughness of the hydrogel, and the concentration of the calcium chloride is reduced, so that the acting force between molecular chains is insufficient, and the crosslinking cannot be completed well. In addition, if the crosslinking time is too short, crosslinking may not be completed, and 2 h is the optimal soaking time.

As a further preferred embodiment, the time of the crosslinking reaction is 5min.

As a further preferred embodiment, the concentration of the tannic acid solution is 0.01% w/v, 0.03% w/v, 0.05% w/v. More preferably, the concentration of the tannic acid solution is 0.03% w/v. According to experimental results of the embodiment of the invention, the composite hydrogel prepared by the invention can obviously close a wound surface, and under the action of TA composite hydrogel with the concentration of 0.03% w/v, the wound surface can be obviously closed in 7 days.

The invention also provides the composite hydrogel which is prepared by the preparation method and can be used for repairing skin wounds. Also provided is a wound protection product comprising the composite hydrogel for repairing skin wound.

The beneficial effects of the invention are as follows:

The preparation method disclosed by the invention is simple, the raw material cost is low, the process flow is stable, and the prepared tannic acid composite hydrogel has high biocompatibility, antioxidation and antibacterial properties and good mechanical properties;

The GelMA/Alg-TA hydrogel prepared by the method has a stable three-dimensional porous structure and proper moisture content, and can continuously release TA for a certain time, so that the GelMA/Alg-TA hydrogel has good antioxidation and antibacterial effects, and can also remarkably promote wound healing of a full-layer skin defect mouse model by adjusting inflammatory reaction and promoting granulation. In addition, the hydrogel has the function of accelerating skin wound repair, and is a candidate material for wound healing with development prospect.

Drawings

FIG. 1 is a schematic diagram of a preparation route of the present invention;

FIG. 2 is a composite hydrogel scanning electron microscope topography;

FIG. 3 is a graph showing the mechanical properties of the composite hydrogel;

FIG. 4 is a graph showing the swelling degree test of the composite hydrogel;

FIG. 5 is a graph showing in vitro release of tannic acid from composite hydrogels;

FIG. 6 is a graph showing in vitro antioxidant activity of the composite hydrogel;

FIG. 7 is a graph showing in vitro antibacterial activity test of composite hydrogels;

FIG. 8 is a graph showing a composite hydrogel cell compatibility test;

FIG. 9 is a graph showing a composite hydrogel hemolytic activity test;

FIG. 10 is a graph showing in vivo evaluation of composite hydrogels for wound healing in mice;

FIG. 11 shows a composite hydrogel histological and hematoxylin-eosin staining and Pinus massoniana staining plot;

FIG. 12 shows a composite hydrogel histological and immunofluorescent staining pattern.

Detailed Description

The conception, specific structure, and technical effects produced by the present application will be clearly and completely described below with reference to the embodiments and the drawings to fully understand the objects, aspects, and effects of the present application. It should be noted that, without conflict, the embodiments of the present application and features of the embodiments may be combined with each other.

Example 1

Step 1, synthesis of methacrylic gelatin (GelMA): respectively dissolving 10 g gelatin and 6 mL methacrylic anhydride in 50 mL deionized water, water-bathing at 50 ℃ and stirring for 1.5 h; then dialyzing (MD 3500) 72 h in ultra-pure water at 50deg.C, adjusting to pH 7.4, and freeze-drying 48: 48 h to obtain GelMA.

Step 2, synthesizing GelMA/Alg hydrogel: gelMA (2 g) and Alg (0.4 g) obtained in step 1 were dissolved in 20 mL deionized water, respectively, and then mixed and stirred to obtain a semitransparent solution. Adding phenyl-2, 4, 6-trimethylbenzoyl lithium phosphinate (LAP) (0.04 g,0.1% w/v) under the condition of avoiding light, then placing the solution into a mould, irradiating with ultraviolet light for 5min, finally soaking the hydrogel in CaCl ₂ (50 mL,3% w/v) solution for 2h to obtain GelMA/Alg double-network hydrogel;

step 3, synthesizing GelMA/Alg-TA composite hydrogel: soaking the GelMA/Alg hydrogel (3 g) obtained in the step 2in 20 mL Tannic Acid (TA) solution containing 2mg to obtain the composite hydrogel (GelMA/Alg-TA 1) for repairing skin wound.

Example 2

A preparation method of a composite hydrogel for skin wound repair is the same as the preparation method of the example, except that the solution containing 2 mg Tannic Acid (TA) in 20mL in step 3 is changed into the solution containing 6 mg Tannic Acid (TA) in 20mL, and the composite hydrogel (GelMA/Alg-TA 2) for skin wound repair is obtained.

Example 3

A preparation method of a composite hydrogel for skin wound repair is the same as the preparation method of the example, except that the solution containing 2 mg Tannic Acid (TA) in 20 mL in step 3 is changed into the solution containing 10 mg Tannic Acid (TA) in 20 mL, and the composite hydrogel (GelMA/Alg-TA 3) for skin wound repair is obtained.

The preparation route and performance test chart of examples 1 to 3 are shown in fig. 1, and the composite hydrogels prepared in examples 1 to 3 were subjected to electron microscopy, the results of which are shown in fig. 2, and as can be seen from fig. 2, the composite hydrogels prepared in examples 1 to 3 all had porous structures, and as the TA concentration increased, the pore diameters decreased and the pore densities gradually increased.

Example 4

In this example, the mechanical properties of the composite hydrogels prepared in examples 1 to 3 were tested to investigate the effect of TA concentration on the mechanical properties of the hydrogels

A tensile sensor of 50N was provided and uniaxial tension and compression tests were performed on the hydrogel samples at room temperature. Hydrogel samples for tensile testing were prepared in strips of length 30 mm x width 5mm x thickness 2mm and were given young's modulus, tensile strength and strain at break at a crosshead speed of 10 mm/min. Samples were fabricated into cylinders 10 in diameter mm x 10 in thickness mm at a crosshead speed of 20 mm/min to obtain Young's modulus, compressive strength and strain at break. The corresponding Young's modulus was determined from the slope of the stress-strain curve at 10% strain.

As a result, as shown in FIG. 3, and as shown in FIG. 3 (a), the maximum tensile stress of the GelMA/Alg-TA hydrogel was increased and the tensile strain was reduced from 100% to 60% as compared with the GelMA/Alg hydrogel; as can be seen from fig. 3 (b), the toughness tends to increase and the elasticity tends to decrease with an increase in TA content. The tensile modulus gradually increased from GelMA/Alg to GelMA/Alg-TA2 hydrogel, but the tensile modulus slightly decreased in GelMA/Alg-TA 3.

Example 5

In this example, the composite hydrogels prepared in examples 1-3 were subjected to swelling test as follows:

The results of the calculation of the swelling degree of the composite hydrogels prepared in examples 1 to 3, respectively, were shown in FIG. 4, which shows that the swelling degree decreased with increasing concentration of TA, when the composite hydrogels were placed in PBS buffer at 37℃for several hours. This indicates that swelling of the hydrogel is significantly suppressed as the degree of crosslinking increases.

Example 6

In this example, the composite hydrogels prepared in examples 1-3 were tested for TA release in vitro as follows:

a column of hydrogel of uniform size (10 mm diameter. Times.10 mm height) was used to soak in a 50 mLPBS shaking bath (37 ℃,60 rpm). Subsequently, 1.0 mL samples were taken at 1 h,2 h,4 h,8 h,12 h,24 h,48 h each and the volume was replenished by adding 1mL fresh PBS. The concentration of TA in PBS was determined by UV absorption at 276 nm using a UV-visible spectrophotometer. As a result, the results are shown in FIG. 5, and it is clear from FIG. 5 that GelMA/Alg-TA1 and GelMA/Alg-TA3 were slightly higher than GelMA/Alg-TA2 in 4h after release. 24 At h, gelMA/Alg-TA1 and GelMA/Alg-TA2 are substantially the same, and GelMA/Alg-TA3 is significantly higher than the other two groups.

Example 7

In this example, the composite hydrogels prepared in examples 1-3 were subjected to in vitro antioxidation tests as follows:

2, 2-diphenyl-1-picrylhydrazine (DPPH) powder was formulated as a DPPH/ethanol solution (0.1 mM). The 20 mg hydrogel was placed in DPPH/ethanol solution (2 mL) and immersed in the dark for 30 min, and the absorbance change at 541 nm wavelength was measured with an ultraviolet-visible spectrophotometer. The DPPH radical scavenging activity is calculated as: clear DPPH activity (%) = (blank-sample)/blank×100

The antioxidant activity of GelMA/Alg and GelMA/Alg-TA hydrogels was evaluated by measuring their ability to scavenge DPPH free radicals. The DPPH has a strong absorption curve at 541 nm and the results in FIG. 6 show no significant change in absorption in the GelMA/Alg hydrogels. However, in GelMA/Alg-TA1 and GelMA/Alg-TA2 and GelMA/Alg-TA3, the absorption gradually decreases. The oxidation resistance of TA was verified and the greater the TA concentration, the greater the oxidation resistance.

Example 8

In this example, the composite hydrogels prepared in examples 1-3 were tested for in vitro antimicrobial properties as follows:

Staphylococcus aureus was used as the experimental species. Dissolving 15 g soytone in 500 mL ultrapure water, sterilizing at high temperature, and cooling to obtain a liquid bacterial culture medium; 15 g soytone and 8 g agar are dissolved in 500 mL ultrapure water, sterilized at high temperature, poured into a culture dish, and cooled to obtain a solid culture medium. Staphylococcus aureus was added to the liquid medium and activated at 37 ℃ at 130 rpm for about 16: 16h, after which it was diluted to the desired concentration for use. Bacterial suspensions were added to well plates where different sets of hydrogels were placed and co-incubated 24 h. And observing turbidity degrees of different groups of bacteria solutions, and primarily judging the antibacterial effect of the hydrogel. The plate was placed in an microplate reader to test the optical density OD ₆₀₀. Then 2 mu L of bacterial liquid is added into 10 mL ultrapure water to dilute 5000 times, and is uniformly mixed by a vortex instrument, 100 mu L of bacterial liquid is taken to a culture plate and uniformly coated. The colony count of the culture plate was observed after culturing in a ventilated incubator at 37℃for 18 h hours. Each group was subjected to 3 replicates and the results were expressed in terms of antimicrobial activity: antibacterial activity (%) = (Cc-Cs)/cc×100, where Cc and Cs represent colony numbers of the control group and the sample group, respectively.

In the co-culture and zone of inhibition test of staphylococcus aureus, the results are shown in fig. 7, and as can be seen from fig. 7 (a), gelMA/Alg-TA hydrogel shows remarkable antibacterial performance; as can be seen from FIG. 7 (b), the proliferation degree of Staphylococcus aureus in GelMA/Alg group was higher than that in the control group; the antibacterial effects of the GelMA/Alg-TA1 group, the GelMA/Alg-TA2 group and the GelMA/Alg-TA3 group are gradually enhanced, and compared with a control group, the in vitro proliferation of staphylococcus aureus is obviously inhibited. This shows that the composite hydrogel prepared by the invention has excellent antibacterial property.

Example 9

In this example, the composite hydrogels prepared in examples 1 to 3 were tested for cytocompatibility and hemolytic activity as follows:

The procedure for the cell compatibility experiments was as follows: hydrogels (10 mm diameter x10 mm height) were incubated in 5 mLDMEM medium with 10% fetal bovine serum for 24 h and supernatants were collected as conditioned medium. L929 fibroblasts were seeded in 96-well plates at a rate of 5X10 ³/well and cells were cultured using normal or leach solutions as different groups of media. Cytotoxicity of the composite hydrogels was assessed 1 day later using cell counting kit-8 (CCK-8). L929 fibroblasts were seeded in 96-well plates at a rate of 2X 10 ³/well and cells were cultured using the normal or leach solutions prepared as described above as different groups of media. Cytotoxicity of the composite hydrogels was detected with CCK-8 at 1, 2,3, and 4 days to obtain culture curves. Cell viability was determined by Calcein acetoxymethyl ester/propidium iodide (Calcein-AM/PI) kit while medium was aspirated from the well plate, stained with the kit, and cell morphology was observed using an inverted fluorescence microscope. Cell viability calculation formula: cell viability (%) =as/ac×100, where As and Ac are the absorbance of the hydrogel sample and control solutions, respectively.

The results of the cell compatibility experiments show that: in FIG. 8 (a), the cell viability of each of the other groups except the GelMA/Alg-TA3 group was more than 85%, indicating good cell compatibility. Whereas the GelMA/Alg-TA3 group was less than 40%, indicating cytotoxicity. In FIG. 8 (b), the blank, gelMA/Alg-TA1 and GelMA/Alg-TA2 groups had higher cell densities and GelMA/Alg-TA3 had lower cell densities. This indicates that the TA composite hydrogel has excellent cell compatibility.

The hemolysis experiment steps are as follows: the hemolytic activity of the composite hydrogel was evaluated with rat erythrocytes (rbc). These hydrogels were incubated with diluted erythrocytes (10% v/v) in suspension at 37℃for 3 h. After centrifugation, the absorbance of the supernatant was measured at 540 nm using an ultraviolet-visible spectrophotometer. Rbc was co-cultured with PBS and triton as negative control group and positive control group, respectively. Hemolysis ratio (%) = (As-An)/(Ap-An) ×100, where As, ap, and An are absorbance of sample supernatant, positive and negative control groups, respectively.

The results of the hemolysis experiment are shown in FIG. 9: after contacting red blood cells at 37 ℃ with the GelMA/Alg-TA hydrogel for 3 h, the hemolysis rate of the GelMA/Alg-TA hydrogel is less than 5%, and meets the requirements of the American society for testing materials.

Example 10

The composite hydrogels prepared in examples 1-3 were used for in vivo evaluation of wound healing in mice by the following procedure:

40 male KM mice (weight 35-40 g) of 6-8 weeks old were used in total. Mice were kept at an ambient temperature of 26 ℃ ± 2 ℃ for one week during which they were fed water normally. The mice were normal in activity and diet. The mice were randomly divided into 5 groups and were intraperitoneally injected at a dose of 30-90mg/kg of pentobarbital sodium, after anesthesia, the lower and middle back hairs were shaved with an electric shaver, the back hairs were removed with a depilatory cream to expose a smooth epidermis portion, and sterilized with 70% alcohol. Circular full-cortical defect wounds of 10mm diameter were then cut out with sterile surgical scissors and a drop of a suitable concentration of staphylococcus aureus suspension (50 μl) was added dropwise to infect the wound with a syringe. The mice are put back into cages for normal feeding for 4-6 h, suppuration reaction is observed on the wounds, which indicates that bacterial infection wounds are successfully molded, and the next treatment of the composite hydrogel dressing can be carried out. Preparing hydrogel dressings, preparing different groups of hydrogels into disc-shaped dressings with the diameter of 10mm and the thickness of 1: 1mm, placing the disc-shaped dressings into a container, soaking the disc-shaped dressings in 70% alcohol for 30: 30min, and irradiating the disc-shaped dressings under an ultraviolet lamp for 30: 30min for sterilization for later use. After the wound conditions of different groups are recorded by mobile phone photographing, hydrogel dressing treatment is started to the mice. Different groups of composite hydrogels were applied to the wound sites of each mouse and fixed with a transparent 3M patch, and the control group was only applied with a 3M patch. The hydrogel dressing was changed every two days and a photograph of the wound was taken with a cell phone and attached to a ruler before the change. The sections were scanned with a digital slide scanner to analyze the inflammatory response, epidermal regeneration and healing quality of the wound and the wound area was statistically and computationally analyzed with ImageJ software.

In this example, the wound repair effect of GelMA/Alg-TA hydrogel was evaluated in a skin wound model as shown in FIGS. 10 (a) and 10 (b). The wound surface has different degrees of festering on the 0 th day after the bacterial infection skin defect modeling. The wound sizes of the control group and the hydrogel group are different on the 7 th day, and the wound closure of the GelMA/Alg-TA2 group is obvious. After 12 days of treatment, there were no open wounds in all but the GelMA/Alg hydrogel group. The result shows that the embedded TA of the GelMA/Alg hydrogel can effectively promote wound closure. The wound size calculations further confirm that the wound healing effect is higher for the GelMA/Alg-TA group than for the other groups.

Example 11

This example is to further verify the mechanism of action of the composite hydrogel on wound healing, using hematoxylin-eosin (H & E) staining, masson (Masson) staining and Immunofluorescence (IF) staining.

The operation steps of the embodiment are as follows: wound skin tissue 15 mm x 15 mm in length was fixed by immersing in 4% paraformaldehyde at 48 h, then embedded in paraffin, and serially sectioned 5 μm thick by a microtome. The sections were subjected to section dewaxing, antigen retrieval and circle drawing. Bovine serum albumin blocking serum was added dropwise. Removing the sealing liquid, dropwise adding the prepared primary antibody, incubating overnight at 4 ℃, then dropwise adding the corresponding horseradish peroxidase-labeled secondary antibody, and incubating at room temperature for 50 min. Finally, the corresponding TSA fluorescent dye was added.

The H & E staining results are shown in FIG. 11 (a). On day 0, each panel of H & E patterns had an inflammatory response. On day 7, the control group also had inflammation on the surface, and the rest of the groups had inflammation relief. On day 12, the control group, gelMA/Alg group, and GelMA/Alg-TA group all had inflammation relieving and skin regenerating effects, and the GelMA/Alg-TA2 and GelMA/Alg-TA3 groups produced new hair follicle tissue.

Masson staining observed the formation and orientation of collagen fibers that play a key role in wound healing, and further analyzed the quality of wound healing for different groups as shown in fig. 11 (b): the GelMA/Alg-TA2 and GelMA/Alg-TA3 groups had thicker, dense, more directional collagen fibers on day 7, while the control group had sparse collagen fibers.

IF staining, immune cell CD45 was performed on wound sections on day 0 and day 7, respectively, and the results are shown in fig. 12 (a): on day 0, the expression levels of CD45 were substantially uniform for each group, with a large number of immune cells (CD 45) infiltrating around the wound. Macrophages in the wound microenvironment are diverse. CD86 is a cell surface marker typical of M1 macrophages, with pro-inflammatory effects and the effect of clearing damaged tissue. CD163 is a typical cell surface marker of M2 macrophages, secreting anti-inflammatory cytokines and growth factors, inhibiting inflammatory responses, and promoting tissue regeneration. Thus, immunofluorescent staining of macrophages expressing CD86 and CD163 was performed on the wound healing days 0 and 7, and the results are shown in fig. 12 (b): on day 0, each group of M1 macrophages had higher CD86 levels and concentrated on the wound surface with little expression of CD163, suggesting an acute inflammatory phase at this time. On day 7, the control and GelMA/Alg groups showed a different decrease in CD86 expression, but it was still evident that the three GelMA/Alg-TA hydrogels showed almost no CD86 expression, with sporadic CD163 expression. This demonstrates that the healing effect of the tannic acid complex hydrogel group is significantly improved compared to the model group and the GelMA/Alg group.

While the present invention has been described in considerable detail and with particularity with respect to several described embodiments, it is not intended to be limited to any such detail or embodiments or any particular embodiment, but is to be construed as providing broad interpretation of such claims by reference to the appended claims in view of the prior art so as to effectively encompass the intended scope of the invention. Furthermore, the foregoing description of the invention has been presented in its embodiments contemplated by the inventors for the purpose of providing a useful description, and for the purposes of providing a non-essential modification of the invention that may not be presently contemplated, may represent an equivalent modification of the invention.

Claims

1. A method for preparing a composite hydrogel for repairing skin wounds, which is characterized by comprising the following steps:

The obtained GelMA/Alg hydrogel is soaked in tannic acid solution with the concentration of 0.01-0.05% w/v for 4 h%, and finally the composite hydrogel for repairing skin wound is obtained.

2. The preparation method according to claim 1, wherein the preparation process of the methacrylic acid gelatin comprises the following steps:

3. The method according to claim 2, wherein the ratio of gelatin to methacrylic anhydride is 10 g:6 mL.

4. The preparation method according to claim 1, wherein the dosage ratio of the methacrylic acid gelatin, the sodium alginate and the phenyl-2, 4, 6-trimethylbenzoyl lithium phosphinate is 1 g:0.2 g:20 mg.

5. The method of claim 1, wherein the concentration of CaCl ₂ solution is 3% w/v.

6. The method of claim 1, wherein the soaking time is 2h.

7. The method of claim 1, wherein the time for the crosslinking reaction is 5 min.

8. The method according to claim 1, wherein the concentration of the tannic acid solution is 0.01% w/v, 0.03% w/v or 0.05% w/v.

9. A composite hydrogel for skin wound repair, prepared by the method of any one of claims 1-8.

10. A wound protection product comprising the composite hydrogel of claim 9 for repair of skin wounds.