CN116585525A - Interleukin-27-based wound healing promoting composite hydrogel and preparation method and application thereof - Google Patents
Interleukin-27-based wound healing promoting composite hydrogel and preparation method and application thereof Download PDFInfo
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L26/00—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form
- A61L26/0061—Use of materials characterised by their function or physical properties
- A61L26/008—Hydrogels or hydrocolloids
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L26/00—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form
- A61L26/0009—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form containing macromolecular materials
- A61L26/0014—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form containing macromolecular materials obtained by reactions only involving carbon-to-carbon unsaturated bonds
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L26/00—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form
- A61L26/0009—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form containing macromolecular materials
- A61L26/0028—Polypeptides; Proteins; Degradation products thereof
- A61L26/0038—Gelatin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L26/00—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form
- A61L26/0009—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form containing macromolecular materials
- A61L26/0028—Polypeptides; Proteins; Degradation products thereof
- A61L26/0047—Specific proteins or polypeptides not covered by groups A61L26/0033 - A61L26/0042
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L26/00—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form
- A61L26/0061—Use of materials characterised by their function or physical properties
- A61L26/0066—Medicaments; Biocides
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
- A61L2300/412—Tissue-regenerating or healing or proliferative agents
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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Abstract
The invention discloses a wound healing promoting composite hydrogel based on interleukin-27, and a preparation method and application thereof, wherein the composite hydrogel comprises hydrogel and interleukin-27, and the interleukin-27 is dispersed in the hydrogel; the hydrogel is polymerized by methacrylic acid acylated gelatin and methacrylic acid acylated polyvinyl alcohol. The composite hydrogel has good biocompatibility, keeps a moist environment around a wound surface, can stably convey the loaded interleukin-27 to the wound surface, and guides fibroblast migration and proliferation in the dermis restoration process, promotes angiogenesis, and further accelerates wound healing.
Description
Technical Field
The invention belongs to the technical field of biomedical dressings, and particularly relates to interleukin-27-based wound healing promoting composite hydrogel and a preparation method and application thereof.
Background
After skin injury, it is often necessary to cover the wound with a dressing to prevent secondary injury and bacterial infection.
Traditional dressing such as absorbent cotton gauze, cotton pad and the like is the dressing with the widest application of clinical skin wound. The traditional dressing has a reticular braided structure, has low price, relatively simple manufacturing process and wide raw material sources, and is still widely applied to skin wounds to date. However, the traditional dressing has obvious defects, such as poor barrier effect after the dressing is permeated by interstitial fluid at the wound, poor effect of promoting wound healing, easy secondary injury of the wound and the like. In recent years, the dressing such as polyurethane film, cellulose hydrocolloid dressing and the like developed is greatly improved compared with the traditional absorbent cotton gauze, but the dressing still cannot play a remarkable role in promoting wound healing (Chen Xiaojie, lv Aifeng, high-crystalline, and the like; theory and practice of wound moist environment healing of functional dressing [ J ]. Biomedical engineering progress, 2013,34 (1): 4.).
Interleukin is a commonly used anticancer drug in the field of cancer immunotherapy. Interleukin-27 is a potential novel preparation, and has been widely studied in cancer immunotherapy because it shows pleiotropic effects of immunopotentiation and immunomodulation.
Maintaining a good local environment of the wound is critical to wound healing. The moist wound environment prevents dehydration of cells, stimulates cell migration and thus promotes repair of the epidermis. There is a need for a bioactive dressing that can absorb wound exudate while maintaining a fluid environment at the wound site and achieve loading and release of drugs or cytokines to promote wound healing.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provides interleukin-27-based wound healing promoting composite hydrogel as well as a preparation method and application thereof. The interleukin-27 composite hydrogel prepared by the invention has good effect of promoting wound healing and has huge application prospect in the aspect of wound healing treatment.
In order to achieve the above purpose, the present invention is realized by the following technical scheme:
an interleukin-27-based wound healing promoting composite hydrogel comprises hydrogel and interleukin-27, wherein the interleukin-27 is dispersed in the hydrogel; the hydrogel is polymerized by methacrylic acid acylated gelatin and methacrylic acid acylated polyvinyl alcohol.
Preferably, the interleukin-27 based wound healing promoting composite hydrogel consists of hydrogel and interleukin-27.
Preferably, the interleukin-27 (IL-27) comprises human, animal interleukin-27.
Preferably, the mass of the methacrylic acid-acylated gelatin is 1 to 5 times that of the methacrylic acid-acylated polyvinyl alcohol;
preferably, the interleukin-27-based wound healing promoting composite hydrogel has a concentration of interleukin-27 of 10ng/mL-200ng/mL.
Preferably, the degree of substitution of the methacrylic acid acylated gelatin is 10% -40%.
Preferably, the degree of substitution of the methacrylic acid acylated polyvinyl alcohol is 0.5% -3%.
The preparation method of the interleukin-27-based wound healing promoting composite hydrogel comprises the following steps:
dissolving methacrylic acid acylated polyvinyl alcohol in deionized water, and heating to dissolve; cooling, adding methacrylic acid acylated gelatin, adding interleukin-27 and photoinitiator to obtain mixed solution, and standing under ultraviolet light to obtain the interleukin-27-based wound healing promoting composite hydrogel.
Preferably, the sum of the mass of the methacrylic acid acylated gelatin and the mass of the methacrylic acid acylated polyvinyl alcohol accounts for 5% -20% of the total mass of the mixed solution;
preferably, the mass of the photoinitiator is 0.02% -0.1% of the total mass of the mixed solution;
preferably, the mass of the interleukin-27 is 10ng/mL-200ng/mL of the total volume of the mixed solution.
Preferably, the cooling is to below 50 ℃;
preferably, the temperature of the heating dissolution is 90-100 ℃;
preferably, the time of the ultraviolet light is 10s-60s.
The application of the composite hydrogel for promoting wound healing in preparing a dressing for promoting wound healing is provided.
Compared with the prior art, the invention has the following advantages and beneficial effects:
the composite hydrogel prepared by the invention is prepared by using two photo-curing hydrogels of interleukin-27, methacrylic acid acylated gelatin and methacrylic acid acylated polyvinyl alcohol, wherein the methacrylic acid acylated gelatin and the methacrylic acid acylated polyvinyl alcohol have good biocompatibility and elastic modulus, and the formed double-network structure can keep the moist environment of a wound, can stabilize the activity of interleukin-27 and gradually convey the interleukin-27 to the surface of the wound to play a role, so that the concentration is prevented from greatly fluctuating. Wherein, interleukin-27 can obviously promote cell migration and angiogenesis in the wound healing process, thereby accelerating the wound healing speed and improving the wound healing quality. Based on this, the composite hydrogel shows great potential in wound healing as a wound dressing.
Drawings
FIG. 1 is a bar graph of composite hydrogels versus L929 cell viability in example 2;
FIG. 2 is a bar graph of IL-27 versus HUVECs cell viability at various concentrations in example 3;
FIG. 3 is a graph showing the ability of varying concentrations of interleukin-27 to repair scratches on HUVECs cells in example 3;
FIG. 4 is a schematic representation of the treatment of skin lesions with interleukin-27 complex hydrogel in the mouse model of example 4;
FIG. 5 is a schematic diagram showing the condition of skin wound surface of each group of mice at each time point in example 4;
FIG. 6 is a graph showing the wound healing rate and the wound healing process at various time points after treatment of each group of wounds in example 4;
FIG. 7 is a schematic representation of a representative cut surface of wound-surface neo-tissue following treatment of each group of wounds in example 4;
FIG. 8 is a graph showing the microscopic morphology of hydrogels formed by crosslinking methacrylic acid-acylated gelatin and methacrylic acid-acylated polyvinyl alcohol in different ratios in example 5.
Detailed Description
The invention is described in detail below in connection with examples which are, however, merely illustrative and are not to be construed as limiting the practice of the invention.
Example 1
The embodiment provides a preparation method of an interleukin-27 composite hydrogel, which comprises the following steps:
(1) 200mg of methacrylic acid acylated polyvinyl alcohol (substitution degree 0.5%) was dissolved in 6mL of deionized water, and heated to 100℃to dissolve completely;
(2) Cooling the solution in the step (1) to below 50 ℃, and adding 400mg of methacrylic acid acylated gelatin (with a substitution degree of 20%) into the solution in the step (1) to be mixed to prepare a mixed solution containing methacrylic acid acylated gelatin-methacrylic acid acylated polyvinyl alcohol;
(3) Adding interleukin-27 (the final concentration is 20 ng/mL) into the solution in the step (2) and mixing to prepare a mixed solution of methacrylic acid acylated gelatin and methacrylic acid acylated polyvinyl alcohol containing interleukin-27;
(4) 3mg of photoinitiator (2959) is added into the solution in the step (3), and the mixture is placed under ultraviolet light for 30 seconds to form gel, and the gel is preserved in a refrigerator at 4 ℃ for standby.
The preparation method of the interleukin 27-free hydrogel is the same as that in the step (3), and interleukin-27 is not added.
The other preparation methods of the interleukin 27 hydrogels with different concentrations are the same as above, except that the concentration of the interleukin-27 added in the step (3) is different.
The interleukin-27 used in this example is human interleukin-27 (Q969H 8, finetest).
Example 2
Described in this example is a toxicity test of an interleukin-27 composite hydrogel (prepared in example 1, human interleukin-27, concentration 20 ng/mL), by which the biocompatibility of the composite hydrogel can be assessed.
The biocompatibility experiment was performed on mouse fibroblasts (L929) by CCK8 method. First, log phase cells were collected, cell suspension concentration was adjusted, inoculated in 96-well plates at a density of 10000 cells/well, after overnight culture, treated with 100uL of complete medium, interleukin 27-free hydrogel extract, interleukin-27 composite hydrogel extract, incubated for 24 hours, 48 hours and 72 hours, and then treated with CCK8 solution, and then cell viability was evaluated by enzyme-labeled assay (see fig. 1).
It can be seen that the interleukin 27-free hydrogel group and the interleukin-27 composite hydrogel group have no significant difference in cell viability compared to the blank group for 72 hours. The results show that the interleukin-27-free hydrogel group and the interleukin-27 composite hydrogel group have good biocompatibility.
Example 3
This example describes an interleukin-27 (concentrations 10ng/mL,20ng/mL and 100 ng/mL) proliferation and repair promoting assay by which the effect of interleukin-27 at various concentrations on proliferation and migration activity of endothelial cells can be assessed.
Human Umbilical Vein Endothelial Cells (HUVECs) were subjected to biocompatibility experiments using CCK8 (see fig. 2). The experimental group is endothelial cell culture medium (the concentration is divided into 10ng/mL,20ng/mL and 100 ng/mL) added with interleukin-27, and the blank group is endothelial cell culture medium. First, log phase cells were collected, cell suspension concentration was adjusted, seeded at a density of 10000 cells/well on 96-well plates, cultured overnight, incubated with 100uL of blank and experimental group media, treated with CCK8 solution after 24 hours, 48 hours and 72 hours, and viability of HUVECs cells was assessed by elisa assay.
Cell migration repair experiments were performed using scratch experiments with HUVECs cells (see fig. 3). Scratch healing rates of 0 hours and 4 hours were observed. The experimental group is endothelial cell culture medium (the concentration is divided into 10ng/mL,20ng/mL and 100 ng/mL) added with interleukin-27, and the blank group is endothelial cell culture medium. First, cells in log phase were collected, cell suspension concentration was adjusted, inoculated on a 48-well plate at a density of 20000 cells/well, after overnight culture, a scratch was scratched at the center of each group of cells, and the size of the scratch at this time was recorded by photographing. After incubation for 4 hours with 200uL of blank and experimental media, the scratch sizes of each group were recorded again by photographing, and the HUVECs mobility was evaluated accordingly.
From this experiment, interleukin-27 promotes endothelial cell proliferation and repair of scratches at low concentrations, while at high concentrations it is detrimental to endothelial cell proliferation repair.
Example 4
The preparation method of the interleukin-27 composite hydrogel in the embodiment is the same as that in the embodiment 1, except that the concentration of interleukin-27 is 200ng/mL; the interleukin-27 used was murine interleukin-27 (Q8K 3I6, finetest).
Healing experiments of interleukin-27 composite hydrogel on full-thickness wounds of FVB mice described in this example (see FIG. 4); the blank control group is 1mL deionized water, the volume of the hydrogel of the interleukin-27-free hydrogel group is the same as that of the interleukin-27 composite hydrogel, and the volume of the hydrogel is 1mL, and the data obtained in the experiment are results obtained by 6 parallel independent experiments, and the method comprises the following specific experimental steps:
(A) Representative photographs of wounds on days 1, 3, 7, and 10 of the blank, no interleukin-27 hydrogel group, and interleukin-27 composite hydrogel group (see fig. 5).
(B) The results of the wound healing rate analysis and comparison of the blank group, the interleukin-27-free hydrogel group and the interleukin-27 composite hydrogel group on days 1, 3, 7 and 10 are shown in a wound healing process chart (see figure 6).
(C) The new granulation tissue sections of the blank group, the interleukin-27-free hydrogel group and the interleukin-27 composite hydrogel group on days 1, 3, 7 and 10 are covered by the broken lines (see FIG. 7).
From experimental results, in (A), the wound treatment effect of the interleukin-27 composite hydrogel group on FVB mice on the 7 th and 10 th days is significantly better than that of the blank group and the interleukin-27-free hydrogel group; in (B), the interleukin-27 composite hydrogel group has better wound healing process, wherein the blank group and the interleukin-27 hydrogel group can see massive crusting, and only punctate wounds are left to be unhealed in the interleukin-27 composite hydrogel group. As can be seen from the quantitative wound healing rate bar graph, the interleukin-27 composite hydrogel group exhibited higher wound healing rates (significantly different from the other two groups) at days 7 and 10. In (C), the new tissue section of the interleukin-27 composite hydrogel group has the most coherent new epidermis with the highest coverage rate, and the epidermis is tightly attached with the underlying tissues.
From the experiment, the interleukin-27 composite hydrogel group is obviously superior to a blank group and an interleukin-27-free hydrogel group in the effect of promoting wound healing.
Example 5
The micro-morphology characterization of the composite hydrogel in this example includes the following steps:
(1) Dissolving methacrylic acid acylated polyvinyl alcohol (substitution degree is 0.5%) in deionized water, and heating to 100 ℃ to completely dissolve;
(2) Cooling the solution in the step (1) to below 50 ℃, and adding the methacrylic acid acylated gelatin (with the substitution degree of 20%) into the solution in the step (1) to be mixed to prepare a mixed solution containing the methacrylic acid acylated gelatin-methacrylic acid acylated polyvinyl alcohol;
(3) Adding 0.05wt% of photoinitiator (2959) into the solution in the step (2), standing for 30 seconds under ultraviolet light to form gel, freeze-drying, and observing microscopic morphology by using SEM.
The preparation methods of the composite hydrogels are the same as those described above, except that the mass ratio of the methacrylic acid-acylated gelatin and the methacrylic acid-acylated polyvinyl alcohol added in the step (1) and the step (2) is different, specifically, three groups of 1:0, 4:1 and 2:1 (see fig. 8).
It can be seen that after the methacrylic acid acylated polyvinyl alcohol is added, an interpenetrating small network structure appears among large network structures in the hydrogel, and the small network density is correspondingly improved along with the improvement of the ratio of the methacrylic acid acylated polyvinyl alcohol. The abundant small network can be used as a sacrificial structure to relieve the damage of strain to the overall structure of the hydrogel.
In summary, the composite hydrogel prepared by the invention is prepared by using two photo-curing hydrogels of interleukin-27, methacrylic acid acylated gelatin and methacrylic acid acylated polyvinyl alcohol, wherein the methacrylic acid acylated gelatin and the methacrylic acid acylated polyvinyl alcohol have good biocompatibility and elastic modulus, the formed double-network structure can keep the moist environment of a wound, and the activity of interleukin-27 can be stabilized to gradually convey the same to the surface of the wound to generate an effect, so that the concentration is prevented from greatly fluctuating. Wherein, interleukin-27 can obviously promote cell migration and angiogenesis in the wound healing process, thereby accelerating the wound healing speed and improving the wound healing quality. Based on this, the composite hydrogel shows great potential in wound healing as a wound dressing.
While the fundamental and principal features of the invention and advantages of the invention have been shown and described, it will be apparent to those skilled in the art that the invention is not limited to the details of the foregoing exemplary embodiments, but may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. Although embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (10)
1. An interleukin-27-based wound healing promoting composite hydrogel, which is characterized by comprising a hydrogel and interleukin-27, wherein the interleukin-27 is dispersed in the hydrogel; the hydrogel is polymerized by methacrylic acid acylated gelatin and methacrylic acid acylated polyvinyl alcohol.
2. The interleukin-27 based wound healing promoting composite hydrogel according to claim 1, wherein the interleukin-27 based wound healing promoting composite hydrogel consists of a hydrogel and interleukin-27.
3. The interleukin-27 based wound healing composite hydrogel of claim 1, wherein the interleukin-27 comprises human, animal interleukin-27.
4. The interleukin-27 based wound healing promoting composite hydrogel according to claim 1, wherein the mass of the methacrylic acid acylated gelatin is 1-5 times that of the methacrylic acid acylated polyvinyl alcohol;
the concentration of interleukin-27 in the interleukin-27 based wound healing promoting composite hydrogel is 10ng/mL-200ng/mL.
5. The interleukin-27-based wound healing promoting composite hydrogel according to claim 1, wherein the degree of substitution of the methacrylic acid acylated gelatin is 10% -40%.
6. The interleukin-27-based wound healing promoting composite hydrogel according to claim 1, wherein the degree of substitution of the methacrylic acid acylated polyvinyl alcohol is 0.5% -3%.
7. The method for preparing the interleukin-27-based wound healing promoting composite hydrogel according to any one of claims 1 to 6, comprising the steps of:
dissolving methacrylic acid acylated polyvinyl alcohol in deionized water, and heating to dissolve; cooling, adding methacrylic acid acylated gelatin, adding interleukin-27 and photoinitiator to obtain mixed solution, and standing under ultraviolet light to obtain the interleukin-27-based wound healing promoting composite hydrogel.
8. The preparation method according to claim 7, wherein the sum of the mass of the methacrylic acid-acylated gelatin and the mass of the methacrylic acid-acylated polyvinyl alcohol is 5% -20% of the total mass of the mixed solution;
the mass of the photoinitiator is 0.02% -0.1% of the total mass of the mixed solution;
the mass of the interleukin-27 is 10ng/mL-200ng/mL of the total volume of the mixed solution.
9. The method of claim 7, wherein the cooling is to below 50 ℃;
the temperature of heating and dissolving is 90-100 ℃;
the time for placing under the ultraviolet light is 10s-60s.
10. Use of a wound-healing promoting composite hydrogel according to any one of claims 1 to 6 for the preparation of a wound-healing promoting dressing.
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