A composition, composite cytokine hydrogel and preparation method and application thereof

Abstract

The present invention provides a composition, a composite cytokine hydrogel, and a preparation method and application thereof, and belongs to the field of biomedical technology. The present invention adds insulin-like growth factor-1, epidermal growth factor, basic fibroblast growth factor-2, human platelet-derived growth factor, vascular endothelial cell growth factor C, and transforming growth factor-β2 in a specific ratio, which can promote fibroblast proliferation, be beneficial to the appearance and function recovery of new skin, and make wounds heal quickly. The composite cytokine hydrogel of the present invention has a strong repair effect and good tissue compatibility, and can form a solid gel after being placed at 37°C for 16 to 17 seconds. The preparation method of the composite cytokine hydrogel of the present invention is simple and can be widely used clinically.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a composition, a composite cytokine hydrogel, and a preparation method and application thereof. Background Art

[0002] When the skin is injured by certain physical, chemical or biological factors, the damage caused by the destruction of the skin and subcutaneous tissue structure is called skin injury. In addition, certain diseases and treatments can also cause skin damage, such as cancerous ulcers, radiation ulcers, vascular ulcers, pressure injuries, diabetic foot, etc., which usually develop into difficult-to-heal wound ulcers. The treatment of these skin injuries and ulcers involves the wound repair process. This process includes three different stages: elimination of inflammation, cell proliferation and repair, and new tissue regeneration, and each stage overlaps with each other. During the inflammatory response period of the wound, the anti-inflammatory factors and growth factors released by platelets and leukocytes stimulate fibroblasts to synthesize collagen and eliminate inflammation; the growth factors released by macrophages during the proliferation and repair period, such as platelet growth factor and transforming growth factor, can accelerate wound healing, stimulate fibroblasts and endothelial cells to synthesize and secrete a variety of growth factors to further promote cell proliferation, and then enter the tissue regeneration period, during which new blood vessels are formed, new epithelium is regenerated, and finally the wound is completely healed. Currently, the use of growth factors to treat skin injuries has become a hot topic, and many researchers have used different growth factors to treat skin injuries.

[0003] When treating skin injuries, topical drugs play a very important role in accelerating wound healing. With the development of medical services and a deeper understanding of growth factors, the types of drugs used in wound repair are becoming more and more abundant. At present, more scholars tend to use combination drugs, but the choice of combination drugs faces great difficulties. How to use multiple growth factors in combination is the focus and difficulty of current research. Although stem cells have the ability to secrete a variety of cytokines, including human epidermal growth factor (EGF), human keratinocyte growth factor (KGF), human basic fibroblast growth factor (FGF), human platelet-derived growth factor (PDGF), fibronectin (FN), etc., the proportion of cytokines secreted by stem cells is limited by the composition of the culture medium and the culture conditions, making it difficult to achieve satisfactory therapeutic effects.

[0004] A wet environment can provide sufficient moisture to the wound, which is beneficial to the release and function of water-soluble substances such as cytokines, thereby reducing wound pain, subsiding the spread of inflammation, reducing exudate, reducing the growth of microorganisms on the wound, and preventing the exposure and death of nerve endings, reducing damage to the wound surface when changing dressings; it is beneficial for phagocytes to perform their phagocytic function. Among them, hydrogel dressings are suitable for various types of burn wounds, which can effectively relieve pain and accelerate wound healing.

[0005] In recent years, the preparation of thermosensitive hydrogels using chitosan and its derivatives as raw materials has achieved remarkable research results. Among them, the pH value of the thermosensitive hydrogel prepared using chitosan and sodium β-glycerophosphate can be maintained within the physiological range of the human body. This thermosensitive hydrogel is in a flowable solution state at low temperatures, so it can be loaded with a certain concentration of drugs or growth factors, etc., and injected into the defects or lesions of the human body through minimally invasive surgery such as intravenous injection. Due to the change in temperature, gel can be formed in situ at the defect or lesion, and can completely fill the defect. This advantage makes it unnecessary to implant it into the lesion through surgery, and the drug can be loaded into the gel through simple mixing. In addition, the gelation process of this system is a physical change, which avoids the disadvantages of using chemical crosslinkers or light radiation crosslinking, so it has broad application prospects in biomedical fields such as tissue engineering and drug release. However, the concentration of sodium β-glycerophosphate must reach a certain value in order for the chitosan / sodium β-glycerophosphate mixed solution to transform into gel at human body temperature. In addition, in order to reduce the gelation time, the dosage of sodium β-glycerophosphate must be increased. When the concentration of sodium β-glycerophosphate exceeds a certain value, it is highly toxic to the body. For example, the osmotic pressure of a sodium β-glycerophosphate solution with a concentration of 800 mg / mL is about 1080 mOsm, which is much higher than the normal osmotic pressure of human cells. Summary of the invention

[0006] The purpose of the present invention is to overcome the defects in the background art and provide a composition for promoting wound healing and a hydrogel that gels quickly at body temperature.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] The present invention provides a composition, which comprises the following components in parts by weight: 20 to 25 parts of insulin-like growth factor-1, 8 to 10 parts of epidermal growth factor, 3 to 5 parts of basic fibroblast growth factor-2, 10 to 16 parts of human platelet-derived growth factor, 5 to 10 parts of vascular endothelial cell growth factor C, and 0.5 to 2.5 parts of transforming growth factor-β.

[0009] The present invention also provides application of the composition in preparing a medicine for treating wound surface repair.

[0010] The present invention also provides a composite cytokine hydrogel, comprising the following raw materials in percentage by weight: comprising the following raw materials in percentage by weight: the composition 2 to 8×10 -3 %, sodium alginate 2-4%, γ-polyglutamic acid 0.5-1.5%, chitosan 4-6%, propylene glycol 0.05-0.2%, polyethylene glycol 0.4-1.6%, β-sodium glycerophosphate 4-8%, and the balance water.

[0011] Preferably, the viscosity-average molecular weight of the chitosan is 570-600 KDa, and the degree of deacetylation is 88-92%.

[0012] Preferably, the polymerization degree of the γ-polyglutamic acid is 3000-5000.

[0013] The present invention also provides a method for preparing the composite cytokine hydrogel, comprising the following steps:

[0014] (1) mixing chitosan with a hydrochloric acid solution to obtain a chitosan solution;

[0015] (2) mixing the chitosan solution with propylene glycol, polyethylene glycol, γ-polyglutamic acid and sodium alginate, stirring, and then adjusting the pH value to 6-6.5 with a NaOH aqueous solution to obtain a solution 1;

[0016] (3) Solution 1, sodium β-glycerophosphate and the composition are mixed, stirred, and ions are removed to obtain the composite cytokine hydrogel solution.

[0017] Preferably, the concentration of the hydrochloric acid solution is 0.08-0.12M.

[0018] Preferably, the mass volume ratio of chitosan to hydrochloric acid solution is 4-6 g:90 mL.

[0019] Preferably, step (2) and step (3) are carried out under ice bath conditions.

[0020] Preferably, the stirring speeds in step (2) and step (3) are independently 1000 to 1500 rpm, and the stirring time is independently 10 to 15 min.

[0021] By adopting the above technical scheme, the present invention has the following beneficial effects: the present invention adds insulin-like growth factor-1, epidermal growth factor, basic fibroblast growth factor-2, human platelet-derived growth factor, vascular endothelial growth factor C, and transforming growth factor-β2 in a specific ratio, which can promote fibroblast proliferation, be beneficial to the appearance and function recovery of new skin, and make wounds heal quickly. The composite cytokine hydrogel of the present invention has a strong repair effect and good tissue compatibility. It is in a liquid state when it is below 10°C, and it turns into a gel when it is above 10°C; it can form a solid gel when placed at 37°C for 16 to 17 seconds. The preparation method of the composite cytokine hydrogel of the present invention is simple and can be widely used in clinical practice. DETAILED DESCRIPTION

[0022] The present invention provides a composition, which comprises the following components in parts by weight: 20 to 25 parts of insulin-like growth factor-1 (IGF-1), 8 to 10 parts of epidermal growth factor (EGF), 3 to 5 parts of basic fibroblast growth factor-2 (bFGF-2), 10 to 16 parts of human platelet-derived growth factor (PDGF), 5 to 10 parts of vascular endothelial growth factor C (VEGF-C), and 0.5 to 2.5 parts of transforming growth factor-β2 (TGF-β).

[0023] In the composition of the present invention, the mass fraction of insulin-like growth factor-1 is 20 to 25 parts, more preferably 22 to 24 parts, and more preferably 23 parts;

[0024] The mass fraction of epidermal growth factor is 8 to 10 parts, more preferably 8.5 to 9.5 parts, and more preferably 9 parts;

[0025] The mass fraction of basic fibroblast growth factor-2 is 3 to 5 parts, more preferably 3.5 to 4.5 parts, and more preferably 4 parts;

[0026] The mass fraction of human platelet-derived growth factor is 10 to 16 parts, more preferably 12 to 14 parts, and more preferably 13 parts;

[0027] The mass fraction of the vascular endothelial growth factor C is 5 to 10 parts, more preferably 7 to 9 parts, and even more preferably 8 parts.

[0028] The mass fraction of the transforming growth factor-β2 is 0.5 to 2.5 parts, more preferably 1 to 2 parts, and more preferably 1.5 parts.

[0029] In the present invention, insulin-like growth factor-1, epidermal growth factor, basic fibroblast growth factor-2, human platelet-derived growth factor, vascular endothelial cell growth factor C and transforming growth factor-β2 are used in combination in a specific ratio, which is beneficial to promoting wound healing and can shorten the wound healing time.

[0030] The present invention also provides application of the composition in preparing a medicine for treating wound surface repair.

[0031] The present invention also provides a composite cytokine hydrogel, comprising the following raw materials in percentage by weight: comprising the following raw materials in percentage by weight: the composition 2 to 8×10 -3 %, sodium alginate 2-4%, γ-polyglutamic acid 0.5-1.5%, chitosan 4-6%, propylene glycol 0.05-0.2%, polyethylene glycol 0.4-1.6%, β-sodium glycerophosphate 4-8%, and the balance water.

[0032] In the composite cytokine hydrogel of the present invention, the mass percentage of the composition is 2 to 8×10 -3 %, more preferably 4 to 6×10 -3 %, more preferably 5×10 -3 %;

[0033] The mass percentage of the sodium alginate is 2-4%, more preferably 2.5-3.5%, and more preferably 3%;

[0034] The mass percentage of the γ-polyglutamic acid is 0.5-1.5%, more preferably 0.8-1.2%, and more preferably 1.0%;

[0035] The mass percentage of the chitosan is 4-6%, more preferably 4.5-5.5%, and more preferably 5%;

[0036] The mass percentage of the propylene glycol is 0.05-0.2%, more preferably 0.1-0.15, more preferably 0.12%;

[0037] The mass percentage of the polyethylene glycol is 0.4-1.6%, more preferably 0.8-1.2%, and more preferably 1.0%;

[0038] The mass percentage of the sodium β-glycerophosphate is 4-8%, more preferably 5-7%, and more preferably 6%.

[0039] The viscosity average molecular weight of the chitosan is 570-600 KDa, more preferably 580-590 KDa, more preferably 585 KDa, and the deacetylation degree of the chitosan is 88-92%, more preferably 89-91%, more preferably 90%;

[0040] The polymerization degree of the γ-polyglutamic acid is 3000 to 5000, more preferably 3500 to 4500, and even more preferably 4000.

[0041] The present invention also provides a method for preparing the composite cytokine hydrogel, comprising the following steps:

[0042] (1) mixing chitosan with a hydrochloric acid solution to obtain a chitosan solution;

[0043] (2) mixing the chitosan solution with propylene glycol, polyethylene glycol, γ-polyglutamic acid and sodium alginate, stirring, and then adjusting the pH value to 6-6.5 with a NaOH aqueous solution to obtain a solution 1;

[0044] (3) Solution 1, sodium β-glycerophosphate and the composition are mixed, stirred, and ions are removed to obtain the composite cytokine hydrogel solution.

[0045] In the present invention, chitosan is first dissolved in a hydrochloric acid solution to obtain a chitosan solution. The concentration of the hydrochloric acid is 0.08 to 0.12 M, more preferably 0.09 to 0.11 M, and more preferably 0.1 M; the mass volume ratio of chitosan to hydrochloric acid is 4 to 6 g:90 mL, more preferably 4.5 to 5.5 g:90 mL, and more preferably 5 g:90 mL.

[0046] In the present invention, propylene glycol, polyethylene glycol, γ-polyglutamic acid and sodium alginate are added to the chitosan solution, stirred, and then the pH value is adjusted to 6 to 6.5, preferably 6.1 to 6.3, and more preferably 6.2 with a NaOH aqueous solution to obtain a solution 1. The concentration of the NaOH aqueous solution is 0.8 to 1.2 M, more preferably 0.9 to 1.1 M, and more preferably 1 M. The stirring is performed under ice bath conditions.

[0047] The invention mixes solution 1, sodium beta-glycerophosphate and the composition, stirs them, removes ions through a semipermeable membrane, and obtains the composite cytokine hydrogel, wherein the stirring is performed under ice bath conditions.

[0048] The rotation speeds of the two stirrings are 1000-1500 rpm, more preferably 1100-1400 rpm, and more preferably 1200 rpm; the stirring time is 10-15 min, more preferably 11-14 min, and more preferably 13 min.

[0049] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0050] The insulin-like growth factor-1 (IGF-1), epidermal growth factor (EGF), basic fibroblast growth factor-2 (bFGF), human platelet-derived growth factor, and vascular endothelial growth factor C (VEGF-C) in the examples of the present invention were purchased from Yisheng Biotechnology (Shanghai) Co., Ltd., and Wistar rats were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd.

[0051] Example 1

[0052] A composition consists of 120g of insulin-like growth factor, 8g of epidermal growth factor, 23g of basic fibroblast growth factor, 10g of human platelet-derived growth factor, 5g of vascular endothelial cell growth factor C and 20.5g of transforming growth factor-β.

[0053] Example 2

[0054] A composition consists of 3g insulin-like growth factor-12, 9g epidermal growth factor, 24g basic fibroblast growth factor-2, 13g human platelet-derived growth factor, 8g vascular endothelial cell growth factor C and 21g transforming growth factor-β.

[0055] Example 3

[0056] A composition comprises 125g of insulin-like growth factor, 10g of epidermal growth factor, 25g of basic fibroblast growth factor, 16g of human platelet-derived growth factor, 10g of vascular endothelial cell growth factor C and 22.5g of transforming growth factor-β.

[0057] Example 4

[0058] A composite cytokine hydrogel, comprising the following raw materials in percentage by weight: 2×10 -3 %, sodium alginate 2%, γ-polyglutamic acid 0.5%, chitosan 4%, propylene glycol 0.05%, polyethylene glycol 0.4%, β-sodium glycerophosphate 4%, and the balance is water. The polymerization degree of γ-polyglutamic acid is 3000, the viscosity-average molecular weight of chitosan is 572.3KDa, and the degree of deacetylation is 88.7%.

[0059] Preparation method: 40 g of chitosan was dissolved in 900 mL of 0.08 M hydrochloric acid solution to obtain a chitosan solution, 0.5 g of propylene glycol, 4 g of polyethylene glycol, 5 g of γ-polyglutamic acid, and 20 g of sodium alginate were added to the chitosan solution, stirred at 1000 rpm for 10 min in an ice bath, and then the pH value was adjusted to 6 with a 0.8 M NaOH aqueous solution to obtain solution 1. Solution 1, 40 g of sodium β-glycerophosphate and 20 mg of the composition of Example 1 were mixed, the volume of the solution was supplemented to 1 L with distilled water, stirred at 1000 rpm for 10 min in an ice bath, and the ions were removed by a semipermeable membrane to obtain the composite cytokine hydrogel solution.

[0060] Example 5

[0061] A composite cytokine hydrogel, comprising the following raw materials in percentage by weight: 5×10 -3 %, sodium alginate 3%, γ-polyglutamic acid 1.0%, chitosan 5%, propylene glycol 0.1%, polyethylene glycol 1.2%, β-sodium glycerophosphate 6%, and the balance is water. The polymerization degree of γ-polyglutamic acid is 4000, the viscosity-average molecular weight of chitosan is 586.5KDa, and the degree of deacetylation is 90.1%.

[0062] Preparation method: 50g chitosan was dissolved in 900mL 0.1M hydrochloric acid solution to obtain chitosan solution, 1g propylene glycol, 12g polyethylene glycol, 10g γ-polyglutamic acid, and 30g sodium alginate were added to the chitosan solution, stirred at 1200rpm for 13min under ice bath, and then the pH value was adjusted to 6.2 with 1M NaOH aqueous solution to obtain solution 1. Solution 1, 60g sodium β-glycerophosphate and 50mg of the composition of Example 2 were mixed, the volume of the solution was supplemented to 1L with distilled water, stirred at 1200rpm for 13min under ice bath, and the ions were removed by semipermeable membrane to obtain the composite cytokine hydrogel solution.

[0063] Example 6

[0064] A composite cytokine hydrogel, comprising the following raw materials in percentage by weight: 8×10 -3 %, sodium alginate 4%, γ-polyglutamic acid 1.5%, chitosan 6%, propylene glycol 0.2%, polyethylene glycol 1.6%, β-sodium glycerophosphate 8%, and the balance is water. The polymerization degree of γ-polyglutamic acid is 5000, the viscosity-average molecular weight of chitosan is 591.4KDa, and the degree of deacetylation is 91.2%.

[0065] Preparation method: 80g chitosan was dissolved in 900mL 0.12M hydrochloric acid solution to obtain chitosan solution, 2g propylene glycol, 16g polyethylene glycol, 10g γ-polyglutamic acid, and 40g sodium alginate were added to the chitosan solution, stirred at 1500rpm for 15min under ice bath, and then the pH value was adjusted to 6.5 with 1M NaOH aqueous solution to obtain solution 1. Solution 1, 80g β-sodium glycerophosphate and 80mg of the composition of Example 3 were mixed, the volume of the solution was supplemented to 1L with distilled water, stirred at 1500rpm for 15min under ice bath, and ions were removed by semipermeable membrane to obtain the composite cytokine hydrogel solution.

[0066] Comparative Example 1

[0067] Different from Example 1, human platelet-derived growth factor is not added to the composition.

[0068] Comparative Example 2

[0069] The difference from Example 1 is that transforming growth factor-β2 is replaced by transforming growth factor-β3.

[0070] Comparative Example 3

[0071] The difference from Example 1 is that the content of insulin-like growth factor-1 in the composition is 15 μg.

[0072] Comparative Example 4

[0073] Different from Example 4, the viscosity-average molecular weight of chitosan is 383 KDa.

[0074] Comparative Example 5

[0075] Different from Example 4, the viscosity-average molecular weight of chitosan is 751 KDa.

[0076] Comparative Example 6

[0077] Different from Example 4, no γ-polyglutamic acid was added when preparing the hydrogel.

[0078] Experimental Example 1

[0079] 70 clean Wistar rats, half male and half female, with a body weight of 200±20g, were anesthetized by intraperitoneal injection of 1% ketamine hydrochloride (3mL / kg). Local skin preparation and routine disinfection were performed, and a circular full-thickness skin wound model with a diameter of 1cm was made on the back of the rats. The model rats were randomly divided into 7 groups, each with 10 rats, and divided into a blank control group, Example 1 group, Example 2 group, Example 3 group, Comparative Example 1 group, Comparative Example 2 group and Comparative Example 3 group. The compositions of Examples 1 to 3 and Comparative Examples 1 to 3 were respectively prepared with deionized water into a solution containing growth factors with a concentration of 50μg / mL.

[0080] The blank control group applied 300 μL of physiological saline on the wound surface every day, and the Example 1-3 group and the Comparative Example 1-3 group were applied with the solution containing growth factors prepared by the composition of Example 1-3 and Comparative Example 1-3, respectively. The wound surface was administered once in the morning every day for 15 consecutive days, and the rats were killed on the 16th day after the wound surface administration. During the administration period, the wound surface of each group was traced once with a translucent weighing paper every 4 days, and the area and healing rate were calculated, and the average value was taken.

[0081] Wound healing rate = (1-wound area after treatment / original wound area) * 100%

[0082] Table 1 Wound healing rates of rats in different groups at 4d, 8d, 12d and 16d after administration

[0083]

[0084] Experimental Example 2

[0085] 70 clean Wistar rats, half male and half female, with a body weight of 200±20g, were anesthetized by intraperitoneal injection of 1% ketamine hydrochloride (3mL / kg). Local skin preparation, routine disinfection, and pressing and scalding the depilatory part of the rat's back with a weight preheated to 100°C for 8s to cause a deep II degree scald wound with a diameter of 1cm on the back. The model rats were randomly divided into 7 groups, each group of 10 was divided into a blank control group, Example 1 group, Example 2 group, Example 3 group, Comparative Example 1 group, Comparative Example 2 group and Comparative Example 3 group. The compositions of Examples 1 to 3 and Comparative Examples 1 to 3 were respectively formulated into a solution containing growth factors with a concentration of 50μg / mL with deionized water.

[0086] The blank control group was smeared with 300 μL of normal saline on the wound surface every day, and the Example 1-3 group and the Comparative Example 1-3 group were smeared with the solution containing growth factors prepared by the composition of the Example 1-3 and the Comparative Example 1-3, respectively. The wound surface was administered once in the morning every day, and the rats were killed in two batches on the 8th day and the 16th day of administration (5 rats in each batch and each group), and 0.5 cm*0.5 cm granulation tissue was cut from the wound edge, fixed in Carnoy's solution for 45 minutes, paraffin embedded, sliced, and stained with conventional HE. The light microscope and MPIAS-500 color pathology graphic analysis system were used for observation, and the number of fibroblasts in the visual field was counted. Five visual fields were observed for each specimen, and the number of fibroblasts in the granulation tissue was counted and the average value was taken. The mean number of fibroblasts in each visual field is shown in Table 2.

[0087] Table 2 Number of fibroblasts in granulation tissue of wound surface in each group

[0088]

[0089] Granulation tissue is mainly composed of new thin-walled capillaries and fibroblasts. Fibroblasts are the most important cells for wound repair. During the cell proliferation and repair period, one of the most critical steps is the formation of granulation tissue. The essence of granulation tissue is a large number of fibroblasts and capillaries. After the wound is formed, fibroblasts proliferate, migrate, synthesize and secrete collagen and extracellular matrix components, and participate in the process of granulation tissue formation, wound contraction and tissue reconstruction. Changes in their number will affect wound healing.

[0090] It can be seen from the test results in Table 1 and Table 2 that, as time goes by, Examples 1 to 3 have significant differences in the wound healing rate and the increase in the number of fibroblasts in rats compared with Comparative Examples 1 to 3, which shows that the composition of the present invention can increase the content of fibroblasts in the granulation tissue of the wound, thereby effectively and quickly promoting wound healing. If the components or contents therein are changed, the therapeutic effect is reduced.

[0091] Experimental Example 3

[0092] The experiment was divided into 6 groups, and hydrogels were prepared according to the methods of Examples 4 to 6 and Comparative Examples 4 to 6, with three replicates in each group. The hydrogel solutions prepared in each group were placed at 37°C, and the time for them to form a solid gel was observed and recorded, and the average value was taken. The time for each group of hydrogel solutions to form a solid gel is shown in Table 3.

[0093] Table 3 The time for each group of hydrogel solutions to form solid gel

[0094]

[0095] As shown in Table 3, the hydrogel solutions prepared according to the preparation methods of Examples 4 to 6 of the present invention can form a solid gel after being placed at 37° C. for 16 to 17 seconds. Changing the viscosity-average molecular weight of chitosan or not adding γ-polyglutamic acid will prolong the time it takes for the hydrogel solution to form a solid gel.

[0096] It can be seen from the above examples that the composition of the present invention can promote fibroblast proliferation, is beneficial to the appearance and function recovery of new skin, and allows wounds to heal quickly. The composite cytokine hydrogel has a strong repair effect and good tissue compatibility, and can form a solid gel after being placed at 37°C for 16 to 17 seconds. The preparation method of the composite cytokine hydrogel of the present invention is simple and can be widely used in clinical practice.

[0097] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A composition, characterized in that The invention is composed of the following components in parts by weight: 20-25 parts of insulin-like growth factor-1, 8-10 parts of epidermal growth factor, 3-5 parts of basic fibroblast growth factor-2, 10-16 parts of human platelet-derived growth factor, 5-10 parts of vascular endothelial cell growth factor C and 0.5-2.5 parts of transforming growth factor-β2.

2. Use of the composition according to claim 1 in the preparation of a medicine for treating wound repair.

3. A composite cytokine hydrogel, characterized in that: The raw materials comprising the following mass percentages: 2 to 8 × 10 -3 %, sodium alginate 2-4%, γ-polyglutamic acid 0.5-1.5%, chitosan 4-6%, propylene glycol 0.05-0.2%, polyethylene glycol 0.4-1.6%, β-sodium glycerophosphate 4-8%, and the balance water; The viscosity average molecular weight of the chitosan is 570-600 KDa, and the deacetylation degree is 88-92%.

4. The composite cytokine hydrogel according to claim 3, characterized in that: The polymerization degree of the gamma-polyglutamic acid is 3000-5000.

5. The method for preparing the composite cytokine hydrogel according to any one of claims 3 to 4, characterized in that: The following steps are involved: (1) mixing chitosan with a hydrochloric acid solution to obtain a chitosan solution; (2) mixing the chitosan solution with propylene glycol, polyethylene glycol, γ-polyglutamic acid and sodium alginate, stirring, and then adjusting the pH value to 6-6.5 with a NaOH aqueous solution to obtain a solution 1; (3) Solution 1, sodium β-glycerophosphate and the composition of claim 1 are mixed, stirred again, and ions are removed to obtain the composite cytokine hydrogel solution.

6. The preparation method according to claim 5, characterized in that: The concentration of the hydrochloric acid solution is 0.08-0.12M.

7. The preparation method according to claim 6, characterized in that: The mass volume ratio of the chitosan to the hydrochloric acid solution is 4-6 g:90 mL.

8. The preparation method according to claim 5, characterized in that: Step (2) and step (3) are carried out under ice bath conditions.

9. The preparation method according to claim 5, characterized in that: The stirring speeds in step (2) and step (3) are independently 1000-1500 rpm, and the stirring time is independently 10-15 min.

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