CN115678048B - Injectable composite hydrogel capable of promoting wound healing and reducing scar formation, and preparation method and application thereof - Google Patents

Abstract

The invention discloses an injectable composite hydrogel capable of promoting wound healing and reducing scar formation, and a preparation method and application thereof. The preparation method comprises the steps of taking an extracellular matrix as a main component to carry out chemical modification, loading mesenchymal stem cells and a verteporfin drug, and forming hydrogel through in-situ crosslinking by Schiff base reaction. The mesenchymal stem cells loaded by the injectable composite hydrogel can secrete anti-fibrosis cytokines to inhibit tissue fibrosis, and the verteporfin can inhibit YAP gene expression to prevent activation of Engrailed-1 (En 1) genes in the wound healing process, so that scar formation is prevented and reduced cooperatively. In addition, the extracellular matrix component in the composite hydrogel can effectively promote wound healing by combining with the immunoregulation and angiogenesis improvement effects of stem cells. The invention can be applied to the fields of scar formation prevention and reduction, wound repair, tissue engineering and the like.

Description

An injectable composite hydrogel that can promote wound healing and reduce scar formation, and its preparation method and application

Technical Field

The invention relates to the fields of biomedical materials and tissue engineering, and in particular to an injectable composite hydrogel capable of promoting wound healing and reducing scar formation, and a preparation method and application thereof.

Background technique

At present, the main methods for treating scars in medicine can be divided into surgical treatment and non-surgical treatment. Among them, non-surgical treatment is more widely used due to its advantages of low invasiveness, small damage, easy operation and short recovery time. Non-surgical treatment methods include physical therapy and drug therapy. Physical therapy mainly includes laser therapy, compression therapy, radiotherapy, cryotherapy and rehabilitation therapy. However, physical therapy takes a long time, among which laser therapy can only remove protruding skin, but cannot fill the concave place, and improper use of laser will also produce new scars or uneven spots. In the drug method, on the one hand, glucocorticoid drugs such as triamcinolone acetonide, compound betamethasone, and prednisolone are injected into the scar. However, this type of drug is prone to cause skin atrophy, capillary dilation, local skin pigmentation or loss at the injection site. In addition, hormone drugs interfere with the normal metabolism of pigments in the body. On the other hand, scar patches and scar removal ointments, such as silicone gel, polysulfonated mucopolysaccharide cream, and compound heparin sodium allantoin ointment, are used to reduce scar formation. However, these drugs usually take effect slowly and are prone to cause skin redness, swelling, pain, and allergies, and sometimes aggravate scar symptoms.

Mesenchymal stem cells (MSCs) can secrete anti-fibrotic cytokines, reduce the expression of α-SMA and type I collagen genes, and have a certain inhibitory effect on tissue fibrosis, thus having the potential to reduce scar formation. However, studies have reported that the effect of MSCs in treating scars is not significant. The main reason behind this is that the microenvironment at the wound is not conducive to the survival of stem cells and the tissue can clear the transplanted stem cells, which is not conducive to the secretion and function of stem cell factors. In addition, it is difficult to completely prevent or reduce scar formation by relying solely on anti-fibrotic cytokines secreted by stem cells. However, there are currently no relevant research reports or patent applications.

Summary of the invention

The purpose of the present invention is to overcome the deficiencies in the prior art and provide an injectable composite hydrogel and its preparation method and application that can promote wound healing and reduce scar formation. The injectable composite hydrogel is composed of a biomimetic extracellular matrix hydrogel scaffold, stem cells and verteporfin drugs. Among them, the biomimetic extracellular matrix hydrogel scaffold uses the main components of the extracellular matrix and its derivatives (hyaluronic acid, collagen or gelatin) as raw materials, and obtains oxidized hyaluronic acid, carbohydrazide collagen or carbohydrazide gelatin after oxidation reaction and amino modification. The biomimetic extracellular matrix hydrogel scaffold has good mechanical properties and is not easy to be enzymatically hydrolyzed, and can provide a compatible environment for stem cells to meet the needs of stem cell growth, proliferation and metabolism. At the same time, it has the effect of reducing scar formation. And because verteporfin can be slowly released in the hydrogel, it is avoided that the concentration of the drug in contact with the wound is too high, thereby prolonging the drug effect, reducing the frequency of administration and clinical burden. In addition, the injectable composite hydrogel can provide a mild oxygen-isolating condition for the wound, and provide a closed and moist environment for the scar, which is conducive to collagen rearrangement. Therefore, the present invention can maximize the effects of the bionic hydrogel scaffold, stem cells and verteporfin, synergistically prevent and reduce scar formation and promote wound healing.

To achieve the above object, the present invention adopts the following technical solution:

The present invention first provides a method for preparing an injectable composite hydrogel that can promote wound healing and reduce scar formation, comprising the following steps:

1) Dissolve hyaluronic acid in ultrapure water, add sodium periodate thereto, and perform oxidation reaction at room temperature for 2-3 hours, then add 0.5 mL of ethylene glycol to terminate the reaction for 1 hour; after the reaction, use ultrapure water as dialyzate, and dialyze the reaction solution for 72 hours using a dialysis bag with a molecular weight of 3500Da; after the dialysis, collect the liquid in the dialysis bag, transfer it to a polytetrafluoroethylene container, place it in a -80°C refrigerator and freeze it for 5 hours, and then freeze-dry it at -50°C and 1-20Pa for 72 hours to obtain an oxidized hyaluronic acid sponge material;

2) Dissolve the natural polymer in ultrapure water, add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide, activate the reaction at room temperature for 2 hours, then add carbohydrazide powder and react at room temperature for 12 hours; after the reaction, use ultrapure water as the dialysate, and dialyze the reaction solution using a dialysis bag with a molecular weight of 8000Da for 72 hours; after the dialysis, collect the liquid in the dialysis bag, transfer it to a polytetrafluoroethylene container, place it in a -80℃ refrigerator and freeze it for 5 hours, and then freeze-dry it at -50℃ and 1~20Pa for 72 hours to obtain the carbohydrazide natural polymer material;

3) preparing a solution of an oxidized hyaluronic acid sponge material with ultrapure water, and preparing a solution of a carbohydrazide natural polymer material with ultrapure water; adding mesenchymal stem cells to the oxidized hyaluronic acid sponge material solution to obtain a hydrogel precursor solution 1; adding verteporfin to the carbohydrazide natural polymer material solution to obtain a hydrogel precursor solution 2; mixing the hydrogel precursor solution 1 and the hydrogel precursor solution 2 evenly, and stirring at room temperature for 10 to 20 seconds until the mixed solution is in a gelatinous state, thereby obtaining the injectable composite hydrogel.

Furthermore, in the above step 1), the amount of hyaluronic acid used is 1 g, and the amount of sodium periodate used is 0.25 g.

Furthermore, in the above step 2), the natural polymer is selected from any one of collagen and gelatin.

Furthermore, in the above step 2), the amount of the natural polymer is 9 g, the amount of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is 8.64 g, the amount of N-hydroxysuccinimide is 0.58 g, and the amount of the carbohydrazide powder is 6.6 g.

Furthermore, in the above step 3), the concentration of the oxidized hyaluronic acid sponge material solution is 2% to 8%, and the concentration of the carbohydrazide natural polymer material solution is 4% to 12%.

Furthermore, in the above step 3), the mesenchymal stem cells are selected from any one of human adipose-derived mesenchymal stem cells and human bone marrow-derived mesenchymal stem cells.

Furthermore, in the above step 3), the volume ratio of the hydrogel precursor solution 1 to the hydrogel precursor solution 2 is 1:1.

Furthermore, in the above step 3), the concentration of mesenchymal stem cells in the injectable composite hydrogel is 1×10 ⁶ to 1×10 ⁷ cells/mL, and the concentration of verteporfin is 0.01 to 10 mg/mL.

The present invention also provides an injectable composite hydrogel prepared by the above preparation method.

The present invention also provides the use of the above-mentioned injectable composite hydrogel in the preparation of a medicine for promoting wound healing and/or reducing scar formation.

The beneficial effects of the present invention are:

1) The present invention provides a biomedical material, which uses a polymer material with good biocompatibility as a substrate to prepare a biodegradable injectable composite hydrogel, which can achieve the controlled release of cytokines and drugs.

2) The injectable composite hydrogel of the present invention can maintain the growth and proliferation of mesenchymal stem cells in vitro for more than 5 days, and the cytokines secreted by the mesenchymal stem cells can be continuously released to promote wound healing.

3) The injectable composite hydrogel of the present invention supports the slow release of verteporfin for more than 7 days, reduces the diffusion area of the drug, reduces the frequency of administration, achieves better scar treatment effect and reduces clinical burden.

4) The injectable composite hydrogel of the present invention can be used for wound healing and prevention and reduction of scar formation.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1: Schematic diagram of the biomimetic hydrogel scaffold prepared in Example 1 through a 30G needle syringe.

Figure 2: Microstructure of the biomimetic hydrogel scaffold prepared in Example 1.

FIG3 : Release curve of verteporfin in the biomimetic hydrogel scaffold prepared in Example 1.

FIG4 : Release curve of human bone marrow mesenchymal stem cells in the bionic hydrogel scaffold prepared in Example 2.

Figure 5: Shear thinning properties of the biomimetic hydrogel scaffold prepared in Example 2.

Figure 6: Live/Dead staining of mesenchymal stem cells in biomimetic hydrogel scaffolds.

Figure 7: Growth distribution of human bone marrow mesenchymal stem cells in biomimetic hydrogels.

Detailed ways

In order to make the contents of the present invention easier to understand, the technical solution of the present invention is further described below in conjunction with specific implementation methods, but the present invention is not limited thereto.

Example 1

Step 1): Weigh 1g hyaluronic acid (30kDa) and dissolve it in 100mL ultrapure water, add 0.25g sodium periodate thereto, and carry out oxidation reaction at room temperature for 2~3h, then add 0.5mL ethylene glycol to terminate the reaction at room temperature for 1h; after the reaction, use ultrapure water as the dialysate, and dialyze the reaction solution using a 3500Da molecular weight dialysis bag for 72h; after the dialysis, collect the liquid in the dialysis bag and transfer it to a polytetrafluoroethylene container, place it in a -80℃ refrigerator and freeze it for 5h, and then freeze-dry it at -50℃ and 1~20Pa for 72h to obtain the oxidized hyaluronic acid sponge material.

Step 2): Weigh 9g collagen (bovine source, type I, 30kDa) and dissolve it in 500mL ultrapure water, add 8.64g 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and 0.58g (N-hydroxysuccinimide (NHS), activate the reaction at room temperature for 2h, then add 6.6g carbohydrazide powder and react at room temperature for 12h; after the reaction, use ultrapure water as the dialysate, and dialyze the reaction solution using a dialysis bag with a molecular weight of 8000Da for 72h; after the dialysis, collect the liquid in the dialysis bag and transfer it to a polytetrafluoroethylene container, place it in a -80℃ refrigerator and freeze it for 5h, and then freeze-dry it at -50℃ and 1~20Pa for 72h to obtain the carbohydrazide collagen material.

Step 3): The oxidized hyaluronic acid sponge material is prepared into a solution with a concentration of 5wt% with ultrapure water, and the carbohydrazide collagen material is prepared into a solution with a concentration of 7wt% with ultrapure water; the 5wt% oxidized hyaluronic acid sponge material solution and the 7wt% carbohydrazide collagen material solution are mixed evenly in a volume ratio of 1:1, and stirred at room temperature for 10 to 20 seconds until the mixed solution becomes gel-like, thereby obtaining a bionic hydrogel scaffold.

Example 2

Step 1): Referring to step 1 of Example 1), prepare an oxidized hyaluronic acid sponge material.

Step 2): Weigh 9g gelatin (porcine source, type A, 300g bloom) and dissolve it in 500mL ultrapure water, add 8.64g 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and 0.58g (N-hydroxysuccinimide (NHS), activate the reaction at room temperature for 2h, then add 6.6g carbohydrazide powder and react at room temperature for 12h; after the reaction, use ultrapure water as the dialysate, and dialyze the reaction solution using a 8000Da molecular weight dialysis bag for 72h; after the dialysis, collect the liquid in the dialysis bag and transfer it to a polytetrafluoroethylene container, place it in a -80℃ refrigerator and freeze it for 5h, and then freeze-dry it at -50℃ and 1~20Pa for 72h to obtain the carbohydrazide gelatin material.

3) The oxidized hyaluronic acid sponge material is prepared into a solution with a concentration of 5wt% with ultrapure water, and the carbohydrazide gelatin material is prepared into a solution with a concentration of 7wt% with ultrapure water; the 5wt% oxidized hyaluronic acid sponge material solution and the 7wt% carbohydrazide gelatin material solution are evenly mixed in a volume ratio of 1:1, and stirred at room temperature for 10 to 20 seconds until the mixed solution becomes gelatinous, thereby obtaining a biomimetic hydrogel scaffold.

Figure 1 is a schematic diagram of the biomimetic hydrogel scaffold prepared in Example 1 through a 30G needle syringe. As can be seen from the figure, the biomimetic hydrogel scaffold prepared in Example 1 can be injected through a 30G needle syringe and can quickly gel for practical application.

Figure 2 shows the microstructure of the biomimetic hydrogel scaffold prepared in Example 1. Scanning electron microscope observation revealed that the biomimetic hydrogel scaffold had a porous structure with uniform pore size, which could absorb wound tissue exudate, facilitate the growth and proliferation of mesenchymal stem cells therein, the secretion of cytokines produced, and the slow release of verteporfin.

FIG3 shows the release curve of verteporfin in the biomimetic hydrogel scaffold prepared in Example 1. 200 μL of the biomimetic hydrogel scaffold prepared in Example 1 was taken and evenly mixed with a 1 mg/mL verteporfin solution, immersed in 1×PBS (PH=7.4) at 37°C, and the drug release was detected at fixed time intervals. The results show that the biomimetic hydrogel scaffold has a porous structure, which supports the slow release of drugs, thereby reducing the frequency of drug administration.

Figure 4 shows the release curve of human bone marrow mesenchymal stem cells in the biomimetic hydrogel scaffold prepared in Example 2. Human bone marrow mesenchymal stem cells were added to the biomimetic hydrogel scaffold to a final concentration of 3×10 ⁶ cells/mL, and the scaffold was cultured in a 37°C incubator. The culture medium was aspirated at fixed time points to detect the total content of cytokines secreted by the stem cells. The results show that the biomimetic hydrogel scaffold supports the release of cytokines secreted by mesenchymal stem cells when they grow therein.

Figure 5 shows the shear thinning properties of the biomimetic hydrogel scaffold prepared in Example 2. The biomimetic hydrogel scaffold was gelled (radius and height of 5 mm) and placed on the stage of the rheometer. The viscosity was investigated with a shear rate (0.01-100/s) at an angular frequency of 10 rad/s. It can be observed that the biomimetic hydrogel scaffold has shear thinning properties. As the shear force increases, the viscosity of the biomimetic hydrogel scaffold decreases, indicating that it is injectable.

FIG6 shows the Live/Dead staining of mesenchymal stem cells in a biomimetic hydrogel scaffold. The biomimetic hydrogel scaffold was extracted with serum-free medium to obtain an extract; the mesenchymal stem cells were planted in a 96-well plate, the biomimetic hydrogel scaffold extract was added, and the cells were cultured at 37°C. After 72 hours, the cells were stained with a Calcein-AM/PI kit for 10 to 20 minutes, and the morphology and growth of the cells were observed under a fluorescent inverted microscope (the biomimetic hydrogel scaffold prepared in Example 1 was loaded with human adipose mesenchymal stem cells, and the biomimetic hydrogel scaffold prepared in Example 2 was loaded with human bone marrow mesenchymal stem cells). It can be seen from the figure that the biomimetic hydrogel scaffolds prepared in Examples 1 and 2 have good cell compatibility, the mesenchymal stem cells have an elongated spindle-shaped morphology, and the number of red dead cells is very small.

Example 3

Step 1): Referring to step 1 of Example 1), prepare an oxidized hyaluronic acid sponge material.

Step 2): Refer to step 2 of Example 2) to prepare carbohydrazide collagen material and carbohydrazide gelatin material.

Step 3): The oxidized hyaluronic acid sponge material is prepared into a solution with a concentration of 5wt% with ultrapure water, and the carbohydrazide gelatin material is prepared into a solution with a concentration of 7wt% with ultrapure water; a human bone marrow mesenchymal stem cell suspension with a concentration of 6×10 ⁶ cells/mL is added to 100 μL of the 5wt% oxidized hyaluronic acid sponge material solution, and then mixed evenly with 100 μL of the 7wt% carbohydrazide gelatin material solution, and stirred at room temperature for 10 to 20 seconds until the mixed solution is gel-like, thereby obtaining an injectable composite hydrogel, wherein the concentration of mesenchymal stem cells in the injectable composite hydrogel is 3×10 ⁶ cells/mL.

FIG7 shows the growth and distribution of human bone marrow mesenchymal stem cells in the injectable composite hydrogel of this embodiment. Live/Dead staining was performed on the hydrogel loaded with mesenchymal stem cells on the first and fifth days after gelation, and the 3D distribution of stem cells in the hydrogel was observed by laser scanning confocal microscopy. It can be seen from the figure that from the first day to the fifth day, the number of human bone marrow mesenchymal stem cells in the injectable composite hydrogel increased.

Example 4

Step 1): Referring to step 1 of Example 1), prepare an oxidized hyaluronic acid sponge material.

Step 2): Referring to step 2 of Example 1), prepare carbohydrazide collagen material.

Step 3): The oxidized hyaluronic acid sponge material is configured with ultrapure water to a solution with a concentration of 5wt%, and the carbohydrazide collagen material is configured with ultrapure water to a solution with a concentration of 7wt%; a human adipose mesenchymal stem cell suspension with a concentration of 6×10 ⁶ cells/mL is added to 100 μL of the 5wt% oxidized hyaluronic acid sponge material solution to obtain a hydrogel precursor solution 1; a 2mg/mL verteporfin solution is added to 100 μL of the 7wt% carbohydrazide collagen material solution to obtain a hydrogel precursor solution 2; the hydrogel precursor solution 1 and the hydrogel precursor solution 2 are mixed uniformly in a volume ratio of 1:1, and stirred at room temperature for 10 to 20 seconds until the mixed solution is gel-like, thereby obtaining an injectable composite hydrogel, wherein the concentration of mesenchymal stem cells in the injectable composite hydrogel is 3×10 ⁶ cells/mL, and the concentration of verteporfin is 1 mg/mL.

Example 5

Step 1): Referring to step 1 of Example 1), prepare an oxidized hyaluronic acid sponge material.

Step 2): Refer to step 2 of Example 2) to prepare carbohydrazide collagen material and carbohydrazide gelatin material.

Step 3): The oxidized hyaluronic acid sponge material is configured with ultrapure water to a solution with a concentration of 5wt%, and the carbohydrazide gelatin material is configured with ultrapure water to a solution with a concentration of 7wt%; a human bone marrow mesenchymal stem cell suspension with a concentration of 6×10 ⁶ cells/mL is added to 100 μL of the 5wt% oxidized hyaluronic acid sponge material solution to obtain a hydrogel precursor solution 1; a 2mg/mL verteporfin solution is added to 100 μL of the 7wt% carbohydrazide gelatin material solution to obtain a hydrogel precursor solution 2; the hydrogel precursor solution 1 and the hydrogel precursor solution 2 are mixed uniformly in a volume ratio of 1:1, and stirred at room temperature for 10 to 20 seconds until the mixed solution is gelatinous, thereby obtaining an injectable composite hydrogel, wherein the concentration of mesenchymal stem cells in the injectable composite hydrogel is 3×10 ⁶ cells/mL, and the concentration of verteporfin is 1 mg/mL.

Example 6

New Zealand rabbits weighing 2.5-3 kg were anesthetized with sodium pentobarbital and isoflurane, and four wounds with a diameter of 10 mm were made on the hairless area inside the rabbit ears. The groups were set as: control group (200 μL of normal saline was injected into the wound, once every three days), simple mesenchymal stem cell group (200 μL of human adipose mesenchymal stem cell suspension with a concentration of 3×10 ⁶ cells/mL was injected into the wound, once every three days), simple verteporfin group (200 μL of verteporfin solution with a concentration of 1 mg/mL was injected into the wound, once every three days), mesenchymal stem cell-loaded hydrogel group (200 μL of the injectable composite hydrogel prepared in Example 3 was attached to the wound after gelation, and a new injectable composite hydrogel identical to the previous one was replaced every 3 days), mesenchymal stem cell- and verteporfin-loaded hydrogel group (200 μL of the injectable composite hydrogel prepared in Example 4 was attached to the wound after gelation, and a new injectable composite hydrogel identical to the previous one was replaced every 3 days), commercial 3M dressing group (commercial 3M dressing was attached to the wound, and a new dressing was replaced every 3 days), silicone gel (silicone gel was applied to the wound, once every 3 days). After the 15th day of treatment, the rabbits were euthanized, and the wound closure rate and SEI (scar elevation index) were statistically compared. The wound healing calculation formula is: Wound closure rate (%) = (A ₀ -A _t )/A ₀ ×100%, where A ₀ represents the wound area on day 0; At represents the wound area on day _t . The formula for calculating the scar elevation index (SEI) is: SEI= (A+B)/B, where A is the scar tissue thickness higher than the normal epidermis, and B is the normal epidermis thickness. The results are shown in the following table.

Table 1 Statistical results of wound closure rate

Table 2 Statistical results of CD31(+) blood vessels in wound healing

Table 3 SEI (Scar Elevation Index) Statistical Results

Note: SEI = 1, indicating that the scar is equal in height to the surrounding uninjured dermis; SEI > 1, indicating that the scar is elevated;

SEI = 2 means that the scar dermis thickness is 100% thicker than that of normal tissue.

SEI>2 means that the scar dermis thickness is more than 100% thicker than that of normal tissue.

As can be seen from the wound closure rate (Table 1), compared with the commercial 3M dressing, the wound closure rate of the hydrogel group loaded with mesenchymal stem cells and the hydrogel group loaded with mesenchymal stem cells and verteporfin was significantly improved. Since the hydrogel substrate is selected from natural polymer materials, it has good biocompatibility itself and the hydrogel is loaded with mesenchymal stem cells. The porous structure of the hydrogel supports the secretion of stem cell factors and promotes wound repair. In addition, in histological staining, on the 15th day, the wound epithelial regeneration completeness rate of the hydrogel group loaded with mesenchymal stem cells and the hydrogel group loaded with mesenchymal stem cells and verteporfin was higher than that of the commercial 3M dressing group. In the process of wound healing, blood vessels need to be generated to promote the restoration of skin structure and function. The statistical results of the number of CD (+) blood vessels in Table 2 show that on the 10th day, the number of CD31 (+) blood vessels in the hydrogel group loaded with mesenchymal stem cells was 22.38% ± 0.69%, which was higher than 11.41% ± 1.00% in the commercial 3M dressing group. The results show that the hydrogel group loaded with mesenchymal stem cells can better promote wound angiogenesis and re-epithelialization.

From the SEI (Scar Elevation Index) in Table 3, it can be seen that the hydrogel group loaded with mesenchymal stem cells and verteporfin has the best scar treatment effect, with an SEI index of 1.26±0.02. This may be due to the fact that the surface is covered with hydrogel to achieve hypoxia, water retention and moisturizing effects, the mesenchymal stem cells secrete cytokines to promote wound healing, and the anti-fibrotic factors secreted in the late stage of wound healing inhibit tissue fibrosis. In addition, it contains verteporfin, an effective scar removal ingredient, and achieves excellent scar removal effects. The values of the hydrogel group loaded with mesenchymal stem cells are 1.78±0.15; the verteporfin group alone is 1.70±0.18; the mesenchymal stem cell group alone is 2.27±0.04; and the silicone gel group is 2.75±0.16. The silicone gel only provides a closed and moist environment for scar tissue, which promotes partial improvement of fibroblasts and collagen in scar tissue, such as the rearrangement of collagen close to the normal skin order. In the wound healing process, the hydrogel group loaded with mesenchymal stem cells and verteporfin, the slow release of verteporfin on the inhibition of YAP gene expression will block En1 activation and promote ENF-mediated repair, resulting in skin regeneration within 30 days, functional hair follicles and sebaceous glands restoration without fibrosis. Specifically, during scar formation, the Engrailed-1 gene in the Engrailed-1 lineage negative fibroblasts (ENF) cells specific to the deep tissue of the skin is activated and expressed under the action of YAP protein, which will initiate a pro-fibrotic cell program in response to local tissue mechanics in the wound. Verteporfin is a YAP inhibitor that can block the action of YAP, thereby inhibiting fibrosis and reducing scar formation.

Example 7

30kg/Duroc red pigs were taken and 5mL/pill was subcutaneously injected with 100 mg/mL dimethylaminothiazide hydrochloride solution. The back of the Duroc red pigs was then cleaned, depilated and disinfected, and then 18 wounds (2cm×2cm) were established symmetrically on both sides of the midline of the pig's back. The groups were set as: control group (200 μL of normal saline was injected into the wound once every three days), simple mesenchymal stem cell group (200 μL of human adipose mesenchymal stem cell suspension with a concentration of 3×10 ⁶ /mL was injected into the wound once every three days), simple verteporfin group (200 μL of verteporfin solution with a concentration of 1 mg/mL was injected into the wound once every three days), mesenchymal stem cell hydrogel group (200 μL of the injectable composite hydrogel prepared in Example 3 was attached to the wound after gelation, and a new injectable composite hydrogel identical to the previous one was replaced every three days), mesenchymal stem cell and verteporfin hydrogel group (200 μL of the injectable composite hydrogel prepared in Example 4 was attached to the wound after gelation, and a new injectable composite hydrogel identical to the previous one was replaced every three days), silicone gel (silicone gel was applied to the wound once every three days). After the 60th day of treatment, the wound tissue of Duroc Red Pig was taken and the SEI (scar elevation index) was calculated.

From the SEI, it can be seen that the scar treatment effect of the hydrogel group loaded with mesenchymal stem cells and verteporfin is closest to that of normal skin, with an SEI index of 1.36±0.05. This may be due to the fact that the surface is covered with hydrogel, which has the effect of hypoxia, water retention and moisturizing. The hydrogel group loaded with mesenchymal stem cells secretes cytokines to promote wound healing, and secretes anti-fibrotic factors to inhibit tissue fibrosis in the later stage of wound healing. It also contains verteporfin, an effective scar removal ingredient, and achieves excellent scar removal effects. The SEI index of the hydrogel group loaded with mesenchymal stem cells is 1.61±0.11; the verteporfin group alone is 1.58±0.04; the mesenchymal stem cell group alone is 2.37±0.13; and the silicone gel group is 2.17±0.08. The silicone gel only provides a closed and moist environment for scar tissue, which promotes partial improvement of fibroblasts and collagen in scar tissue, such as the rearrangement of collagen close to the order of normal skin.

The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention should fall within the scope of the present invention.

Claims (9)

1. A method for preparing injectable composite hydrogel capable of promoting wound healing and reducing scar formation, which is characterized by comprising the following steps: the method comprises the following steps:

1) Dissolving hyaluronic acid in ultrapure water, adding sodium periodate into the solution, performing oxidation reaction at room temperature for 2-3 hours, and adding 0.5mL of ethylene glycol to terminate the reaction for 1 hour; after the reaction, using ultrapure water as a dialyzate, and dialyzing the reaction solution for 72 hours by using a dialysis bag with molecular weight of 3500 Da; after the dialysis is finished, collecting liquid in a dialysis bag, transferring the liquid into a polytetrafluoroethylene container, freezing the container in a refrigerator at the temperature of minus 80 ℃ for 5 hours, and then freeze-drying the container at the temperature of minus 50 ℃ under the pressure of 1-20 Pa for 72 hours to obtain the oxidized hyaluronic acid sponge material;

2) Dissolving natural high polymer in ultrapure water, adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide, performing activation reaction at room temperature for 2 hours, adding carbohydrazide powder, and performing reaction at room temperature for 12 hours; after the reaction, using ultrapure water as a dialyzate, and dialyzing the reaction solution for 72 hours by using a dialysis bag with a molecular weight of 8000 Da; after the dialysis is finished, collecting liquid in a dialysis bag, transferring the liquid into a polytetrafluoroethylene container, freezing the container in a refrigerator at the temperature of minus 80 ℃ for 5 hours, and then freeze-drying the container at the temperature of minus 50 ℃ under the pressure of 1-20 Pa for 72 hours to obtain the natural high polymer carbohydrazide material;

3) Preparing an oxidized hyaluronic acid sponge material into a solution by using ultrapure water, and preparing a carbohydrazide natural high polymer material into a solution by using ultrapure water; adding mesenchymal stem cells into the oxidized hyaluronic acid sponge material solution to obtain hydrogel precursor solution 1; adding verteporfin into a carbohydrazide natural high polymer material solution to obtain a hydrogel precursor solution 2; uniformly mixing the hydrogel precursor solution 1 and the hydrogel precursor solution 2, and stirring at room temperature for 10-20 s until the mixed solution is gelatinous, thus obtaining the injectable composite hydrogel;

the natural polymer is selected from any one of collagen and gelatin.

2. The method of manufacturing according to claim 1, characterized in that: in the step 1), the amount of the hyaluronic acid is 1g, and the amount of the sodium periodate is 0.25g.

3. The method of manufacturing according to claim 1, characterized in that: in the step 2), the amount of the natural polymer is 9g, the amount of the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride is 8.64g, the amount of the N-hydroxysuccinimide is 0.58g, and the amount of the carbohydrazide powder is 6.6g.

4. The method of manufacturing according to claim 1, characterized in that: in the step 3), the concentration of the oxidized hyaluronic acid sponge material solution is 2% -8%, and the concentration of the carbohydrazide natural high polymer material solution is 4% -12%.

5. The method of manufacturing according to claim 1, characterized in that: in the step 3), the mesenchymal stem cells are selected from any one of human adipose-derived mesenchymal stem cells and human bone marrow-derived mesenchymal stem cells.

6. The method of manufacturing according to claim 1, characterized in that: in the step 3), the volume ratio of the hydrogel precursor solution 1 to the hydrogel precursor solution 2 is 1:1.

7. The method of manufacturing according to claim 1, characterized in that: in the step 3), the concentration of the injectable composite hydrogel mesenchymal stem cells is 1X 10 ⁶~1×10⁷/mL, and the concentration of the verteporfin is 0.01-10 mg/mL.

8. An injectable composite hydrogel prepared by the method of claim 1.

9. Use of the injectable composite hydrogel according to claim 8, for the following purposes: the application of the injectable composite hydrogel in preparing medicaments for promoting wound healing and/or reducing scar formation.