CN118750659A - Preparation method of hydrogel with tissue repair and anti-oxidation functions and hydrogel thereof - Google Patents

Abstract

The present invention provides a preparation method of a hydrogel with tissue repair and anti-oxidation functions and the hydrogel thereof, wherein the hydrogel is a solution comprising: recombinant human type I collagen, recombinant human type III collagen, or a mixture of recombinant human type I collagen and recombinant human type III collagen, a solution of methacryloyl chitosan containing a photoinitiator and total saponins of Panax notoginseng; the mass volume concentration of the total saponins of Panax notoginseng is not more than 0.6%. The present invention proposes a hydrogel with tissue repair and anti-oxidation functions, which is suitable for a variety of tissue repair, antibacterial and fibrosis prevention, and can also exhibit the functions of anti-oxidation and free radical scavenging.

Description

Preparation method of hydrogel with tissue repair and anti-oxidation functions and hydrogel thereof

Technical Field

The invention relates to the field of materials, and in particular to a preparation method of a hydrogel with tissue repair and anti-oxidation functions and the hydrogel.

Background Art

One of the main causes of infertility in women of childbearing age is the damage to the endometrium due to congenital or acquired diseases, surgery, etc., which leads to reduced endometrial receptivity. Intrauterine adhesion (IUA), also known as Asherman’s syndrome (AS), is usually caused by mechanical damage such as artificial abortion, curettage or infection, and manifests as damage to the basal layer of the endometrium, which in turn causes tissue adhesion and fibrosis, ultimately leading to decreased fertility.

At present, the main clinical treatment method is hysteroscopic surgery, which is to separate the adhesion site through surgery. This method has a high recurrence rate and is prone to secondary injury. The treatment methods of drug infusion or oral medication also have obvious defects, such as: the effective ingredients are easily lost, which is only suitable for immediate injury treatment and cannot solve the subsequent healing and repair problems; at the same time, it is also easy to cause late wound adhesion and wound infection.

After injury, the wound site will cause the accumulation of reactive oxygen and angiogenesis disorders, which will make it difficult to heal. Scavenging oxygen free radicals, inhibiting inflammatory responses, and inducing angiogenesis are the key to treating the wound site. Therefore, a new type of material that can resist oxidation and scavenge free radicals is needed to better promote wound healing.

Summary of the invention

In order to solve the above problems, the present invention provides a method for preparing a hydrogel with tissue repair and antioxidant functions, wherein the hydrogel comprises the following solution: recombinant human type I collagen, recombinant human type III collagen, or a mixture of recombinant human type I collagen and recombinant human type III collagen, a solution of methacryloylated chitosan containing a photoinitiator and notoginseng total saponins, wherein when the solution is subjected to a viscosity test at 37° C. and a shear rate of 1/s, the viscosity is 3000±3000*15% mPa·s to 15000±15000*15% mPa·s, preferably, the viscosity is 3000±3000*10% mPa·s to 15000±15000*10% mPa·s, and more preferably, the viscosity is 3000±3000*5% mPa·s to 15000±15000*5% mPa·s. mPa·s; when the solution is applied to the corresponding tissue, it is irradiated externally with ultraviolet light or visible blue light, and the irradiation is stopped after the solution becomes a gel, so as to prepare the hydrogel; the mass volume concentration of the total saponins of Panax notoginseng is not greater than 0.6%, that is, the mass of the total saponins of Panax notoginseng in 100 ml of the solution is not greater than 0.6 g.

Human collagen is divided into more than 28 types of collagen according to different tissue locations, physiological functions, and molecular structures. Among them, type I collagen and type III collagen are closely related to the skin damage repair process and repair quality. From the distribution point of view, type I collagen is mainly found in adult skin, tendons, and bone tissue; type III collagen is mainly found in infant skin or vascular endothelium and intestines. Type I and type III collagen in the human body maintain the normal tissue structure of the skin, constitute the extracellular matrix network structure, play a role in supporting organs and protecting the body, and are also related to cell attachment and cell migration. The balanced ratio of the two also determines the hardness and tenderness of the skin, as well as the degree of wound healing and scarring. Therefore, when recombinant human collagen is used in tissue repair, both type I and type III collagen can effectively promote repair and tissue regeneration.

In one embodiment, the mass volume concentration of the total saponins of Panax notoginseng is 0.4% to 0.6%.

In one embodiment, the photoinitiator is lithium phenyl-2,4,6-trimethylbenzoylphosphite or (2-hydroxy-2-methyl-1-[4-(2-hydroxyethoxy)phenyl]-1-propanone).

In one embodiment, when the viscosity of the hydrogel is tested at 37° C. and a shear rate of 1/s, the viscosity is 10025.85 mPa·s to 3163.85 mPa·s.

In one embodiment, when the mass volume concentration of the methacryloylated chitosan is 8%, that is, 100 ml of the solution includes 8 g of methacryloylated chitosan, and when the mass volume concentration of recombinant human type I collagen, recombinant human type III collagen, or a mixture of recombinant human type I collagen and recombinant human type III collagen is 30%, that is, 100 ml of the solution includes 30 g of recombinant human type I collagen, recombinant human type III collagen, or a mixture of recombinant human type I collagen and recombinant human type III collagen, the volume ratio of the methacryloylated chitosan and the recombinant human type I collagen, recombinant human type III collagen, or the mixture of recombinant human type I collagen and recombinant human type III collagen is 3:1-1:1.

In one embodiment, the volume ratio of the methacryloylated chitosan and the recombinant human type I collagen, recombinant human type III collagen, or a mixture of recombinant human type I collagen and recombinant human type III collagen is 1:1, and the mass volume concentration of the notoginseng total saponins is 0.6%.

In one embodiment, a hydrogel for preventing and treating endometrial damage is provided, wherein the hydrogel is prepared by the above method.

In one embodiment, a hydrogel for preventing and treating intrauterine adhesions and thin endometrium is provided, wherein the hydrogel is prepared by the above method.

In one embodiment, a hydrogel for preventing and treating intrauterine adhesions and thin endometrium is provided, wherein the hydrogel comprises a solution of recombinant human type I collagen, recombinant human type III collagen, or a mixture of recombinant human type I collagen and recombinant human type III collagen, methacryloyl chitosan containing a photoinitiator, and notoginseng total saponins. When the solution is applied to a corresponding tissue, it is irradiated externally with ultraviolet light or visible blue light, and the irradiation is stopped after the solution becomes a gel state to prepare the hydrogel. When the solution is subjected to a viscosity test at 37°C and a shear rate of 1/s, the viscosity is 3000 mPa·s to 15000 mPa·s. The mass volume concentration of notoginseng total saponins is not greater than 0.6%, that is, the mass of notoginseng total saponins in 100 ml of solution is not greater than 0.6 g.

In one embodiment, when the mass volume concentration of the methacryloylated chitosan is 8%, when the mass volume concentration of recombinant human type I collagen, recombinant human type III collagen, or a mixture of recombinant human type I collagen and recombinant human type III collagen is 30%, the volume ratio of the methacryloylated chitosan and the recombinant human type I collagen, recombinant human type III collagen, or a mixture of recombinant human type I collagen and recombinant human type III collagen is 1:1-1:1, and the mass volume concentration of the notoginseng total saponins is 0.6%.

Tissue damage can occur in various organs or tissues of the human body, usually including external trauma, bacterial infection, surgical side effects, etc. If it is not treated promptly and effectively, it may lead to wound infection, slow healing, scar hyperplasia, and even necrosis. Based on methacrylylated chitosan, collagen, and Panax notoginseng total saponins, the present invention proposes a preparation method of a hydrogel with tissue repair and antioxidant functions and its hydrogel, which can be injected into the corresponding tissue in its liquid state, and can be photocrosslinked and cured to form a gel under the irradiation of ultraviolet light or visible light, firmly adhere to the wound, promote wound healing, and at the same time exhibit antioxidant free radical scavenging functions. The present invention can be applied to a variety of tissue repair, antibacterial and prevention of fibrosis, including visceral damage repair, endometrial repair, cartilage repair, superficial skin repair, etc.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

FIG1 is a diagram showing the repair effect of a hydrogel of the present invention on endometrial damage in rats.

DETAILED DESCRIPTION

In order to enable those skilled in the art to better understand the technical solutions in the present application, the present invention will be further described below in conjunction with the following embodiments. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in the field without creative work should belong to the scope of protection of this application. The present invention will be further described below in conjunction with the accompanying drawings and embodiments.

Example 1. Preparation of raw materials of the present invention

1. Preparation of methacryloyl chitosan (CSMA)

Chitosan powder was added to 0.1 mol/L acetic acid solution with a mass volume fraction of 0.5%–5%, and stirred at room temperature until completely dissolved. The chitosan solution was placed in a 60 ℃ water bath, and methacrylic anhydride was added dropwise, wherein the weight ratio of chitosan to methacrylic anhydride was 1:2-1:3. After the addition was completed, the reaction was stirred at 60 ℃ for 6-12 h. After the reaction was completed, 0.5 mol/L NaOH solution was slowly added dropwise to the reaction solution to adjust the pH of the reaction solution to neutral. The neutral reactant solution was dialyzed in deionized water for 72 h, and the water was changed every 6 h. The dialyzed solution was frozen at -80 ℃ and vacuum freeze-dried for 72 h, and the freeze-dried powder product obtained was methacrylylated chitosan.

2. Preparation of recombinant human collagen type I or type III (RC) solution

Dissolve recombinant human type I collagen or type III collagen powder in PBS buffer and stir at 37°C until completely dissolved.

3. Preparation of Panax notoginseng saponins (PNS) solution

Dissolve the total saponin powder of Panax notoginseng in PBS buffer and stir at room temperature until it is completely dissolved.

4. Preparation of Methacrylylated Chitosan-Collagen+Panax Notoginseng Saponins Hydrogel Precursor Solution

Take methacryloyl chitosan powder, dissolve it in PBS buffer, add 0.25% photoinitiator at the same time, and stir at room temperature for 12 h until it is completely dissolved. Then add collagen solution and Panax notoginseng total saponin solution, and stir at 4°C in the dark. Among them, the mass volume concentration of methacryloyl chitosan is 8% (w/v), the mass volume concentration of collagen is 30% (w/v), and the mass volume concentration of Panax notoginseng total saponin solution is 10% (w/v).

5. Hydrogel Preparation Method

When the methacrylylated chitosan-collagen + Panax notoginseng total saponin hydrogel precursor solution is used for injury repair, the solution is first injected into the corresponding tissue, and then the corresponding tissue is irradiated externally with ultraviolet light (365 nm) or visible blue light (405 nm), and the filling solution is stopped after it becomes a gel. At this time, the hydrogel is highly adherent in a gel state to prevent tissue adhesion; at the same time, the repair effect of various components can be better exerted to promote the repair and regeneration of damaged tissues. The material and application method are suitable for the repair of various wounds, especially irregularly shaped wounds, such as epidermal damage, endometrial damage, cartilage regeneration, vaginal mucosal damage, etc., and can remove excessive free radicals in the site of oxidative stress of the trauma. The mixed solution can quickly change from liquid to gel under the irradiation of ultraviolet light (365 nm) or visible blue light (405 nm).

Example 2. Viscosity test of methacrylylated chitosan-collagen solution at different volume ratios

Methacryloyl chitosan (CSMA)-collagen (RC) solutions with different volume ratios were dripped onto the sample stage of the rheometer, and the viscosity was tested at 37 °C and a shear rate of 1/s.

Table 1 Viscosity of CSMA:RC solutions with different ratios

Viscosity test data show that changing the volume ratio of methacryloyl chitosan-collagen will directly affect the viscosity of the solution. When the ratio of CSMA:RC is greater than 3:1, the viscosity is lower than 3000 mPa·s, and its viscosity is small and fluidity is strong, which is not conducive to staying in the uterine cavity; when the ratio of CSMA:RC is less than 1:1, the viscosity is higher than 15000 mPa·s, and its viscosity is too high to achieve injectable type, and it is difficult to completely cover the damaged tissue and achieve the therapeutic effect. Therefore, it is believed that when CSMA:RC=1~3:1, the solution viscosity is suitable for use as a drug delivery platform in subsequent experiments.

Example 3 Experiment on the Cytocompatibility of Composite Hydrogels Loaded with Different Ratios of Total Panax Notoginseng Saponins

In a methacryloyl chitosan-collagen hydrogel precursor solution with a volume ratio of CSMA:RC=1:1, several different proportions of 10% Panax notoginseng total saponin solutions are loaded to form several different proportions of methacryloyl chitosan-collagen + Panax notoginseng total saponin hydrogel precursor solutions. The loading ratios of the 10% Panax notoginseng total saponin solution are 2%, 4%, 6%, 8%, and 10%, that is, a total of 100 ml of the solution contains 2 ml, 4 ml, 6 ml, 8 ml and 10 ml of the 10% Panax notoginseng total saponin solution by mass volume.

Each group of precursor solutions was injected into a six-well plate, and after ultraviolet irradiation sterilization for 30 min, methacrylylated chitosan-collagen + Panax notoginseng total saponin hydrogels (respectively denoted as 2% hydrogel, 4% hydrogel, 6% hydrogel, 8% hydrogel, and 10% hydrogel) were formed. Each group of hydrogels and DMEM complete culture medium (DMEM basal medium, 10% fetal bovine serum and 1% double antibody, the double antibody is streptomycin and penicillin) were placed in a 50 ml sterile centrifuge tube to obtain a hydrogel concentration of 0.1g/mL. The centrifuge tube containing the sample was placed in a 37°C, 5% CO ₂ incubator for 72 h to obtain a hydrogel extract. Human endometrial stromal cells HESCs were seeded in a 96-well plate at a density of 1×10 ³ /100 μL cell suspension per well and cultured in a 37°C, 5% CO ₂ incubator for 24 h. The culture medium was discarded, and 200 μL of extract was added to each well to set up the experimental group. At the same time, a control group was set up to continue culturing with 200 μL of DMEM complete culture medium per well. Each group was cultured for 24 h, 48 h, and 72 h at the same time for observation, and 5 replicate wells were set up for each time point in each group. During the measurement, the old culture medium was discarded, 100 μL of DMEM complete culture medium and 10 μL of CCK8 solution were added to each well, pipetted evenly, and incubated at 37°C for 1.5 h, and then the absorbance was measured at 450 nm using an enzyme reader.

The calculation formula of cell viability is:

The calculated average cell survival rate is shown in the table below. According to the data in the table, the cell survival rate of the 8% hydrogel group was lower than 95% after 72 h, and the cell survival rate of the 10% hydrogel group was lower than 95% after 48 h. Therefore, it is considered that when the total saponin loading ratio of Panax notoginseng is greater than 8%, it is not suitable for normal cell growth and subsequent in vivo experiments.

It is believed that when the loading ratio of Panax notoginseng total saponins is no more than 8%, the methacrylylated chitosan-collagen + Panax notoginseng total saponins hydrogel has good cell compatibility, is suitable for use on wounds, and is beneficial to wound healing.

Table 2 Effects of hydrogels with different loading ratios of Panax notoginseng total saponins on cell survival rate

Example 4 Effect of different concentrations of total saponins of Panax notoginseng on the antioxidant capacity of hydrogel

Oxidative stress refers to a state in which the body produces excessive amounts of highly active molecules such as reactive oxygen and reactive nitrogen free radicals when it is subjected to various harmful stimuli, the degree of oxidation exceeds the ability to remove oxides, and the oxidation system and antioxidant system are dynamically unbalanced, leading to cell and tissue damage. Studies have shown that oxidative stress caused by an imbalance in the body's redox level is the pathophysiological basis of many diseases, and the defense against oxidative stress mainly relies on the body's antioxidant system. DPPH, or diphenylpicrylhydrazine, is a stable nitrogen-centered chromogenic free radical that produces a characteristic absorption peak at a wavelength of 515 nm. The antioxidants in the sample will reduce the absorbance of DPPH until it disappears, and the change in its absorbance is linearly related to the content of antioxidants within a certain range.

In a methacryloyl chitosan-collagen hydrogel precursor solution with a volume ratio of CSMA:RC=1:1, several different proportions of Panax notoginseng total saponin solutions were loaded to form several different proportions of methacryloyl chitosan-collagen + Panax notoginseng total saponin hydrogel precursor solutions, and the loading ratios of the Panax notoginseng total saponin solutions were 2%, 4%, and 6%.

Each group of precursor solutions was injected into a six-well plate, and after UV curing and sterilization for 30 min, methacryloyl chitosan-collagen + Panax notoginseng total saponin hydrogels (respectively recorded as 2% hydrogel, 4% hydrogel, and 6% hydrogel) were formed. Each group of hydrogels was taken out and placed in a -80℃ refrigerator for quick freezing for 1 hour, and then placed in a freeze vacuum dryer for vacuum drying for 48-72 hours to prepare freeze-dried samples. 0.1 g of dry sample was placed at the bottom of a centrifuge tube, 1 mL of PBS buffer was added, and it was placed in a 37℃ incubator for incubation for 0.5-1 h, and then the extract was taken out and centrifuged (6000 rmp, 10 min), and the supernatant was taken. The concentration of DPPH reagent was prepared to be 0.04 mg/mL. 950 μL of DPPH reagent and 50 μL of sample extract supernatant were added to the EP tube. The blank group was set up with 950 μL of DPPH reagent and 50 μL of PBS buffer, and the control group was set up with 950 μL of DPPH reagent and 50 μL of anhydrous ethanol. After thorough mixing, 100 μL was added to a 96-well plate, and the plate was tested at a wavelength of 515 nm using an ELISA reader, and the OD value was recorded. Five replicate wells were set up for each group.

DPPH free radical scavenging rate = (OD blank - OD sample + OD control) / OD blank * 100%

The DPPH free radical scavenging rate of each group is shown in the table below. It can be seen that the average DPPH free radical scavenging rate of the 2% hydrogel group is only 1.1904%, which is difficult to improve the state of oxidative stress at the wound site. The 4% hydrogel and 6% hydrogel groups have a more significant free radical scavenging effect and can defend against oxidative stress to a large extent. Therefore, it is believed that when the total saponin loading ratio of Panax notoginseng is greater than 4%, the composite hydrogel can have a better antioxidant effect.

Table 3 DPPH radical scavenging rate of hydrogels with different loading ratios of Panax notoginseng total saponins

Example 4 Effects of different ratios of methacrylylated chitosan-collagen hydrogels on the repair ability of endometrial damage in rats

The rat endometrial injury repair experiment was established. Female SD rats aged 8 weeks, weighing about 250 g, and sexually mature were selected and cultured for one week. After the rats were anesthetized with isoflurane, the abdominal skin was prepared, the abdominal cavity was cut open, and the Y-shaped uterus was found. The upper and lower ends of the uterus were clamped with hemostatic clamps, and a 1 mL syringe was inserted into the rat uterus. 95% alcohol was injected until it was filled. After 60 seconds, the uterus was rinsed several times with normal saline. At this time, different concentrations of methacryloyl chitosan-collagen + Panax notoginseng total saponin hydrogel precursor solutions (CRP1, CRP2, CRP3) were injected into the injured uterus, and then irradiated externally with ultraviolet light or blue light for about 60 seconds, which showed that it was in a gel state. After suturing and disinfection, the rats were kept and observed. At the same time, an alcohol injury control group (referred to as Control) was set up. All rats were killed 7 days after surgery, and the uterine tissue was taken for photographs and fixed in 4% paraformaldehyde for 48 hours. After gradient dehydration with ethanol, paraffin embedding, and section staining, the observation was observed.

As shown in Figure 1, obvious congestion can be seen in the uterus of the alcohol-injured control group, and there are no obvious wounds in the uterus of the CRP1, CRP2, and CRP3 groups. There is a very small amount of material remaining in the uterus, which is in a gel-like state. From the results of H&E staining, it can be seen that the rat uterine epithelium is lost after alcohol injury, the number of stromal cells is small, the arrangement is loose, and it shows symptoms of thin endometrium, and the repair effect is poor. Each of the CRP1, CRP2, and CRP3 groups has a certain degree of repair, the thickness of the endometrium increases, the number of stromal cells increases, obvious glands can be seen, and the uterine cavity is completely covered with epithelium. Among them, the repair effect of the CRP1 group is more significant. The above results show that the CRP1 group, i.e., CSMA:RC=1:1, methacrylylated chitosan-collagen + Panax notoginseng total saponin hydrogel loaded with 6% Panax notoginseng total saponin, has good antioxidant capacity and also has the ability to promote endometrial damage repair and wound healing.

It should be understood that the disclosed invention is not limited only to the specific method, scheme and material of description, because these all can change.It should also be understood that the terminology used herein is only for the purpose of describing specific embodiment scheme, rather than being intended to limit the scope of the present invention, and the scope of the present invention is only limited to the appended claims.

Those skilled in the art will also recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are also intended to be encompassed by the appended claims.

Claims (10)

1. The preparation method of the hydrogel with tissue repair and antioxidation functions is characterized in that the hydrogel is a solution comprising the following components: recombinant human type I collagen, recombinant human type III collagen or a mixture of recombinant human type I collagen and recombinant human type III collagen, a solution containing a photoinitiator of methacryloylated chitosan and total saponins of panax notoginseng, wherein when the solution is subjected to viscosity test under the conditions of 37 ℃ and a shear rate of 1/s, the viscosity is 3000+/-3000 x 15% mPas-15000+/-15000 x 15% mPas; when the solution is applied to corresponding tissues, ultraviolet light or visible blue light is used for external irradiation, and the hydrogel is prepared after the solution becomes gel; the mass and volume concentration of the total saponins of panax notoginseng is not more than 0.6 percent, namely the mass of the total saponins of panax notoginseng in the 100 ml solution is not more than 0.6: 0.6 g.

2. The method for preparing the hydrogel according to claim 1, wherein the mass and volume concentration of the total saponins of panax notoginseng is 0.4% -0.6%.

3. The method of preparing a hydrogel according to claim 1, wherein the photoinitiator is phenyl-2, 4, 6-trimethylbenzoyl lithium phosphite or (2-hydroxy-2-methyl-1- [4- (2-hydroxyethoxy) phenyl ] -1-propanone).

4. The method for preparing a hydrogel according to claim 1, wherein the viscosity of the hydrogel is 10025.85 mPa s to 3163.85 mPa s when the hydrogel is subjected to a viscosity test at 37 ℃ and a shear rate of 1/s.

5. The method according to claim 1, wherein the 100 ml solution contains 8% of the methacryloylated chitosan, and the 100 ml solution contains 30% of the recombinant human type i collagen, the recombinant human type iii collagen, or the mixture of the recombinant human type i collagen and the recombinant human type iii collagen, and the volume ratio of the methacryloylated chitosan to the recombinant human type i collagen, the recombinant human type iii collagen, or the mixture of the recombinant human type i collagen and the recombinant human type iii collagen is 3:1 to 1:1.

6. The method according to claim 5, wherein the volume ratio of the methacryloylated chitosan to the recombinant human type I collagen, the recombinant human type III collagen, or the mixture of the recombinant human type I collagen and the recombinant human type III collagen is 1:1, and the total saponins of panax notoginseng mass and volume concentration is 0.6%.

7. A hydrogel for controlling endometrial damage, characterized in that it is prepared by the method of any one of claims 1-6.

8. A hydrogel for the prevention and treatment of intrauterine adhesions and thin endometrium, characterized in that it is prepared by the method according to any one of claims 1-6.

9. The hydrogel for preventing and treating intrauterine adhesion and thin endometrium is characterized by comprising recombinant human type I collagen, recombinant human type III collagen or a mixture of the recombinant human type I collagen and the recombinant human type III collagen, a solution containing photoinitiator of methacryloyl chitosan and total saponins of pseudo-ginseng, and when the solution is applied to corresponding tissues, ultraviolet light or visible blue light is used for irradiation outside, and the hydrogel is prepared after the solution becomes gel; when the viscosity of the solution is tested under the conditions of 37 ℃ and a shear rate of 1/s, the viscosity is 3000 mPa & s-15000 mPa & s; the mass and volume concentration of the total saponins of panax notoginseng is not more than 0.6 percent, namely the mass of the total saponins of panax notoginseng in the 100ml solution is not more than 0.6: 0.6 g.

10. The hydrogel of claim 9, wherein when the methacryloylated chitosan has a mass to volume concentration of 8%, the methacryloylated chitosan and the recombinant human type i collagen, the recombinant human type iii collagen, or the mixture of the recombinant human type i collagen and the recombinant human type iii collagen has a mass to volume concentration of 30%, the ratio of the methacryloylated chitosan to the recombinant human type i collagen, the recombinant human type iii collagen, or the mixture of the recombinant human type i collagen and the recombinant human type iii collagen is 1:1, and the total saponins of panax notoginseng has a mass to volume concentration of 0.6%.