WO2024109777A1

WO2024109777A1 - Artificial skin stent, biological printing method, and method for culturing artificial skin

Info

Publication number: WO2024109777A1
Application number: PCT/CN2023/133081
Authority: WO
Inventors: 赵晓丽; 边少荃; 朱昊
Original assignee: 中国科学院深圳先进技术研究院
Priority date: 2022-11-23
Filing date: 2023-11-21
Publication date: 2024-05-30
Also published as: CN115737936A; CN115737936B

Abstract

Provided are an artificial skin stent and a printing method. The artificial skin stent comprises hydrogel layers (10) and fiber membrane layers (20). The hydrogel layers (10) include a lower hydrogel layer (11), a middle hydrogel layer (12), and an upper hydrogel layer (13). The lower hydrogel layer (11) and the middle hydrogel layer (12) are of a stacked grid structure, and the upper hydrogel layer (13) is of a stacked dense structure. The fiber membrane layers (20) are disposed between adjacent hydrogel layers (10). The fiber membrane layers (20) are fiber membranes having a porous structure. According to the described artificial skin stent and a method for culturing an artificial skin, the artificial skin is provided with enhanced mechanical performance by means of adding the fiber membrane material, and can be fixed to a skin tissue at the edge of a wound by means of suture, thus promoting the repair and regeneration of skin tissue defects.

Description

Artificial skin scaffold, bioprinting method and artificial skin cultivation method

Technical Field

The present application relates to the technical field of biomedical materials, and in particular to an artificial skin scaffold, a bioprinting method, and an artificial skin cultivation method.

Background technique

As the largest organ in the human body, the skin mainly acts as a barrier to protect the human body. It can effectively shield the body from harmful substances from the outside world, ensure the balance of water and electrolytes in the human body, and maintain body temperature. For some minor skin injuries, the skin can quickly repair itself and regenerate. However, large-area skin injuries caused by accidents and diseases will destroy the barrier protection of the skin, seriously threatening human life and health.

At present, autologous skin transplantation is an effective means of clinical treatment of large-area skin injuries and is also the gold standard. However, it will cause new skin damage to patients, and if the area of skin damage is too large, there will be a problem of insufficient donors. In addition, autologous transplantation using other people's skin is greatly restricted due to factors such as insufficient donors, immune rejection, and medical ethical issues. The xenotransplantation technology using animals such as pigs has the risk of immune rejection and viral infection and cannot fully meet clinical treatment needs. Traditional dressing products cannot quickly establish epidermal barriers and vascular networks at the site of skin defects, cannot mention real skin tissue, and have limited actual treatment effects.

In recent years, the development of tissue engineering technology is expected to provide a new and effective way for the repair and regeneration of large-area skin injuries. Tissue-engineered skin usually involves planting skin cells cultured in vitro on a tissue engineering scaffold and culturing them in vitro to form a skin substitute with an epidermis-dermis double-layer structure. Although tissue-engineered skin is a major improvement over wound dressings, it still lacks the bionics of real skin and cannot fully realize the functions of dermal skin, such as multi-layer anisotropic structure, vascular network, epidermal barrier, and reproduction of accessory structures such as sweat glands and hair follicles. Recently, bioprinting technology has shown great advantages in the manufacture of tissue-engineered artificial skin. It can achieve precise arrangement of cells and scaffold materials, and can better bionic the complex structure and cell distribution of real skin tissue, thereby obtaining an artificial skin substitute that is close to the function of real skin.

At present, the design concept of bioprinted artificial skin is mainly based on the biomimetic structure and composition of real skin, which usually includes two layers of structure, epidermis and dermis, and some designs will add a layer of subcutaneous tissue. The artificial dermis layer is constructed by loading dermal fibroblasts with printing ink, and the artificial epidermis layer is constructed by planting epidermal keratinocytes on the surface of the artificial dermis. The printing ink used is usually hydrogel, which includes natural polymer materials such as gelatin, hyaluronic acid, sodium alginate and chitosan, as well as synthetic polymers with good biosafety such as polyethylene glycol. However, the bioprinted artificial skin made of hydrogel ink material is limited by the mechanical properties of hydrogel, and the mechanical properties of artificial skin are weak, which is easy to be damaged during actual use. In addition, bioprinted artificial skin cannot be fixed to the skin tissue at the edge of the wound by suturing like transplanted skin tissue, because the mechanical properties of hydrogel cannot support the pulling of sutures, which causes the artificial skin to move easily on the wound surface during use, affecting the final treatment effect.

technical problem

In view of this, it is necessary to provide an artificial skin scaffold, a bioprinting method and an artificial skin culture method that promote the repair and regeneration of skin tissue defects in order to address the defects in the prior art.

Technical Solutions

To solve the above problems, this application adopts the following technical solutions:

One of the purposes of the present application is to provide an artificial skin scaffold, including a hydrogel layer and a fiber membrane layer, wherein the hydrogel layer includes a bottom hydrogel layer, a middle hydrogel layer and a top hydrogel layer, the bottom gel layer and the middle gel layer are in a grid structure of multiple layers stacked together, the top gel layer is in a dense structure of multiple layers stacked together, the fiber membrane layer is arranged between adjacent hydrogel layers, and the fiber membrane layer has a fiber membrane with a porous structure.

The second object of the present application is to provide a bioprinting method of the artificial skin scaffold, comprising the following steps:

printing the bottom hydrogel layer;

Disposing the fiber membrane layer on the surface of the bottom hydrogel layer;

Printing the middle hydrogel layer on the surface of the fiber membrane layer;

Arranging the fiber membrane layer on the surface of the middle hydrogel layer;

The top hydrogel layer is printed on the surface of the fiber membrane layer.

In some embodiments, the step of printing the bottom hydrogel layer specifically includes the following steps:

The printer is used to extrude hydrogel ink strips, wherein the hydrogel ink strips are alternately arranged in a cross shape to form a multi-layer superimposed grid structure, so as to print the bottom hydrogel layer.

In some embodiments, the step of printing the middle hydrogel layer on the surface of the fiber membrane layer specifically includes the following steps:

A printer is used to extrude hydrogel ink strips, wherein the hydrogel ink strips are alternately arranged in a cross shape to form a multi-layer superimposed grid structure, so as to print a middle hydrogel layer on the surface of the fiber membrane layer.

In some embodiments, the step of printing the top hydrogel layer on the surface of the fiber membrane layer specifically includes the following steps:

A printer is used to extrude hydrogel ink strips, wherein the hydrogel ink strips are closely arranged in parallel to form a multi-layered dense structure, so as to print a top hydrogel layer on the surface of the fiber membrane layer.

In some of the embodiments, it is characterized in that the hydrogel ink is prepared by the following method: dissolving methacryloyl gelatin and methacryloyl hyaluronic acid in deionized water, adding a photoinitiator and human dermal fibroblasts, cooling to 4-10° C. for storage, and obtaining the hydrogel ink.

In some of the embodiments, the methacryloyl gelatin is prepared by the following steps: dissolving gelatin in deionized water, adding dropwise a tetrahydrofuran solution containing methacrylic anhydride, and adjusting the pH value to 8-10 at the same time; after the reaction is completed, precipitating with ethanol, and collecting the precipitate by centrifugation; dissolving the collected precipitate with deionized water, dialyzing it in deionized water using a dialysis bag, and then freeze-drying it to obtain the methacryloyl gelatin.

In some embodiments, the methacryloyl hyaluronic acid is prepared by the following steps: adding methacrylic anhydride solution to a hyaluronic acid solution in deionized water and adjusting the pH to 8-10 at the same time; after the reaction is completed, precipitating with ethanol and collecting the precipitate by centrifugation; dissolving the collected precipitate with deionized water, dialyzing it in deionized water using a dialysis bag, and then freeze-drying it to obtain the methacryloyl hyaluronic acid.

In some of the embodiments, the fiber membrane layer is prepared by the following steps: dissolving polylactic acid polyhydroxyacetic acid polymer and gelatin in a hexafluoroisopropanol solution, using a stainless steel plate with a porous structure as a receiving substrate, preparing an electrospun fiber membrane material with an evenly distributed porous structure, and obtaining the fiber membrane layer.

The third object of the present application is to provide a method for culturing artificial skin of the artificial skin scaffold, comprising the following steps:

Planting HKCs cells on the surface of the top hydrogel layer;

After the artificial skin scaffold was immersed in a special culture medium and cultured for 3-7 days, the artificial skin scaffold was transferred to a Transwell device, and the top hydrogel layer was exposed to the air and cultured at the air-liquid interface for 14 days to obtain an artificial skin with an epidermal structure.

In some embodiments, the special culture medium formula is: DMEM/F12=3/1 (v/v), supplemented with 1% FBS and 1% HKGS.

Beneficial Effects

This application adopts the above technical solution, and its beneficial effects are as follows:

The artificial skin scaffold and printing method provided in the present application include a hydrogel layer and a fiber membrane layer, wherein the hydrogel layer includes a bottom hydrogel layer, a middle hydrogel layer and a top hydrogel layer, wherein the bottom gel layer and the middle gel layer are in a grid structure of multiple layers stacked together, and the top gel layer is in a dense structure of multiple layers stacked together, and the fiber membrane layer is arranged between adjacent hydrogel layers, wherein the fiber membrane layer has a fiber membrane with a porous structure. The above-mentioned artificial skin scaffold and artificial skin cultivation method obtain enhanced mechanical properties by adding fiber membrane materials, and can be fixed to the skin tissue at the edge of the wound by suturing, thereby promoting the repair and regeneration of skin tissue defects.

BRIEF DESCRIPTION OF THE DRAWINGS

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

FIG1 is a schematic diagram of an artificial skin support structure provided in an embodiment of the present application.

FIG2 is a flow chart of the steps of the bioprinting method of the artificial skin scaffold provided in an embodiment of the present application.

FIG3 is a fluorescence photograph of live cell staining after printing with cells loaded with hydrogel ink provided in Example 1 of the present application.

FIG4 is a schematic diagram of an electrospun fiber membrane with a porous structure provided in Example 1 of the present application.

FIG5 is a diagram showing the superior tear resistance of the fiber membrane-added hydrogel scaffold provided in Example 1 of the present application compared with the pure hydrogel scaffold.

FIG6 is a diagram showing the fiber membrane hydrogel scaffold provided in Example 1 of the present application being tightly integrated with the skin at the edge of the wound through suturing.

FIG. 7 is a schematic diagram of an artificial skin scaffold cultured in a Transwell device provided in Example 1 of the present application.

Embodiments of the present invention

The embodiments of the present application are described in detail below, and examples of the embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to be used to explain the present application, and should not be construed as limiting the present application.

In the description of the present application, it should be understood that the terms "upper", "lower", "horizontal", "inside", "outside", etc., indicating orientations or positional relationships, are based on the orientations or positional relationships shown in the accompanying drawings, and are only for the convenience of describing the present application and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore should not be understood as a limitation on the present application.

In addition, the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of the features. In the description of this application, the meaning of "plurality" is two or more, unless otherwise clearly and specifically defined.

In order to make the objectives, technical solutions and advantages of the present application more clearly understood, the present application is further described in detail below in conjunction with the accompanying drawings and embodiments.

Please refer to Figure 1, which is a structural schematic diagram of an artificial skin scaffold provided by an embodiment of the present application, including: a hydrogel layer 10 and a fiber membrane layer 20, the hydrogel layer 10 includes a bottom hydrogel layer 11, a middle hydrogel layer 12 and a top hydrogel layer 13, the bottom gel layer 11 and the middle gel layer 12 are in a grid structure of multiple layers stacked together, the top gel layer 13 is in a dense structure of multiple layers stacked together, the fiber membrane layer 20 is arranged between adjacent hydrogel layers, and the fiber membrane layer 20 has a fiber membrane with a porous structure.

The artificial skin scaffold provided in the above-mentioned embodiment of the present application obtains enhanced mechanical properties by adding fiber membrane materials, and can be fixed to the skin tissue at the edge of the wound by suturing, thereby promoting the repair and regeneration of skin tissue defects.

Please refer to FIG. 2 , which is a bioprinting method for an artificial skin scaffold provided in another embodiment of the present application, including the following steps S110 to S150 , and the implementation method of each step is described in detail below.

Step S110: printing the bottom hydrogel layer.

In some of the embodiments, the step of printing the bottom hydrogel layer specifically includes the following steps: using a printer to extrude hydrogel ink strips, wherein the hydrogel ink strips are arranged alternately in a cross shape to form a multi-layer superimposed grid structure to print the bottom hydrogel layer.

Step S120: disposing the fiber membrane layer on the surface of the bottom hydrogel layer.

Step S130: printing the middle hydrogel layer on the surface of the fiber membrane layer.

In some of the embodiments, the step of printing the middle hydrogel layer on the surface of the fiber membrane layer specifically includes the following steps: using a printer to extrude hydrogel ink strips, wherein the hydrogel ink strips are arranged alternately in a cross shape to form a multi-layer superimposed grid structure, so as to print the middle hydrogel layer on the surface of the fiber membrane layer.

Step S140: disposing the fiber membrane layer on the surface of the middle hydrogel layer.

Step S150: printing the top hydrogel layer on the surface of the fiber membrane layer.

In some of the embodiments, the step of printing the top hydrogel layer on the surface of the fiber membrane layer specifically includes the following steps: using a printer to extrude hydrogel ink strips, wherein the hydrogel ink strips are parallel and closely arranged to form a multi-layer stacked dense structure, so as to print the top hydrogel layer on the surface of the fiber membrane layer.

In some of the embodiments, it is characterized in that the hydrogel ink is prepared by the following method: dissolving methacryloyl gelatin and methacryloyl hyaluronic acid in deionized water, adding a photoinitiator and human dermal fibroblasts, cooling to 4°C-10°C for storage, and obtaining the hydrogel ink.

The bioprinting method of the artificial skin scaffold provided in the above-mentioned embodiment of the present application obtains enhanced mechanical properties by adding fiber membrane materials, and can be fixed to the skin tissue at the edge of the wound by suturing, thereby promoting the repair and regeneration of skin tissue defects.

The present application also provides a method for culturing artificial skin of the artificial skin scaffold, comprising the following steps:

Step S210: planting HKCs cells on the surface of the top hydrogel layer.

Step S220: After immersing the artificial skin scaffold in a special culture medium for 3-7 days, the artificial skin scaffold is transferred to a Transwell device, and the top hydrogel layer is exposed to the air for air-liquid interface culture for 14 days to obtain an artificial skin with an epidermal structure.

It can be understood that the hydrogel ink is composed of methacryloyl gelatin (Gel-MA) and methacryloyl hyaluronic acid (HA-MA), and is loaded with human dermal fibroblasts. The three hydrogel layers serve as the artificial skin dermis. Human epidermal keratinocytes are planted on the surface of the top dense hydrogel layer as the artificial skin epidermis. The fiber membrane between the two adjacent hydrogel layers is prepared by electrospinning and has an evenly distributed pore structure.

The artificial skin cultivation method provided in the above embodiment obtains enhanced mechanical properties by adding fiber membrane materials, and can be fixed to the skin tissue at the edge of the wound by suturing, thereby promoting the repair and regeneration of skin tissue defects.

The present invention is described in detail below based on examples.

Embodiment 1

1) Preparation of bioprinting ink

A certain amount of methacryloyl gelatin (Gel-MA), methacryloyl hyaluronic acid (HA-MA) and photoinitiator LAP were fully dissolved in PBS buffer (0.0067 M, pH = 7.2-7.4), and then a certain amount of human dermal fibroblasts (HDFs) were added and evenly dispersed into the solution. The final content of each component in the bioprinting ink was: Gel-MA 10 wt%, HA-MA 2 wt%, LAP 0.2 wt%, HDFs 1 million/mL.

2) Preparation of electrospun fiber membrane with porous structure

First, polylactic acid polyglycolic acid polymer (PLGA) and gelatin are dissolved in a hexafluoroisopropanol solution to obtain a spinning solution with a PLGA content of 15 wt% and a gelatin content of 1 wt%. Then, a stainless steel plate with a porous structure is used as a receiving substrate, and an electrospinning apparatus is used to prepare a fiber membrane with a porous structure. The specific parameters are: 5 mL syringe, spinning propulsion speed of 1.5 cm/hour, voltage of 15 kV, receiving distance of 15 cm, spinning time of 5 min, and pore structure diameter of 2 mm. Please refer to Figure 3, which is a schematic diagram of an electrospinning fiber membrane with a porous structure provided in this embodiment.

3) Culture of human keratinocytes (HKCs)

HKCs cells were cultured in epidermal differentiation medium, the composition of which was EpLife + 1% epidermal differentiation supplement (HKGS). After the cells were cultured to full growth in the culture dish, a cell suspension was prepared.

4) 3D printed artificial skin scaffold

The artificial skin scaffold is composed of three layers of hydrogel and two layers of fiber membrane stacked in sequence. The hydrogel layer is divided into a bottom layer, a middle layer and a top layer, and a fiber membrane with a porous structure is added between two adjacent hydrogel layers. The overall dimensions of the scaffold are: 2 cm long, 2 cm wide, and 2.5 mm thick.

The hydrogel layer was prepared by extruding ink strips using a 3D printer. Before printing, the bioprinting ink was cooled at 4°C for 30 minutes and then loaded into the printer. The printing parameters were: printing temperature 19°C, ink extrusion speed 0.8 μL/s, print head movement speed 6 mm/s, triggering light source 405 nm, and printing ink strip height 0.15 mm.

For the bottom hydrogel layer, the spacing between the strips is 1.5 mm. First, multiple parallel strips are extruded to form the first layer, and then multiple parallel strips are extruded perpendicular to the direction of the first layer strips to form the second layer. Repeat this process to print out a bottom hydrogel layer with 6 layers layer by layer, with a height of 0.9 mm.

A porous fiber membrane with a length of 2 cm and a width of 2 cm is placed on the bottom hydrogel layer.

For the middle hydrogel layer, printing was performed on the surface of the porous fiber membrane in the same way as the bottom hydrogel layer. Six layers were printed with a strip spacing of 1.2 mm and a height of 0.9 mm.

A porous fiber membrane with a length of 2 cm and a width of 2 cm is placed on the middle hydrogel layer.

For the top hydrogel layer, printing was performed on the porous fiber membrane surface. First, multiple parallel strips were extruded to form the first layer, and then multiple parallel strips were extruded in the same direction as the first layer of strips to form the second layer. This process was repeated to print out a bottom hydrogel layer with 4 layers, with a strip spacing of 0.8 mm and a height of 0.6 mm.

After printing, HKCs cells were planted on the surface of the top hydrogel layer at a number of 1 million/cm2. The artificial skin scaffold was then immersed in a special culture medium for culture. The special culture medium formula is: DMEM/F12=3/1 (v/v), with 1% FBS and 1% HKGS added.

5) Subsequent culture of artificial skin scaffold

After 3 days of culturing the artificial skin scaffolds immersed in a special culture medium, the scaffolds were transferred to the Transwell device, and the top hydrogel layer was exposed to the air and cultured at the air-liquid interface for 14 days to obtain artificial skin with epidermal structure. Please refer to Figure 4 for an artificial skin scaffold cultured in a Transwell device. Figure 5 is a H&E staining of a tissue section after 14 days of air-liquid interface culture of the artificial skin scaffold, showing the appearance of epidermal structure.

6) Anti-stretching properties of artificial skin

An artificial skin scaffold with a length of 3 cm, a width of 1 cm, and a thickness of 2.5 mm was prepared, and the tensile properties of the scaffold were tested using a universal material tester. The test conditions were: tensile speed 10 mm/min.

The pure hydrogel scaffold without porous fiber membrane was used as the control group.

7) Tear resistance of artificial skin

An artificial skin scaffold with a length of 3 cm, a width of 1 cm, and a thickness of 2.5 mm was prepared, and a 2 mm long slit was cut in the center. The tear resistance of the scaffold was tested using a universal material tester. The test conditions were: tensile speed 10 mm/min.

A pure hydrogel scaffold without porous fiber membrane was used as the control group.

8) Sutureability of artificial skin

An artificial skin scaffold with a length of 3 cm, a width of 2 cm, and a thickness of 2.5 mm was prepared, and the two ends were sutured to the pig skin with surgical sutures, and a weight of 100 g was pulled up for testing.

The artificial skin provided in the above embodiment obtains enhanced mechanical properties by adding fiber membrane materials, and can be fixed to the skin tissue at the edge of the wound by suturing, thereby promoting the repair and regeneration of skin tissue defects.

It can be understood that the technical features of the above-described embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

The above are only preferred embodiments of the present application, and only specifically describe the technical principles of the present application. These descriptions are only for explaining the principles of the present application and cannot be interpreted as limiting the scope of protection of the present application in any way. Based on the explanation here, any modifications, equivalent substitutions and improvements made within the spirit and principles of the present application, and other specific implementation methods of the present application that can be associated with the technicians in this field without creative work, should be included in the scope of protection of the present application.

Claims

An artificial skin scaffold, characterized in that it includes a hydrogel layer and a fiber membrane layer, the hydrogel layer includes a bottom hydrogel layer, a middle hydrogel layer and a top hydrogel layer, the bottom gel layer and the middle gel layer are in a grid structure of multiple layers stacked together, the top gel layer is in a dense structure of multiple layers stacked together, the fiber membrane layer is arranged between adjacent hydrogel layers, and the fiber membrane layer is a fiber membrane with a porous structure.
A bioprinting method for an artificial skin scaffold as claimed in claim 1, characterized in that it comprises the following steps:

printing the bottom hydrogel layer;

Disposing the fiber membrane layer on the surface of the bottom hydrogel layer;

Printing the middle hydrogel layer on the surface of the fiber membrane layer;

Arranging the fiber membrane layer on the surface of the middle hydrogel layer;

The top hydrogel layer is printed on the surface of the fiber membrane layer.
The bioprinting method of the artificial skin scaffold according to claim 2, characterized in that, in the step of printing the bottom hydrogel layer, the following steps are specifically included:

The printer is used to extrude hydrogel ink strips, wherein the hydrogel ink strips are alternately arranged in a cross shape to form a multi-layer superimposed grid structure, so as to print the bottom hydrogel layer.
The bioprinting method of the artificial skin scaffold according to claim 2, characterized in that in the step of printing the middle hydrogel layer on the surface of the fiber membrane layer, the following steps are specifically included:

A printer is used to extrude hydrogel ink strips, wherein the hydrogel ink strips are alternately arranged in a cross shape to form a multi-layer superimposed grid structure, so as to print a middle hydrogel layer on the surface of the fiber membrane layer.
The bioprinting method of the artificial skin scaffold according to claim 2, characterized in that in the step of printing the top hydrogel layer on the surface of the fiber membrane layer, the following steps are specifically included:

A printer is used to extrude hydrogel ink strips, wherein the hydrogel ink strips are closely arranged in parallel to form a multi-layered dense structure, so as to print a top hydrogel layer on the surface of the fiber membrane layer.
The bioprinting method of an artificial skin scaffold as described in claim 3, 4 or 5, characterized in that the hydrogel ink is prepared by the following method: dissolving methacryloyl gelatin and methacryloyl hyaluronic acid in deionized water, adding a photoinitiator and human dermal fibroblasts, and cooling to 4°C-10°C for storage to obtain the hydrogel ink.
The bioprinting method of an artificial skin scaffold as described in claim 6 is characterized in that the methacryloyl gelatin is prepared by the following steps: dissolving gelatin in deionized water, adding a tetrahydrofuran solution containing methacrylic anhydride, and adjusting the pH = 8-10 at the same time; after the reaction is completed, precipitating with ethanol, and collecting the precipitate by centrifugation; dissolving the collected precipitate with deionized water, dialyzing it in deionized water using a dialysis bag, and then freeze-drying it to obtain the methacryloyl gelatin.
The bioprinting method of an artificial skin scaffold as described in claim 6 is characterized in that the methacryloyl hyaluronic acid is prepared by the following steps: adding a methacrylic anhydride solution to a hyaluronic acid solution in deionized water and adjusting the pH to 8-10 at the same time; after the reaction is completed, precipitating with ethanol and collecting the precipitate by centrifugation; dissolving the collected precipitate with deionized water, dialyzing it in deionized water using a dialysis bag, and then freeze-drying it to obtain the methacryloyl hyaluronic acid.
The bioprinting method of an artificial skin scaffold as described in claim 6 is characterized in that the fiber membrane layer is prepared by the following steps: dissolving polylactic acid polyglycolic acid polymer and gelatin in a hexafluoroisopropanol solution, using a stainless steel plate with a pore structure as a receiving substrate, preparing an electrospun fiber membrane material with an evenly distributed pore structure, and obtaining the fiber membrane layer.
A method for culturing artificial skin based on the artificial skin scaffold according to claim 1, characterized in that it comprises the following steps:

Planting HKCs cells on the surface of the top hydrogel layer;

After the artificial skin scaffold was immersed in a special culture medium and cultured for 3-7 days, the artificial skin scaffold was transferred to a Transwell device, and the top hydrogel layer was exposed to the air and cultured at the air-liquid interface for 14 days to obtain an artificial skin with an epidermal structure.
The method for culturing artificial skin of an artificial skin scaffold according to claim 10, characterized in that the special culture medium formula is: DMEM/F12=3/1 (v/v), and 1% FBS and 1% HKGS are added.