KR102321505B1 - Manufacturing method of 3D artificial skin with wound and 3D artificial skin with wound - Google Patents

Abstract

The present invention relates to a method for manufacturing 3D artificial skin with a wound part, comprising: a dermal layer forming step of forming a dermal layer containing biocompatible polymers and fibroblasts; a sacrificial layer forming step of forming a sacrificial layer by applying a sacrificial material to a portion of an upper surface of the dermal layer; an epidermal layer forming step of dispensing and culturing keratinocytes on the dermal layer on which the sacrificial layer is formed to form the epidermal layer; and a sacrificial layer removing step of removing the sacrificial layer, and to a 3D artificial skin with a wound part. According to the present invention, there is an advantage that the wound part of the artificial skin can be formed more precisely and uniformly.

Description

A method of manufacturing a three-dimensional artificial skin with a defect site and a three-dimensional artificial skin with a defect site {Manufacturing method of 3D artificial skin with wound and 3D artificial skin with wound}

The present invention relates to a method for manufacturing a three-dimensional artificial skin having a defect site and a three-dimensional artificial skin having a defect site, and more particularly, to a three-dimensional artificial skin capable of uniformly and precisely forming a skin defect due to a wound. It relates to a manufacturing method of skin and three-dimensional artificial skin.

[Project unique number] 2017-0-01982

[Name of Ministry] Ministry of Science and ICT

[Research and Management Specialized Institution] Information and Communication Technology Promotion Center

[Research project name] ICT convergence industry source technology development

[Research project name] Development and commercialization of artificial skin models for animal experiments using 3D bioprinting

[Organizer] Korea Polytechnic University Industry-Academic Cooperation Foundation

[Research period] 2017.12.01. ~ 2019.11.30. (24 months)

The skin is an organ that covers the outside of the body and is largely composed of the epidermis and dermis.

The epidermal layer is located on the outside of the skin and consists of the stratum corneum, clear layer, granular layer, stratum corneum, and basal layer. replace it with

The dermal layer is located below the epidermal layer, and the extracellular matrix, such as a biocompatible polymer, is formed as a layer (about 1500 μm) that is 15 to 40 times thicker than the epidermis, and includes various structures such as blood vessels, nerves, and sweat glands.

Since the skin acts as a barrier to protect the internal organs from ultraviolet rays, bacteria, and various pollutants, reactions such as inflammation, allergy, and hypersensitivity may occur due to these substances. Therefore, in the development of cosmetics or drugs applied in a percutaneous absorption method, experiments for evaluating their effectiveness and safety are required, and currently, efficacy and safety evaluations are mainly made through animal experiments using animals.

However, in recent years, in addition to the social atmosphere emphasizing animal ethics, there is a problem that the reliability of experimental results is lowered due to the difference between animal skin and human skin. It is currently being used commercially.

On the other hand, when performing an efficacy or safety evaluation test of a transdermal formulation in a skin defect due to a wound using artificial skin, a biopsy punch is used on the artificial skin to simulate the defect, and in this case, a wound is induced. The cut surface was cut irregularly, and the epidermis and dermis were separated according to the skill of the experimenter, making it difficult to produce a uniform model.

Accordingly, it is necessary to develop a three-dimensional artificial skin in which a new defect site having a uniform defect site for each individual is formed.

Registered Patent No. 10-1621288 (Registered on May 10, 2016)

An object of the present invention is to provide a method for manufacturing a three-dimensional artificial skin having a defect region capable of uniformly and precisely forming a skin defect region due to a wound, and a three-dimensional artificial skin having a defect region.

One embodiment of the present invention for achieving the object as described above, the dermal layer forming step of forming a dermal layer containing a biocompatible polymer and fibroblasts; forming a sacrificial layer by applying a sacrificial material to a portion of the upper surface of the dermal layer to form a sacrificial layer; an epidermal layer forming step of dispensing and culturing keratinocytes on the dermal layer on which the sacrificial layer is formed to form an epidermal layer; and a sacrificial layer removing step of removing the sacrificial layer.

The epidermal layer forming step may be a step of dispensing and culturing keratinocytes on the upper surface of the dermal layer except for the area where the sacrificial layer is formed.

The sacrificial material may be at least one selected from the group consisting of alginate and gelatin.

The step of removing the sacrificial layer may be performed through a degrading enzyme, a physical external force, a phase transition, or a natural decomposition.

The biocompatible polymer is collagen, chitosan, hyaluronic acid, silk protein, silk dermatoin, keratin, cellulose, fibronectin, elastin, fibrinogen, fibromodulin, laminin, tenacin, vitronectin, polylactic acid (PLA). ), polyglycolic acid (PGA), copolymer of polylactic acid and polyglycolic acid (PLGA), polycaprolactone (PCL), poly{poly(ethylene oxide) terephthalate-co-butylene terephthalate } (PEOT/PBT), polyphosphoester (PPE), polyphosphazene (PPA), polyanhydride (PA), polyorthoester {poly(ortho ester; POE}, poly(propylene) It may be at least one selected from the group consisting of fumarate)-diacrylate {poly(propylene fumarate)-diacrylate; PPF-DA} and polyethylene glycol diacrylate {poly(ethylene glycol) diacrylate; PEG-DA} have.

The sacrificial layer may be formed in one region of the dermal layer or two or more regions spaced apart by a predetermined distance.

Another embodiment of the present invention is the dermal layer; a defect formed on a portion of the upper surface of the dermal layer; and an epidermal layer formed on the remaining upper surface of the dermal layer except for the region where the defect portion is formed.

The defect site may be formed by applying a sacrificial material on the dermal layer, dispensing keratinocytes to form an epidermal layer, and then removing the sacrificial material.

The sacrificial material may be at least one selected from the group consisting of alginate and gelatin.

The sacrificial material may be removed through a degrading enzyme, a physical external force, a phase transition, or natural decomposition.

The epidermal layer may be formed by dispensing and culturing keratinocytes on the upper surface of the dermal layer except for the area where the sacrificial material is applied.

The dermal layer may include a biocompatible polymer and fibroblasts.

The biocompatible polymer is the biocompatible polymer is collagen, chitosan, hyaluronic acid, silk protein, silk skin loin, keratin, cellulose, fibronectin, elastin, fibrinogen, fibromodulin, laminin, tenacin, vitronectin, polylactic acid (polylactic acid; PLA), polyglycolic acid (PGA), copolymer of polylactic acid and polyglycolic acid (PLGA), polycaprolactone (PCL), poly{poly(ethylene oxide)terephthalate-co -Butylene terephthalate} (PEOT/PBT), polyphosphoester (PPE), polyphosphazene (PPA), polyanhydride (PA), polyorthoester {poly(ortho ester; POE) }, poly(propylene fumarate)-diacrylate {poly(propylene fumarate)-diacrylate; PPF-DA} and polyethylene glycol diacrylate {poly(ethylene glycol) diacrylate; PEG-DA} It may be at least one or more.

At least one defect portion may be formed on the dermal layer.

According to the manufacturing method of the three-dimensional artificial skin with a defect site of the present invention, it is possible to manufacture artificial skin with a uniform defect site for each artificial skin object, and it is possible to form a plurality of defect sites on one artificial skin object. There is an advantage in that the screening speed can be dramatically increased.

In addition, there is no risk of loss of the manufactured sample, it is possible to reduce the experimental error according to the skill of the experimenter, and it is possible to enable a more convenient experiment.

In addition, since the depth of the defect can be adjusted more precisely, the reliability of the screening test results can be improved.

1 is a view showing the sequence of the manufacturing method of the three-dimensional artificial skin formed with a defect according to an embodiment of the present invention.
2 to 4 are photographs taken of the upper surface and cross-section of the three-dimensional artificial skin in which the defect region manufactured according to the conventional method and the method of the present invention is formed.
5 is a photograph showing the results of Experimental Example 2.
6 is a photograph showing the results of Experimental Example 3.

Hereinafter, before describing in detail through preferred embodiments of the present invention, terms or words used in the present specification and claims should not be construed as being limited to conventional or dictionary meanings, and meanings consistent with the technical spirit of the present invention and should be interpreted as a concept.

Throughout this specification, when a part "includes" a certain component, it means that other components may be further included rather than excluding other components unless otherwise stated.

In each step, the identification code is used for convenience of description, and the identification code does not describe the order of each step, and each step may be performed differently from the specified order unless the specific order is clearly stated in the context. have. That is, each step may be performed in the same order as specified, may be performed substantially simultaneously, or may be performed in the reverse order.

Hereinafter, the present invention will be described in detail with reference to embodiments and drawings of the present invention. It will be apparent to those of ordinary skill in the art that these examples are only presented by way of example to explain the present invention in more detail, and that the scope of the present invention is not limited by these examples. .

First, the present invention relates to a method for manufacturing a three-dimensional artificial skin having a defect site and a three-dimensional artificial skin having a defect site formed therein. It has an object to provide artificial skin.

1 is a view showing the sequence of the manufacturing method of the three-dimensional artificial skin formed with a defect according to an embodiment of the present invention.

A method of manufacturing a three-dimensional artificial skin having a defect region according to an embodiment of the present invention includes a dermal layer forming step (S1) of forming a dermal layer 100 including a biocompatible polymer 110 and fibroblasts 120; a sacrificial layer forming step (S2) of forming a sacrificial layer 200 by applying a sacrificial material to a portion of the upper surface of the dermal layer 100; an epidermal layer forming step (S3) of dispensing and culturing keratinocytes 310 on the dermal layer 100 on which the sacrificial layer 200 is formed to form an epidermal layer 300; and a sacrificial layer removing step (S4) of removing the sacrificial layer 200 .

First, the dermal layer forming step (S1) is a step of forming the dermal layer 100 including the biocompatible polymer 110 and fibroblasts 120 .

The method for forming the dermal layer is not particularly limited, and for example, a solution containing the biocompatible polymer 110 and fibroblasts 120 may be dispensed or performed by 3D printing. As another example, it may be performed by first preparing a polymer layer using the biocompatible polymer 110 and inoculating the fibroblasts 120 therein.

The biocompatible polymer 110 is a support for supporting the dermal layer 100, has mechanical properties of a predetermined strength, and is a polymer showing biocompatibility characteristics, for example, collagen, chitosan, hyaluronic acid, silk protein, silk skin. Phosphorus, keratin, cellulose, fibronectin, elastin, fibrinogen, fibromodulin, laminin, tenacin, vitronectin, polylactic acid (PLA), polyglycolic acid (PGA), polylactic acid and polyglycolic acid of copolymer (PLGA), polycaprolactone (PCL), poly{poly(ethylene oxide) terephthalate-co-butylene terephthalate} (PEOT/PBT), polyphosphoester (PPE), poly Phosphazene (PPA), polyanhydride (PA), polyorthoester {poly(ortho ester; POE}), poly(propylene fumarate)-diacrylate {poly(propylene fumarate)-diacrylate; PPF- DA} and polyethylene glycol diacrylate {poly(ethylene glycol) diacrylate; PEG-DA} may be used, and preferably, collagen is used as the biocompatible polymer 110. can

Next, the sacrificial layer forming step (S2) is a step of forming the sacrificial layer 200 by applying a sacrificial material to a portion of the upper surface of the dermal layer 100, and the sacrificial material is any one of alginate and gelatin. More than one can be used.

This step may be performed by applying, dropping, or 3D printing a sacrificial material solution in which the sacrificial material is solubilized on a portion of the upper surface of the dermal layer 100 . In particular, in the case of forming the sacrificial layer 200 through the 3D printing method, the thickness of the sacrificial layer 200 can be adjusted and the sacrificial layer 200 can be formed in various shapes, so that the sacrificial layer 200 is formed thereafter. There is an advantage in that the shape or depth of the removed portion 400 can be precisely and variously adjusted.

Since cells are not attached to the sacrificial material, adhesion, diffusion, differentiation, etc. of cells does not occur in the area of the sacrificial layer 200, so that the area of the sacrificial layer 200 made of the sacrificial material corresponds to a wound on the skin thereafter. A defective portion 400 may be formed.

The sacrificial layer 200 may be formed in one region of the dermal layer 100 , or may be formed in two or more regions spaced apart from each other by a predetermined distance. When the sacrificial layer 200 is formed in a plurality of regions as described above, since a plurality of defective regions 400 may be formed in one three-dimensional artificial skin object in which the defective region 400 is formed, the efficacy or safety evaluation test of the transdermal formulation There is an advantage in that the screening speed can be dramatically increased.

At this time, as an example for changing the depth of the defect region 400 between the dermal layer forming step ( S1 ) and the sacrificial layer forming step ( S2 ), the dermal layer 100 in the region where the sacrificial layer 200 is to be formed is partially removed. This step may be further performed as needed.

As another example for changing the depth of the defect region 400, between the sacrificial layer forming step (S2) and the epidermal layer forming step (S3), an auxiliary dermal layer is placed on the upper surface of the dermal layer 100 except for the region where the sacrificial layer 200 is formed. The forming step may be further performed as needed.

After the sacrificial layer forming step (S2), the epidermal layer forming step (S3) of forming the epidermal layer 300 is performed, and this step includes keratinocytes 310 on the dermal layer 100 on which the sacrificial layer 200 is formed. It can be carried out by dispensing and culturing.

Referring to the top view of the sacrificial layer forming step (S2) of FIG. 1 , the keratinocytes 310 are dispensed on the upper surface of the sacrificial layer 200 (A); and the upper surface (B) of the dermal layer 100 except for the region where the sacrificial layer 200 is formed Keratinocytes 310 may be dispensed and cultured.

Since the keratinocytes 310 do not adhere to the region where the sacrificial layer 200 is formed, the epidermal layer 300 is not formed in the region where the sacrificial layer 200 is formed. Therefore, when the keratinocytes 310 are dispensed on the upper surface (A) of the sacrificial layer 200 , the keratinocytes 310 dispensed on the upper surface (A) of the sacrificial layer 200 are applied to the sacrificial layer 200 . Some of them are not attached and move to the surface of the dermal layer 100 to form the epidermal layer 300, but a problem may occur that some of them aggregate with each other on the sacrificial layer 200, except for the region where the sacrificial layer 200 is formed. It is preferable that the keratinocytes 310 are dispensed and cultured only on the upper surface (B) of the dermal layer 100 .

After the skin layer 300 is formed through the skin layer forming step S3 , the sacrificial layer removing step S4 of removing the sacrificial layer 200 may be performed.

The removal of the sacrificial layer 200 may be performed through a decomposing enzyme, a physical external force, a phase transition, or natural decomposition.

In particular, in the case of alginate, it may be removed through an alginate degrading enzyme such as (poly(beta-D-mannuronate) lyase or poly(alpha-L-guluronate) lyase), but is not limited thereto.

In addition, since gelatin is a temperature-sensitive material, it may be removed using a phenomenon of becoming a liquid at a high temperature, but is not limited thereto.

When the sacrificial layer 200 is removed through this step, the dermal layer 100 under the sacrificial layer 200 and the epidermal layer 300 surrounding the sacrificial layer 200 are cross-sections in the region where the sacrificial layer 200 is removed. A defect region 400 corresponding to a wound on the skin is formed by being exposed.

Another embodiment of the present invention is a three-dimensional artificial skin in which a defect region is formed, except for the dermal layer 100, the defect region 400 formed on a part of the upper surface of the dermal layer 100, and the region in which the defect region 400 is formed. and an epidermal layer 300 formed on the remaining upper surface of the dermal layer 100 .

The defect site 400 may be formed by applying a sacrificial material on the dermal layer 100, dispensing keratinocytes 310 to form the epidermal layer 300, and then removing the sacrificial material, Specifically, the defect region 400 may be formed through the sacrificial layer forming step (S2), the epidermal layer forming step (S3) and the sacrificial layer removing step (S4) described above in the embodiment of the present invention.

In the defect region 400 , cross-sections of the dermal layer 100 and the epidermal layer 300 are exposed to form a structure corresponding to a skin wound.

The dermal layer 100 includes a biocompatible polymer 110 and fibroblasts 120, and as the biocompatible polymer 110, the same type of biocompatible polymer 110 as mentioned in an embodiment of the present invention will be used. can

The sacrificial material for forming the defect region 400 may be any one or more selected from the group consisting of alginate and gelatin, and the removal of the sacrificial material is performed through a decomposing enzyme, a physical external force, a phase transition or natural decomposition. can be done

At least one defect region 400 may be formed on the dermal layer 100 . Specifically, when the sacrificial material is applied, a plurality of defects are formed by applying the sacrificial material to a plurality of regions spaced apart from each other by a predetermined distance on the dermal layer 100 . A region 400 may be formed. In this case, a plurality of defective regions 400 are formed in one three-dimensional artificial skin object in which the defective region 400 is formed, so that the screening speed can be dramatically improved when performing an evaluation test for efficacy or safety of a transdermal formulation.

In addition, since the depth or shape of the plurality of defective regions 400 can be individually adjusted, various screening experiments can be performed through one three-dimensional artificial skin object in which the defective region 400 is formed.

In addition, in the conventional case, when manufacturing a three-dimensional artificial skin with a defect site, artificial skin including a dermal layer and an epidermal layer is manufactured, and the defect site is formed using a biopsy punch, and then the defect site is formed separately under the artificial skin Artificial skin was prepared by placing and culturing the dermal layer of

In this case, when the defect site is formed, the shape of the defect site is formed differently for each individual depending on the skill level of the operator, and thus there may be a problem that the reliability of the experiment is lowered.

In addition, there is a problem that an additional dermal layer is required, and an epidermal layer is formed on the surface of the defect site, so there is a possibility that the defect site simulation fails.

On the other hand, in the case of the present invention, it is possible to imitate the defect site 400 of a uniform and constant shape, and depending on the shape of the sacrificial layer 200, the form of the defect site 400 can be changed, and one artificial skin Since a plurality of defective regions 400 can be formed on the surface, there is an advantage that the screening speed can be dramatically increased.

Hereinafter, specific actions and effects of the present invention will be described through an embodiment of the present invention. However, this is presented as a preferred example of the present invention, and the scope of the present invention is not limited according to the embodiments.

[ manufacturing example ]

Example Produce

First, a dermal layer is formed by printing a collagen solution containing fibroblasts (1×10 ⁶ cells/mL) in a culture vessel, and alginate is printed only on the center of the upper surface of the dermal layer to form a sacrificial layer, and then the alginate is not printed. Keratinocytes were dispensed and printed on the remaining area of the upper surface of the dermal layer, and keratinocytes growth media were used to incubate for 1 day in an environment of 5% _{CO 2 and 37°C.} Thereafter, in the air-liquid interface condition, for about 7 days, CaCl ₂ ^{was added to KGM Gold medium (KGM TM} Gold Keratinocyte Growth Medium BulletKit ^TM ) purchased from Lonza at a concentration of 1.8 mM. At this time, the culture medium was replaced on the basis of 2 days.

Next, by administering poly(beta-D-mannuronate) lyase, which is an alginate degrading enzyme, the alginate sacrificial layer is removed for 1 day, and the alginate degrading enzyme is removed by washing with a buffer solution. Artificial skin was prepared.

comparative example Produce

First, a dermal layer is formed by printing a collagen solution containing fibroblasts (1×10 ⁶ )cells/mL in a culture vessel, keratinocytes are dispensed on the upper surface of the dermal layer, and then cultured under the same conditions as in the above Example to artificial skin. was prepared.

Next, the three-dimensional artificial skin in which the defect region of the comparative example was formed was manufactured by forming a penetration part using a biopsy punch in the center of one artificial skin.

[ Experimental example One]

The upper surface of each of Examples and Comparative Examples prepared in Preparation Example was photographed and shown in FIG. 2 .

In addition, in order to distinguish the epidermal layer from the dermal layer, each of Examples and Comparative Examples were stained using H&E staining method, and cross-sectional photos were taken and shown in FIG. A cross-section of the was taken using an OCT device and is shown in FIG. 4 .

2 to 4 , in the case of the embodiment, it can be seen that the dermal layer is not completely penetrated at the defect site, and a concave shape is uniformly formed. On the other hand, in the case of the comparative example, it can be confirmed that the dermal layer is completely penetrated at the defect site, so that an additional dermal layer is required under the artificial skin when evaluating the efficacy or safety of the transdermal formulation.

[ Experimental example 2]

A cross-linked alginate mattress was prepared by mixing _{100 mM CaCl 2} solution in 4% alginate solution, ^{1 × 10 5} cells/mL of keratinocytes were dispensed on the top surface of the prepared mattress, and then cultured for 1 day to prepare a sample. .

Thereafter, an optical photograph of the prepared sample was taken using an optical microscope equipment, and is shown in FIG. 5 .

Referring to FIG. 5 , it can be seen that the keratinocytes do not adhere to the alginate matrix and aggregate with each other, so that the epidermal layer is not formed.

[ Experimental example 3]

The three-dimensional artificial skin sample having the defect region of the Example prepared through Preparation Example and the three-dimensional artificial skin sample having the defect region of the Comparative Example were cultured in an air-liquid interface condition for 4 days in an environment of 5% _{CO 2 , 37 ° C.} At this time, both types of samples were cultured by adding CaCl ₂ ^{at a concentration of 1.8 mM to KGM Gold medium (KGM TM} Gold Keratinocyte Growth Medium BulletKit ^TM ) purchased from Lonza, and the media was replaced once every 2 days. Thereafter, each of Examples and Comparative Examples were stained using the H&E staining method, and cross-sectional photos were taken using a microscope, and are shown in FIG. 6 .

Referring to FIG. 6 , in the case of Example, it can be seen that the keratinocytes do not migrate to the surface of the defect site after formation of the defect site, so that the dermal layer is maintained at the defect site as it is.

On the other hand, in the case of the comparative example, since the keratinocytes continuously move along the surface of the defect site after formation of the defect site, when a predetermined time elapses after the defect site is formed, the exposed state of the dermal layer at the defect site is not maintained, so it is not easy to observe the progress. It is not predicted that

The present invention is not limited to the specific embodiments and descriptions described above, and without departing from the gist of the present invention claimed in the claims, anyone with ordinary skill in the art to which the invention pertains can implement various modifications and such modifications shall fall within the protection scope of the present invention.

100: dermal layer
110: biocompatible polymer
120: fibroblasts
200: sacrificial layer
300: epidermal layer
310: keratinocytes
400: defect site

Claims (13)

A dermal layer forming step of forming a dermal layer containing biocompatible polymers and fibroblasts;
forming a sacrificial layer by applying a sacrificial material to a portion of the upper surface of the dermal layer to form a sacrificial layer;
an epidermal layer forming step of dispensing and culturing keratinocytes on the dermal layer on which the sacrificial layer is formed to form an epidermal layer; and
A method of manufacturing a three-dimensional artificial skin having a defect, including; a sacrificial layer removing step of removing the sacrificial layer. According to claim 1,
The epidermal layer forming step comprises dispensing and culturing keratinocytes on the upper surface of the dermal layer except for the area where the sacrificial layer is formed. According to claim 1,
The sacrificial material is at least one selected from the group consisting of alginate and gelatin. According to claim 1,
The step of removing the sacrificial layer is a method of manufacturing a three-dimensional artificial skin with a defect, characterized in that it is performed through a decomposing enzyme, a physical external force, a phase transition, or a natural decomposition. According to claim 1,
The biocompatible polymer is collagen, chitosan, hyaluronic acid, silk protein, silk dermatoin, keratin, cellulose, fibronectin, elastin, fibrinogen, fibromodulin, laminin, tenacin, vitronectin, polylactic acid (PLA). ), polyglycolic acid (PGA), polylactic acid and polyglycolic acid copolymer (PLGA), polycaprolactone (PCL), poly{poly(ethylene oxide) terephthalate-co-butylene terephthalate }(PEOT/PBT), polyphosphoester (PPE), polyphosphazene (PPA), polyanhydride (PA), polyorthoester {poly(ortho ester; POE}, poly(propylene) Fumarate)-diacrylate {poly(propylene fumarate)-diacrylate; PPF-DA} and polyethylene glycol diacrylate {poly(ethylene glycol) diacrylate; PEG-DA} A method of manufacturing a three-dimensional artificial skin with a defect, characterized in that. According to claim 1,
The sacrificial layer is a method of manufacturing a three-dimensional artificial skin with a defect, characterized in that formed in one region of the dermal layer or two or more regions spaced apart by a predetermined interval. dermal layer;
a defect formed on a portion of the upper surface of the dermal layer; and
Including; and an epidermal layer formed on the remaining upper surface of the dermal layer except for the region where the defect is formed.
The defect site is formed by applying a sacrificial material on the dermal layer, dispensing keratinocytes to form an epidermal layer, and then removing the sacrificial material. 8. The method of claim 7,
The sacrificial material is a three-dimensional artificial skin with a defect, characterized in that at least one selected from the group consisting of alginate and gelatin. 9. The method of claim 8,
The sacrificial material is a three-dimensional artificial skin with a defect, characterized in that it is removed through a decomposing enzyme, a physical external force, a phase transition or natural decomposition. 8. The method of claim 7,
The epidermal layer is a three-dimensional artificial skin with a defect, characterized in that it is formed by dispensing and culturing keratinocytes on the upper surface of the dermal layer except for the area where the sacrificial material is applied. 8. The method of claim 7,
The dermal layer is a three-dimensional artificial skin with a defect, characterized in that it contains a biocompatible polymer and fibroblasts. 12. The method of claim 11,
The biocompatible polymer includes collagen, gelatin, chitosan, hyaluronic acid, silk protein, silk dermatoin, keratin, cellulose, fibronectin, elastin, fibrinogen, fibromodulin, laminin, tenacin, vitronectin, Polylactic acid (PLA), polyglycolic acid (PGA), copolymer of polylactic acid and polyglycolic acid (PLGA), polycaprolactone (PCL), poly{poly(ethylene oxide) terephthalate -co-butylene terephthalate} (PEOT/PBT), polyphosphoester (PPE), polyphosphazene (PPA), polyanhydride (PA), polyorthoester {poly(ortho ester) ; POE}, poly(propylene fumarate)-diacrylate {poly(propylene fumarate)-diacrylate; PPF-DA} and polyethylene glycol diacrylate {poly(ethylene glycol) diacrylate; PEG-DA} from the group consisting of A three-dimensional artificial skin with a defect, characterized in that at least one selected. 8. The method of claim 7,
The defect site, characterized in that at least one or more is formed on the dermal layer, a three-dimensional artificial skin with a defect site.

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