Patent Application
20230279360
The present invention relates to artificial skin having structure and properties by age, and more specifically to artificial skin having structure and properties, for which effective analysis and application are possible on the basis of age of the skin, as the human skin can be replicated according to age by simple control in the internal structure of properties such as height and width, and a method for manufacturing the same.
Currently, there is a trend toward banning animal testing worldwide, and artificial skin is a key technology that can replace existing animal testing and lead the market for customized pharmaceutical compositions and cosmetics.
As illustrated in
As such, the related art has problems in that it is a flat artificial skin that does not simulate the concave-convex structure of the dermal-epidermal junction like real skin, and it is manufactured such that the mechanical properties do not differ according to age. It is difficult for the human skin models used in the existing animal replacement test method to have the same characteristics for each manufacturing company, and age-specific simulations have not been performed.
Since human cells have different behaviors depending on the type and physical properties of applied substrates, it is important to model human skin models by age through controlling the physical properties of the dermis layer. In addition, since the structure of human skin varies according to age, the behavior of the epidermis layer also varies according to the structure. Therefore, it is necessary to develop age-specific artificial skin with controlled physical properties and structure for cosmetics research, where formulations and active ingredients vary according to age.
In addition, if artificial skin that simulates the structure of simple skin is referred to as the first generation and artificial skin that implements a full-layer skin model is referred to as the second generation, it is necessary to develop a third-generation artificial skin that can be effectively analyzed and applied according to skin age. Since the structure of human skin, particularly, the concave-convex structure of the dermal-epidermal junction (DEJ) flattens as it ages, itis necessary to manufacture artificial skin that simulates the DEJ concave-convex structure.
In addition, depending on the age (the degree of aging), the skin shows a difference in the degree of crosslinking and bonding strength of the extracellular matrix. The extracellular matrix, which provides a physical environment in which cells can attach and grow in the human body, not only plays a structural role as a cell scaffold, but also serves as a very important cell physiological regulator for cell growth, proliferation and differentiation. Therefore, not only structural simulation but mechanical properties should be controlled.
Accordingly, as the internal structure of properties such as height, width and the like can be controlled very easily and human skin can be simulated by age, the situation is that there is an urgent need to develop artificial skin that can be effectively analyzed and applied according to skin age.
The present invention has been devised to solve the above-described problems, and an object of the present invention is to provide artificial skin having structure and properties, for which effective analysis and application are possible on the basis of age of the skin, as the human skin can be replicated according to age by simple control in the internal structure of properties such as height and width, and a method for manufacturing the same.
In addition, the present invention was researched with the support of the following national research and development projects, and specific information of the national research and development projects is as follows.
In order to solve the above-described problems, the present invention provides a method for manufacturing artificial skin, including the steps of (1) controlling mechanical properties by introducing ECM derived substances into a dermis layer; (2) culturing until a dermal-epidermal junction derived from epidermal cells is formed on a surface of the dermis layer into which the ECM derived substances have been introduced, by seeding human epidermis cells on the dermis layer into which the ECM derived substances have been introduced; and (3) manufacturing full-thickness artificial skin having structure and properties by age by differentiating epidermal cells to form a stratum corneum on epidermal cells.
The ECM derived substances may include at least one selected from the group consisting of polysaccharide, elastin, chitosan, hyaluronic acid (HA) and collagen that mimic the structure of glycosaminoglycan (GAG).
In addition, the dermis layer may include a concave-convex structure on a surface thereof.
In addition, the average difference in height between peaks and valleys of the concave-convex structure may be 100 μm or more to 200 μm or less, in the case of artificial skin at the age of 20 or more to less than 40, wherein the average difference in height between peaks and valleys of the concave-convex structure may be 50 μm or more to less than 100 μm, in the case of artificial skin at the age of 40 or more to less than 60, and wherein the average difference in height between peaks and valleys of the concave-convex structure may be less than 50 μm, in the case of artificial skin at the age of 60 or more.
In addition, the distance between a valley of the concave-convex structure and other valleys adjacent to the valley may be 250 μm or less.
In addition, the modulus of elasticity may be 20 to 30 kPa, in the case of artificial skin at the age of 20 or more to less than 40, wherein the modulus of elasticity may be 10 to 20 kPa, in the case of artificial skin at the age of 40 or more to less than 60, and wherein the modulus of elasticity may be 10 kPa or less, in the case of artificial skin at the age of 60 or more.
In addition, the concave-convex structure of the dermis layer may be manufactured in a shape-changing hydrogel mold prepared by at least one method selected from the group consisting of a shape-changing ductility technique, a three-dimensional lithography technique and a 3D printing technique.
In addition, the culturing of step (2) may be performed by submerged culture.
In addition, the epidermal cells in step (3) may be differentiated through at least one selected from the group consisting of air exposure and air-liquid interface culture (ALIC).
In addition, the epidermal cells may include keratinocytes.
Meanwhile, the present invention provides artificial skin having structure and properties by age, including a dermis layer into which ECM derived substances are introduced; an epidermis layer including sequentially stacked stratum corneum and human epidermis cell layers and provided on top of the dermis layer; and a dermal-epidermal junction provided between the dermis layer and the epidermis layer.
According to an exemplary embodiment of the present invention, the dermis layer may include a concave-convex structure on a surface thereof, and wherein the epidermal cell layer and the dermal-epidermal junction may have a shape corresponding to the concave-convex structure of the dermis layer.
According to an exemplary embodiment of the present invention, the shape-changing hydrogel mold may include an acrylic compound including a first acrylic compound and a second acrylic compound.
According to an exemplary embodiment of the present invention, the acrylic compound may include the first acrylic compound and the second acrylic compound at a weight ratio of 5:5 to 1:9.
The artificial skin having structure and properties by age according to the present invention and the method for manufacturing the same can very easily control the internal structure of properties such as height, width and the like to simulate human skin by age, and accordingly, it has effects that effective analysis and application according to the skin age are possible.
(a) to (c) of
(a) to (b) of
(a) of
Hereinafter, exemplary embodiments of the present invention will be described in detail so that those of ordinary skill in the art to which the present invention pertains can easily practice the present invention. The present invention may be embodied in many different forms and is not limited to the exemplary embodiments described herein.
As illustrated in
First, step (1) of controlling mechanical properties by introducing ECM derived substances into the dermis layer will be described.
The dermis layer may be used without limitation, as long as it is a material that can be used to form the dermis layer of artificial skin conventionally in the art, and preferably, it may be implemented by including human dermal fibroblasts.
Meanwhile, the ECM derived substances perform a function of controlling the mechanical properties of the artificial skin.
According to age (the degree of aging), the skin shows differences in the degree of crosslinking and bonding strength of the extracellular matrix. The extracellular matrix, which provides a physical environment for cells to attach and grow in the human body, serves not only as a structural support for cells, but also as a very important cell physiology regulator for cell growth, proliferation and differentiation. Therefore, mechanical properties as well as structural simulation must be controlled, which can be achieved through the introduction of ECM derived substances.
The ECM derived substances may be used without limitation, as long as these are substances that can be used to form ECM derived substances that can be introduced into the dermis layer of artificial skin conventionally in the art, and preferably, these substances may include at least one selected from the group consisting of polysaccharide, elastin, chitosan, hyaluronic acid (HA) and collagen that mimic the structure of glycosaminoglycan (GAG), and more preferably, these substances may be implemented by including collagen.
Meanwhile, the ECM derived substances may preferably include collagen, or collagen and methacrylic alginate, and in the case of including the collagen and methacrylic alginate, the collagen and methacrylic alginate may be included at a concentration ratio of 1:0.2 to 2.0, and preferably, at a concentration ratio of 1:0.5 to 1.5.
Since the structure of the human skin, particularly the concave-convex structure of the dermis-epidermal junction (DEJ), becomes flatter as it ages, it is necessary to manufacture artificial skin by age in which the structure of the DEJ is simulated.
Accordingly, the dermis layer of the artificial skin having structure and properties by age according to an exemplary embodiment of the present invention may include a concave-convex structure on a surface thereof as illustrated in (a) to (c) of
In addition, the concave-convex structure of the dermis layer may be manufactured in a shape-changing hydrogel mold prepared by at least one method selected from the group consisting of ductility and three-dimensional lithography techniques and 3D printing, and preferably, it may be manufactured in a shape-changing hydrogel mold which is capable of controlling the concave-convex structure of the collagen gel of the dermis layer in which the shape changes depending on temperature.
Meanwhile, the shape-changing hydrogel mold may be manufactured through a shape-changing hydrogel mold composition including an acrylic compound including a first acrylic compound and a second acrylic compound.
The acrylic compound constitutes a random copolymer when manufacturing a shape-changing hydrogel mold, and it may include a first acrylic compound and a second acrylic compound as described above.
The first acrylic compound serves as a main structural function of the shape-changing hydrogel mold, and it may be used without limitation as long as it is a first acrylic compound which is polymerizable and has a polymer chain structure, and preferably, it may include at least one selected from the group consisting of acrylamide, poly(ethylene glycol)diacrylate, methacrylated alginate, polyacrylamide, poly(ethylene glycol), methacrylate, sodium polyacrylate, polyacrylate, glyceryl acrylate/acrylic acid copolymer, styrene/acrylate copolymer, sodium acrylate, crosspolymer-2, methyl acrylate, ethyl acrylate, normal butylacrylate and 2-hydroxyethyl methacrylate, and preferably, acrylamide may be used.
In addition, the second acrylic compound functions to express the sensitivity of a shape-changing hydrogel mold, and it may be used without limitation as long as it is a second acrylic compound which is capable of expressing sensitivity to external stimuli in the art, and preferably, it may include at least one selected from the group consisting of N-isopropylacrylamide (NIPAM), acrylic acid, polyvinyl alcohol, poly N-isopropylacrylamide (pNIPAM), poly 2-(dimethylamino)ethyl methacrylate (pDMAEMA), hydroxypropylcellulose (HPC), polyvinylcaprolactame, polyvinyl methyl ether, poly(N,N-dimethylacrylamide), methacrylic acid-methylmethacrylate copolymer, methacrylic acid-methylacrylate copolymer, methacrylic acid-methylacrylate-methylmethacrylate and poly(2,2-dimethoxy nitrobenzyl methacrylate-r-methyl methacrylate-r-poly(ethylene glycol) methacrylate (PDMP), and preferably, N-isopropyl acrylamide may be used.
In this case, when acrylamide is used as the first acrylic compound and N-isopropyl acrylamide is used as the second acrylic compound, the shape and size of the structure to be manufactured may be easily controlled because the diameter and depth of the mold can be easily controlled, and as the change rate of the diameter and depth of the mold is large, it may be more advantageous to express excellent separability even when manufacturing a structure having low strength.
The acrylic compound may include the first acrylic compound and the second acrylic compound at a weight ratio of 5:5 to 1:9, and preferably, it may include the first acrylic compound and the second acrylic compound at a weight ratio of 3:7 to 1:9. If the weight ratio of the first acrylic compound and the second acrylic compound included in the acrylic compound is less than 5:5, it is not easy to control the diameter and depth of the mold, and thus, it is not easy to control the shape and size when manufacturing a predetermined structure, and there may be a problem in that the change rate of the diameter and depth of the mold becomes small. In addition, if the weight ratio is more than 1:9, there may be a problem in shape control due to rapid contraction and expansion.
Meanwhile, for specific descriptions other than the above-described description of the hydrogel mold, Korean Patent Application No. 10-2018-0147510, which was filed by the same applicant and inventors as the present application, may be incorporated herein by reference, and thus, the detailed description thereof will be omitted.
Meanwhile, when the age of the target artificial skin is 20 or more to less than 40, the average difference in height between peaks and valleys of the concave-convex structure may be 100 μm or more to 200 μm or less, and preferably, it may be 110 μm or more to 190 μm or less. In addition, when the age of the target artificial skin is 40 or more to less than 60, the average difference in height between peaks and valleys of the concave-convex structure may be 50 μm or more to less than 100 μm, and preferably, it may be 60 μm or more to 90 μm or less. In addition, when the age of the target artificial skin is 60 or more, the average difference in height between peaks and valleys of the concave-convex structure may be less than 50 μm, and preferably, it may be less than 40 μm.
In addition, the distance between a valley of the concave-convex structure and other valleys adjacent to the valley may be 250 μm or less, and preferably, it may be 220 μm or less, and by satisfying these, artificial skin by age which is similar to the internal structure of human skin by age may be implemented. As such, when the concave-convex structure is implemented in this way according to age, the same effects and functions as can be seen at the DEJ marker level of
Next, step (2) of culturing until the dermal-epidermal junction derived from epidermal cells is formed on the surface of the dermis layer into which the ECM derived substances are introduced, by seeding human epidermis cells on the dermis layer into which the ECM derived substances are introduced will be described.
The epidermal cells may be used without limitation as long as they are materials that can be used as epidermal cells of artificial skin conventionally in the art, and may preferably be implemented by including keratinocytes.
In addition, the culturing in step (2) may be used without limitation as long as it is a method that can be used for cell culture conventionally in the art, and preferably, it may be performed by submerged culture, and more preferably, by submerged culture including DMEM culture media.
Meanwhile, in step (2), the epidermal cells are cultured until the dermal-epidermal junction derived from epidermal cells is formed on the surface of the dermis layer into which the ECM derived substances are introduced. In this case, the dermal-epidermal junction (DEJ) may serve not only as a mechanical support, but also help the proliferation and differentiation of the epidermal cells, facilitate the movement of metabolites and improve the adhesion between the dermis and the epidermis.
Next, step (3) of manufacturing artificial skin having structure and properties by age by differentiating epidermal cells to form a stratum corneum on the epidermal cells will be described.
In step (3), the epidermal cells may be differentiated through any one or more methods selected from air exposure and air-liquid interface culture (ALIC), and preferably, it may be differentiated through air-liquid interface culture (ALIC) using a medium inducing the differentiation of epidermal cells including a certain amount of Ca2+.
By forming a stratum corneum on the epidermal cell layer through the differentiation, artificial skin having structure and properties by age may be finally implemented.
Meanwhile, when the age of the target artificial skin is 20 or more to less than 40, the elastic modulus of the artificial skin may be 20 to 30 kPa, and preferably, 22 to 28 kPa. In addition, when the age of the target artificial skin is 40 or more to less than 60, the elastic modulus of the artificial skin may be 10 to 20 kPa, and preferably, 12 to 18 kPa. In addition, when the age of the target artificial skin is 60 or more, the elastic modulus of the artificial skin may be 10 kPa or less, and preferably, 8 kPa or less. By satisfying these ranges, it is possible to implement artificial skin by age similar to the physical properties of actual human skin by age.
The artificial skin having structure and properties by age according to the present invention may be implemented by including: a dermis layer into which ECM derived substances are introduced; an epidermis layer including sequentially stacked stratum corneum and human epidermis cell layers and provided on top of the dermis layer; and a dermal-epidermal junction provided between the dermis layer and the epidermis layer.
In this case, as described above, the dermis layer may include a concave-convex structure on a surface thereof, and the epidermal cell layer and the dermal-epidermal junction may have a shape corresponding to the concave-convex structure of the dermis layer.
The artificial skin having structure and properties by age according to the present invention and the method for manufacturing the same can very easily control the internal structure of properties such as height and width to simulate human skin by age, and accordingly, it has effects that effective analysis and application according to the skin age are possible.
The present invention will be described in more detail through the following examples, but the following examples are not intended to limit the scope of the present invention, which should be construed to aid understanding of the present invention.
First, a shape-changing hydrogel mold was prepared by radical polymerization reaction through a shape-changing hydrogel mold composition including an acrylic compound including acrylamide as a first acrylic compound and N-isopropyl acrylamide as a second acrylic compound at a weight ratio of 2:8, 0.1 parts by weight of N,N′-methylenebisacrylamide as a crosslinking agent, 1 part by weight of ammonium persulfate as an initiator, 0.1 parts by weight of tetramethylethylenediamine as a catalyst and 88.8 parts by weight of water as a solvent, based on 100 parts by weight of the acrylic compound.
In this case, the shape-changing hydrogel mold was respectively prepared in Preparation Examples 1 to 3 according to the concave-convex structure of artificial skin by age.
In this case, Preparation Example 1 was prepared to have holes having a diameter and interval of 100 μm or more to 200 μm or less, and an average difference in height between peaks and valleys of the concave-convex structure of 150 μm.
In addition, Preparation Example 2 was prepared to have holes having a diameter and interval of 50 μm or more to 100 μm or less, and an average difference in height between peaks and valleys of the concave-convex structure of 50 μm.
In addition, Preparation Example 3 was prepared to have holes having a diameter and interval of less than 0 to less than 50 μm, and an average difference in height between peaks and valleys of the concave-convex structure of 0 μm.
The shape-changing hydrogel molds prepared in Preparation Examples 1 to 3 were simulated and evaluated for the degree of change in the average diameter and average depth of the surface according to the expansion ratio through
R=R
i−(Rv−2Ri)×S [Relationship Formula 1]
In Relationship Formula 1, R is the changed radius of the concave-convex structure, Ri is the radius of the concave-convex structure immediately after being manufactured, Rv is the length from the center of the concave-convex structure to the nearest concave-convex structure, and S is the expansion ratio of the hydrogel.
D=D
i
+D
i
×S [Relationship Formula 2]
Herein, D is the changed depth of the concave-convex structure, Di is the depth of the concave-convex structure immediately after being manufactured, and S is the expansion ratio of the hydrogel.
When the operating temperature of the shape-changing mold increased, the bulk gel contracted while the radius of the concave-convex structure increased and the depth decreased. Conversely, when the operating temperature decreased, the gel expanded while the radius of the concave-convex structure decreased and the depth increased.
In spite of the relatively low physical properties of the dermis layer, not only can the depth be automatically controlled, but also after the dermis layer with the controlled concave-convex structure was prepared, the depth of the mold structure was finally lowered, and thus, the dermis layer was naturally separated from the mold without external force.
An artificial skin having structure and physical properties by age was manufactured through the method shown in
Specifically, first, the dermis layer was formed by using human dermal fibroblasts as the dermis layer-forming cells, the concave-convex structure was introduced, and collagen was introduced as the ECM derived substances.
In addition, as the ECM derived substances to control the strength, methacrylic alginate (MA) in which a methacrylate functional group was introduced into alginate was applied, and it was used at 4.8 mg/mL based on the concentration of collagen at 4.8 mg/mL.
In this case, the mechanical strength was manufactured to be formed to be 25 (±5) kPa, and for the introduction of the concave-convex structure, in order to increase the roughness, the mold prepared in Preparation Example 1 above having holes with a diameter and interval of 100 to 200 μm and a depth of 100 μm was used when the dermis layer was manufactured (
In addition, it was cultured by submerged culture until the dermal-epidermal junction derived from epidermal cells was formed on the surface of the dermis layer by seeding keratinocytes with human epidermis cells on the dermis layer into which the concave-convex structure was introduced.
Thereafter, epidermal cells were differentiated through air-liquid interface culture (ALIC) to form a stratum corneum on epidermal cells, and finally, artificial skin having structure and properties by age, which included a dermis layer into which ECM derived substances were introduced; an epidermis layer including sequentially stacked stratum corneum and human epidermis cell layers and provided on top of the dermis layer; and a dermal-epidermal junction provided between the dermis layer and the epidermis layer, was manufactured.
Artificial skin was manufactured in the same manner as in Example 1, except that methacrylic alginate (MA) was used at 2.4 mg/mL based on collagen at a concentration of 4.8 mg/mL, and the mechanical strength was manufactured to be formed at a concentration of 15 (±5) kPa, and Example 2 was carried out by using the mold prepared in Preparation Example 2.
Artificial skin was manufactured in the same manner as in Example 1, except that methacrylic alginate (MA) was used at 0 mg/mL based on collagen at a concentration of 4.8 mg/mL, and the mechanical strength was manufactured to be formed at a concentration of 5 (±5) kPa, and Example 3 was carried out by using the mold prepared in Preparation Example 3.
The concave-convex structure of the artificial skin was evaluated for the artificial skin having structure and properties by age manufactured according to Example 1.
As a result, as illustrated in
In addition, as shown in (a) to (b) of
In addition, (a) of
For the artificial skins having structure and properties by age manufactured according to Examples 1 to 3, the structure and physical property behavior by age were evaluated.
As a result, as illustrated in
In addition, as the concave-convex structure of the dermal-epidermal junction helps the proliferation and differentiation of epidermal cells, it can be confirmed that the degree of proliferation and differentiation of epidermal cells decreased toward Example 3 simulating relatively old skin.
In addition, when the biosynthetic amounts (procollagen) of the labeling materials in each of the artificial skins having structure and properties by age according to Examples 1 to 3 were compared, as shown in (b) of
In addition, as can be seen from (c) of
The amount of pro-collagen type I expressed before and after wounding was evaluated for the artificial skins having structure and properties by age manufactured according to Examples 1 to 3.
As a result, it can be confirmed that the expression level of pro-collagen type I after manufacturing artificial skins implemented by age was higher in Example 3 (E-SKIN3) with less extracellular matrix, that is, in artificial skin implemented with a higher age. Since there was no extracellular matrix, it can be considered that the active metabolism of fibroblasts to create the same had occurred. However, it was confirmed that the expression of pro-collagen type I in Example 1 (E-SKIN1), that is, young skin, was significantly increased when the artificial skins by age were wounded. Conversely, in the case of Example 3 (E-SKIN3) which is old skin, there was not much difference in the amounts of expression after manufacturing. Therefore, as in real skin, it can be demonstrated by the expression level of pro-collagen type I that the replication of young skin had better recovery than the replication of old skin among the artificial skins by age.
Although an exemplary embodiment of the present invention has been described above, the spirit of the present invention is not limited to the exemplary embodiment presented herein, and those skilled in the art who understand the spirit of the present invention may easily suggest other exemplary embodiments by modifying, changing, deleting or adding components within the scope of the same spirit, but this will also fall within the scope of the present invention.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10-2020-0163297 | Nov 2020 | KR | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/KR2021/000716 | 1/19/2021 | WO |
