Composition containing nidogen for improving skin condition, treating skin damage or maintaining stem cell function

Abstract

The present invention relates to a composition for improving skin, treating skin damage, or maintaining epidermal stem cell function, which contains nidogen as an active ingredient. According to one embodiment, a composition containing nidogen as an active ingredient strengthens the basement membrane-extracellular matrix bond to improve basement membrane function, maintains the proliferation of epidermal progenitor cells and the stem cell function of epidermal stem cells, improves keratinocyte turnover, promotes collagen production and binding, and thereby affects the epidermal and dermal layers by strengthening the basement membrane. This can be useful for skin improvement, such as strengthening the skin barrier, inhibiting or preventing skin pigmentation, moisturizing, improving skin elasticity, anti-aging, or tissue regeneration through wound treatment.

Description

[Technical Field]

[0001] The present invention relates to a composition containing nidogen as an active ingredient for improving skin, treating skin damage, or maintaining epidermal stem cell function. [Background technology]

[0002] The skin is composed of the epidermis, dermis, and subcutaneous fat layer, and the epidermis is divided into the stratum corneum, granular layer, spinous layer, and basal layer from the outside in. The cells in the epidermis act like bricks, and the intercellular lipids between the epidermal cells form the skin barrier. In addition, the epidermal cells of healthy people contain high concentrations of natural moisturizing factors (NMFs), which help retain moisture in the skin. For example, substances such as amino acids are water-soluble and effectively bind with moisture to prevent the skin from drying out.

[0003] The stratum corneum, the outermost layer of the skin, is in direct contact with the external environment and plays an important barrier function to protect our bodies from external physical and chemical stress. This barrier function is maintained by epidermal homeostasis. Epidermal homeostasis involves the differentiation process of basal layer keratinocytes, which undergoes terminal differentiation through the growth, division, and migration of cells, thereby maintaining a continuous skin barrier function by forming a skin barrier called the stratum corneum (Korean J. Food. Sci. Technol. 43:458-463, 2011).

[0004] Nidogen is one of the key components of basement membranes, along with type IV collagen, laminin, and heparan sulfate proteoglycan 2 (Perlecan). Nidogen mediates the binding between laminin and type IV collagen and is involved in the formation of the characteristic three-dimensional structure of basement membranes. In addition to these structural functions, nidogen has also been reported to be involved in cell attachment, neutrophil chemotaxis, and nerve development.

[0005] As a result, the inventors have confirmed that treatment with nidogen or nidogen and epidermal progenitor cell culture medium together has skin improving effects, skin damage treatment effects, and effects of maintaining epidermal stem cell function, thereby completing the present invention. Summary of the Invention [Problem to be solved by the invention]

[0006] One aspect is to provide a cosmetic composition, an external skin composition, or a pharmaceutical composition for improving skin, treating skin damage, or maintaining epidermal stem cell function, which comprises a nidogen protein as an active ingredient.

[0007] Another aspect is to provide a medium composition for maintaining epidermal stem cell function, which comprises Nidogen protein as an active ingredient.

[0008] Another aspect is to provide an artificial skin comprising a dermal layer containing fibroblasts and an epidermal layer containing keratinocytes and nidogen protein.

[0009] Another aspect is to provide a method for producing artificial skin, comprising the steps of culturing fibroblasts to produce a dermal layer, applying nidogen protein onto the dermal layer, and applying keratinocytes onto the dermal layer to which the nidogen protein has been applied to produce an epidermal layer.

[0010] Another aspect is to provide a method for maintaining epidermal stem cell function in skin cells, comprising culturing the skin cells in a medium containing isolated nidogen protein.

[0011] Another aspect is to provide a method of improving skin conditions or preventing, ameliorating, or treating skin damage comprising administering to an individual in need thereof an effective amount of a nidogen protein.

[0012] Another aspect is to provide the use of a nidogen protein for use in the manufacture of a preparation for improving skin conditions or preventing, improving or treating skin damage. [Means for solving the problem]

[0013] One embodiment provides a composition comprising Nidogen as an active ingredient.

[0014] In one embodiment, the composition may be a cosmetic composition, a topical skin composition, a pharmaceutical composition, or a medium composition.

[0015] The term "nidogen" as used herein refers to one of the basement membrane extracellular matrix proteins present in vivo beneath epithelial cells. Also known as entactin, this protein interacts with laminin and collagen and plays a role in bridging the basement membrane components of the skin. The nidogen may affect the skin basement membrane, epidermis, or dermis. The nidogen may be nidogen-1 or nidogen-2.

[0016] The nidogen may be a wild-type nidogen protein or a recombinant nidogen protein. Specifically, the recombinant nidogen protein may include a fragment of the wild-type nidogen protein, or may include a protein in which some amino acids contained in the wild-type nidogen protein are substituted with other amino acids.

[0017] In one embodiment, the nidogen protein may comprise the amino acid sequence of SEQ ID NO:1 or a sequence having at least about 95%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO:1.

[0018] In one embodiment, the composition may further comprise epidermal progenitor cell-conditioned media (EPC-CM).

[0019] Skin consists of the epidermis, dermis, and subcutaneous fat layer. The basal layer is located at the bottom of the epidermis, and cells (e.g., skin tissue cells or keratinocytes) that make up the new epidermis are formed in this basal layer through cell division. The formed cells that make up the epidermis subsequently replace dead cells in the upper part of the epidermis. This process induces skin cell regeneration. The epidermal progenitor cells may be cells that make up the epidermal layer, for example, the basal layer, and are used interchangeably with epidermal stem cells. The epidermal progenitor cells may be precursor cells of keratinocytes.

[0020] The epidermal progenitor cells can be derived from stem cells. The epidermal progenitor cells can be cells whose differentiation potential and self-renewal ability are limited compared to stem cells. For example, the stem cell-derived epidermal progenitor cells can be cells with 100% differentiation potential, or cells converted from stem cells with any percentage of differentiation potential to cells with 0% differentiation potential, or cells with a lower percentage of differentiation potential than any percentage. The stem cell-derived epidermal progenitor cells can be used interchangeably with epidermal stem cells, differentiated epidermal stem cells, differentiated epidermal progenitor cells, or differentiated keratinocytes.

[0021] The epidermal progenitor cells may have increased expression of one or more selected from the group consisting of keratin 5 (Keratin 5: KRT5), keratin 1 (Keratin 1: KRT1), and keratin 14 (Keratin 14: KRT14) compared to stem cells before culturing in a differentiation medium. That is, as the stem cells differentiate into epidermal progenitor cells, expression of one or more selected from the group consisting of KRT5, KRT1, and KRT14 may increase. The stem cell-derived epidermal progenitor cells may exhibit a specific circular morphology of a certain size.

[0022] The epidermal progenitor cell culture medium (EPC-CM) is a conditioned medium obtained after culturing differentiated epidermal progenitor cells in a medium, and may contain proteins secreted extracellularly through cell-to-cell interactions during the culture process of differentiated stem cell-derived epidermal progenitor cells. The proteins may include useful proteins such as cytokines and growth factors. The epidermal progenitor cell culture medium may contain a large number of useful proteins at high concentrations in the culture medium, and may include conventional media suitable for cell culture and used in the art.Specifically, the epidermal progenitor cell culture medium contains thrombospondin (TSP), tissue inhibitor of metalloproteinases 1 (TIMP1), tissue inhibitor of metalloproteinases 2 (TIMP2), ectodysplasin-A2 (EDA-A2), X-linked ectodysplasin-A receptor (XEDAR), angiopoietin-1, secreted protein acidic and rich in cysteine ​​(SPARC), and transmembrane protein with EGF-like and two follistatin-like domains / tomoregulin-1. The antibody may comprise one or more selected from the group consisting of TMEFF1 / Tomoregulin-1, Nidogen-1, Insulin-like growth factor-binding protein-3 (IGFBP-3), Thrombospondin-2, Tumor necrosis factor-related activation-induced cytokine (TRANCE), and Interleukin-15 receptor alpha (IL-15Rα).

[0023] The epidermal progenitor cell culture may refer to a medium, differentiated epidermal progenitor cells, or a mixture thereof, obtained during or after culturing differentiated epidermal progenitor cells in a medium.

[0024] Compositions containing Nidogen as an active ingredient or nidogen-coated in-plate cultures of the present invention have been shown to improve skin by increasing the adhesion and proliferation ability of keratinocytes in a concentration-dependent manner. Furthermore, synergistic effects were confirmed when these compositions were treated with epidermal progenitor cell culture medium or other extracellular matrix proteins (e.g., collagen). Specifically, compositions containing Nidogen as an active ingredient can enhance the basement membrane and its induced functions, promoting the proliferation and differentiation of epidermal progenitor cells adjacent to the basement membrane and improving epidermal layer and keratinocyte turnover. Nidogen can also regulate melanocyte activity, controlling pigment production. It also functions as a support for fixing collagen bundles in the dermis, and when treated with epidermal progenitor cell culture medium, it can promote collagen production in the dermis. Therefore, compositions containing Nidogen as an active ingredient have been shown to improve skin and maintain epidermal stem cell function.

[0025] The composition containing nidogen as an active ingredient or the culture in a plate coated with nidogen can be used not only for cell adhesion or proliferation, but also for adhesion or proliferation of artificial skin, organoids, tissues, organs, and embryos composed of the cells to a specific substrate or site. Specifically, by pre-coating or co-treating a specific substrate or site, it can have the effect of promoting the engraftment of artificial skin, organoids, tissues, and organs.

[0026] In one embodiment, the cosmetic composition may be for improving skin or maintaining epidermal stem cell function.

[0027] The term "improvement" as used herein means any action that at least reduces the parameters associated with the condition, e.g., the severity of symptoms, of the disease being treated by administration of a composition according to the present invention.

[0028] The term "prevention" as used herein means any action of suppressing or delaying the onset of a disease by administering a composition according to the present invention.

[0029] The skin improvement may be one or more selected from the group consisting of skin barrier improvement, skin pigmentation improvement, prevention or improvement of skin wrinkles, skin moisturizing, promotion of skin anti-aging, promotion of skin resistance, promotion of skin elasticity, skin soothing, prevention or improvement of skin damage, and prevention or improvement of skin diseases.

[0030] The skin improvement can be skin regeneration or skin protection from external stimuli. The term skin regeneration can be used interchangeably with terms such as "skin recovery," "skin reconstruction," or "skin repair."

[0031] The external stimulus may be, but is not limited to, ultraviolet (UV) rays, scratching, friction, trauma, inflammation, heat, temperature, acid, base, surfactant, toxic substance, viral infection, particulate matter, etc., and may include any form of physical or chemical stimulus that can cause skin damage. The external stimulus may damage cells that constitute the human epidermis layer (e.g., keratinocytes), cells that constitute the dermis layer (e.g., fibroblasts), or the basement membrane. The UV rays may be one or more of UVA, UVB, and UVC.

[0032] The term "skin regeneration" refers to the broad concept of skin regeneration, including the ability to generate new keratinocytes in response to general skin damage. The skin damage may include any type of skin wound, scar, or burn caused by external physical or chemical stimuli. For example, the skin damage may be, but is not limited to, a cut, abrasion, puncture, burn, ulcer, bedsore, contusion, or dehiscence.

[0033] The term "skin protection" means suppressing or preventing the above-mentioned skin damage.

[0034] The nidogen may inhibit epidermal-dermal separation caused by external stimuli, more specifically, inhibit the formation of blisters caused by epidermal-dermal separation caused by external stimuli.

[0035] The nidogen may restore the arrangement of basal layer keratinocytes and the structure of the basement membrane damaged by external stimuli. More specifically, the nidogen may restore or prevent damage to structures such as hemidesmosomes, anchoring fibrils, and dermal collagen at the dermo-epidermal junction caused by external stimuli.

[0036] The skin improvement can be suppression or prevention of skin pigmentation.

[0037] Melasma skin exhibits a series of structural and functional changes in the epidermis, basement membrane, and upper dermis, which interact to induce and maintain a local hypermelanogenic phenotype. In facial pigmentation, the basement membrane is thinned and discontinuous, and more basement membrane disruptions and gaps are observed in pigmented skin compared to healthy skin. Therefore, damage to the basement membrane is associated with melanogenesis in pigmented skin and photoaged skin. In the present invention, nidogens as defined above can suppress melanogenesis in pigmented skin or photoaged skin by maintaining the structure of the basement membrane and inducing extracellular matrix bonds within the basement membrane, thereby regulating melanocyte activity and improving pigmentation, including pigmentation and / or dullness, and / or whitening effects. Thus, the nidogen herein can further provide a composition for inhibiting melanin production and / or inhibiting pigmentation (or for lightening blemished or photoaging induced skin).

[0038] The nidogen may restore proliferation and differentiation of the epidermal layer due to external stimuli. More specifically, the nidogen may alleviate the phenomenon of suppression of keratinocyte proliferation in the basal layer of the epidermis due to external stimuli, or may induce the expression of Ki67. The nidogen may also alleviate the phenomenon of suppression of differentiation of keratinocytes in the upper layer of the epidermis, or may induce the expression of filaggrin (FLG).

[0039] In one embodiment, the protein may improve extracellular matrix junctions within the basement membrane or dermal density.

[0040] In one embodiment, the protein may promote the production of collagen in the dermis or Ki67, KRT14, or filaggrin in the epidermis.

[0041] The term "skin barrier improvement" as used herein encompasses both skin barrier strengthening and protective function. Here, the skin barrier refers to the stratum corneum, the outermost layer of the epidermis, which is primarily composed of anuclear, flat keratinocytes. The keratinocytes of the skin barrier, which are maintained through the division and differentiation processes of cells that make up the normal epidermis, synthesize a multilamellar lipid layer of intercellular lipids, such as ceramides, cholesterol, and fatty acids, which acts as a protective barrier to prevent moisture from evaporating from within the skin.

[0042] The improvement of the skin barrier may mean any action of alleviating psoriasis, contact dermatitis, eczematous dermatitis, photodermatitis, seborrheic dermatitis, herpetic dermatitis, lichen planus, lichen sclerosus, pityriasis gangrenosum, pemphigus, bullous epidermolysis, systemic sclerosis, or leprosy, which are skin diseases caused by weakened skin barrier function, or improving damaged skin barrier function.

[0043] As used herein, the term "preventing or improving skin wrinkles" refers to preventing, suppressing or inhibiting the formation of wrinkles on the skin, or alleviating wrinkles that have already formed.

[0044] As used herein, the term "skin elasticity improving effect" refers to preventing, suppressing or inhibiting a decrease in skin elasticity, or improving already decreased skin elasticity.

[0045] In one embodiment, the protein may improve basement membrane function, specifically, improving the basement membrane-extracellular matrix junction or the epidermis-dermis junction, or improving dermal density.

[0046] In one embodiment, the protein may promote the production of collagen in the dermis or Ki67, KRT14, or filaggrin in the epidermis.

[0047] The term "stem cell function" as used herein is a term commonly used in the art to collectively refer to pluripotency, the ability to generate all types of cells, such as embryonic stem cells, and self-renewal, the ability to infinitely generate cells similar to the self. In other words, stem cell function can refer to the ability to maintain stem cell properties.

[0048] As used herein, the term "maintaining stem cell function" can include suppressing cellular senescence of stem cells, increasing the cell proliferation capacity of stem cells, increasing stem cell functionality, increasing telomerase activity of stem cells, increasing the expression of stem cell factors, allowing undifferentiated cells to proliferate while maintaining an undifferentiated state, increasing protein homeostasis in stem cells, or exhibiting characteristics that increase cell migration activity.

[0049] The term "stem cell factor" refers to a gene that is highly expressed in stem cells and is known to play an important role in maintaining the full differentiation potential of stem cells, i.e., stem cell characteristics, and examples thereof include, but are not limited to, SOX2, OCT4, NANOG, KLF4, C-MYC, Oct3 / 4, SSEA-1, SSEA-3, SSEA-4, TRA1-60, TRA1-81, Lin28, Fbx15, etc. Therefore, when expression of at least one of the stem cell factors is observed, the stem cell can be determined to be an undifferentiated stem cell, and the higher the expression level, the higher the full differentiation potential of the stem cell can be determined.

[0050] The term "enhancing functionality" refers to functional maturation and refers to genes that are expressed in stem cells and are known to play an important role in maintaining the mature functional characteristics of stem cells, such as, but not limited to, CYP3A4, CYP3A7, ALB, AFP, TTR, CK19, etc. Therefore, when the expression of at least one factor that increases stem cell functionality is observed, it can be determined that stem cell functionality can be increased, and the greater the expression of these factors, the greater the ability to increase stem cell functionality.

[0051] The term "proliferation" refers to an increase in cell number and can be used interchangeably with "growth." In particular, the term "undifferentiated proliferation" refers to the proliferation of stem cells into cells that have the same properties as the original cell without differentiating into specific cells, i.e., pluripotency (which can be used interchangeably with the term "total differentiation potential").

[0052] The term "culture media" refers to a substance that can support the growth and survival of cells in 2D or 3D culture of various cells, including stem cells and keratinocytes, in vitro, as well as in artificial skin, organoid, tissue, organ or embryo culture.

[0053] In one embodiment, the nidogen can upregulate the expression of a group of genes consisting of Ki67, KRT14 (Keratin 14), ITGA6 (Integrin alpha-6), TP63, and SOX2 (SRY-Box Transcription Factor 2). Specifically, the nidogen is provided in the form of a medium composition or coated thereon, and can upregulate the expression of genes or proteins consisting of the group of Ki67, KRT14 (Keratin 14), ITGA6 (Integrin alpha-6), TP63, and SOX2 (SRY-Box Transcription Factor 2) in keratinocytes.

[0054] In one embodiment, the nidogen was added to a stem cell culture medium and was confirmed to inhibit stem cell senescence, improve cell proliferation, and induce the expression of proteins related to stem cell function, thereby significantly enhancing stem cell function. Therefore, the protein may improve stratum corneum turnover.

[0055] The composition may be provided in the form of a medium composition containing nidogen or in a form coated with nidogen to improve skin (e.g., inhibit epidermal-dermal separation, maintain basement membrane structure, alleviate inhibition of epidermal differentiation, and regulate proliferation, differentiation, and migration of melanocytes in the basal layer of the epidermis) and / or enhance stem cell function of epidermal stem cells. Specifically, the composition may be provided in the form of a plate for culturing cells coated with nidogen, or in the form of a plate coated with nidogen between the epidermis and dermis in artificial skin. The nidogen can be added to a conventional medium used for stem cell culture to achieve the effects related to enhancing stem cell function disclosed herein.

[0056] The nidogen may be contained in an amount of 0.00001 to 90% by weight relative to the total weight of the cosmetic composition, for example, 0.00001 to 80% by weight, 0.00001 to 70% by weight, 0.00001 to 60% by weight, 0.00001 to 50% by weight, 0.00001 to 40% by weight, 0.00001 to 30% by weight, 0.00001 to 20% by weight, 0.00001 to 10% by weight, 0.00001 to 5% by weight, 0.00001 to 3% by weight, 0.00001 to 1% by weight, 0.0001 to 90% by weight, 0.0001 to 70% by weight, 0.0001 to 60% by weight, 0.0001 to 50% by weight, 0.0001 to 40% by weight, 0.00001 to 5 ... The composition may contain 0.0001 to 30% by weight, 0.0001 to 20% by weight, 0.0001 to 10% by weight, 0.001 to 80% by weight, 0.001 to 70% by weight, 0.001 to 60% by weight, 0.001 to 50% by weight, 0.001 to 40% by weight, 0.001 to 30% by weight, 0.001 to 20% by weight, 0.001 to 10% by weight, 0.01 to 10% by weight, 0.01 to 5% by weight, 0.01 to 3% by weight, 0.01 to 1% by weight, 0.1 to 10% by weight, 0.1 to 5% by weight, 0.1 to 3% by weight, or 0.1 to 1% by weight. The composition may further contain an epidermal progenitor cell culture medium.

[0057] The cosmetic composition may be prepared in any formulation commonly used in the art. For example, it may be prepared in the form of a skin lotion, skin soap, skin toner, skin booster, astringent, lotion, milk lotion, moisturizing lotion, nourishing lotion, serum, massage cream, nourishing cream, moisturizing cream, hand cream, hand wash, foundation, essence, nourishing essence, pack, mascara, soap, foam cleanser, cleansing lotion, cleansing cream, body lotion, body cleanser, suspension, gel, powder, paste, mask pack, sheet, stick product, balm-type product, spray, lyophilized, powder, syringe, or pre-filled syringe. Such formulations may be prepared according to conventional methods in the art. The amount of additional ingredients, such as the moisturizer, can be easily determined by those skilled in the art within a range that does not impair the objectives and effects of the present invention.

[0058] In addition to the active ingredients disclosed herein, the cosmetic composition may further contain functional additives and ingredients commonly found in cosmetic compositions, such as purified water, thickeners, preservatives, stabilizers, solubilizers, surfactants, carriers, fragrances, or combinations thereof. The functional additives may include a component selected from the group consisting of water-soluble vitamins, oil-soluble vitamins, polymeric peptides, polymeric polysaccharides, sphingolipids, and seaweed extracts. Examples of the carrier include alcohols, oils, surfactants, fatty acids, silicone oils, humectants, moisturizers, viscosity modifiers, emulsifiers, stabilizers, UV scattering agents, UV absorbers, color formers, and fragrances. Compounds / compositions that can be used as the alcohols, oils, surfactants, fatty acids, silicone oils, humectants, moisturizers, viscosity modifiers, emulsifiers, stabilizers, UV scattering agents, UV absorbers, color formers, and fragrances are already known in the art, and those skilled in the art can select and use appropriate substances / compositions. Furthermore, the cosmetic composition may further contain, as needed, ingredients such as ultraviolet screening agents, antioxidants (butylhydroxyanisole, propyl gallate, erythorbic acid, tocopherol acetate, butylated hydroxytoluene, etc.), preservatives (methylparaben, butylparaben, propylparaben, phenoxyethanol, imidazolidinyl urea, chlorphenesin, etc.), colorants, pH adjusters (triethanolamine, citric acid, citric acid, sodium citrate, malic acid, sodium malate, fumaric acid, sodium fumarate, succinic acid, sodium succinate, sodium hydroxide, sodium monohydrogen phosphate, etc.), moisturizers (glycerin, sorbitol, propylene glycol, butylene glycol, hexylene glycol, diglycerin, betaine, glycereth-26, methyl gluceth-20, etc.), lubricants, etc.

[0059] In addition, in the cosmetic composition of each formulation, appropriate ingredients can be selected and blended depending on the formulation or intended use of the cosmetic. The blending ingredients and methods can be in accordance with conventional techniques, and therefore a detailed description thereof will be omitted in this specification.

[0060] Another embodiment provides a skin topical or pharmaceutical composition comprising Nidogen as an active ingredient.

[0061] In one embodiment, the composition may further comprise epidermal progenitor cell-conditioned media (EPC-CM).

[0062] In one embodiment, the topical skin preparation may be for skin improvement, maintaining epidermal stem cell function, or wound treatment and tissue repair.

[0063] The "Nidogen," "epidermal progenitor cell culture medium," "skin improvement," and "epidermal stem cell function" are as described above.

[0064] The nidogen may be contained in an amount of 0.00001 to 90% by weight relative to the total weight of the external skin preparation composition, for example, 0.00001 to 80% by weight, 0.00001 to 70% by weight, 0.00001 to 60% by weight, 0.00001 to 50% by weight, 0.00001 to 40% by weight, 0.00001 to 30% by weight, 0.00001 to 20% by weight, 0.00001 to 10% by weight, 0.00001 to 5% by weight, 0.00001 to 3% by weight, 0.00001 to 1% by weight, 0.0001 to 90% by weight, 0.0001 to 70% by weight, 0.0001 to 60% by weight, 0.0001 to 50% by weight, 0.0001 to 40% by weight, 0.00001 to 5 ... The composition may contain 0.0001 to 30% by weight, 0.0001 to 20% by weight, 0.0001 to 10% by weight, 0.001 to 80% by weight, 0.001 to 70% by weight, 0.001 to 60% by weight, 0.001 to 50% by weight, 0.001 to 40% by weight, 0.001 to 30% by weight, 0.001 to 20% by weight, 0.001 to 10% by weight, 0.01 to 10% by weight, 0.01 to 5% by weight, 0.01 to 3% by weight, 0.01 to 1% by weight, 0.1 to 10% by weight, 0.1 to 5% by weight, 0.1 to 3% by weight, or 0.1 to 1% by weight. The composition may further contain an epidermal progenitor cell culture medium.

[0065] The topical skin preparation can be formulated in any form, such as a cream, gel, ointment, foam, spray, powder, freeze-dried product, liquid, skin emulsion, skin suspension, transdermal patch, drug-containing bandage, lotion, serum, roll-on, paste, balsam, stick, balm, syringe, pre-filled syringe, etc. The topical skin preparation can be appropriately blended with ingredients typically used in topical skin preparations such as cosmetics, pharmaceuticals, and medical devices, such as aqueous ingredients, oily ingredients, powder ingredients, alcohols, moisturizers, thickeners, UV absorbers, whitening agents, preservatives, antioxidants, surfactants, fragrances, colorants, various skin nutrients, or combinations thereof, as needed. The topical skin preparations may also be appropriately formulated with sequestering agents such as edetate disodium, edetate trisodium, sodium citrate, sodium polyphosphate, sodium metaphosphate, and gluconic acid; drugs such as caffeine, tannin, verapamil, licorice extract, glabridin, Chinese quince fruit hot water extract, various herbal medicines, tocopherol acetate, glycyrrhizic acid, tranexamic acid and derivatives thereof or salts thereof; vitamin C, magnesium ascorbyl phosphate, ascorbyl glucoside, arbutin, kojic acid, sugars such as glucose, fructose, and trehalose; natural polymer materials such as alginic acid, chitosan, collagen, and hyaluronic acid; and synthetic polymer materials such as poly(vinyl alcohol) (PVA), poly(N-vinyl-2-pyrrolidone) (PVP), poly(ethylene glycol) (PEG), and carboxymethylcellulose (CMC).

[0066] The skin includes all skin areas of the body, including the face, hands, arms, legs, feet, chest, abdomen, back, buttocks, and scalp.

[0067] Another aspect provides a method of maintaining epidermal stem cell function in skin cells, comprising culturing the skin cells in a medium containing isolated nidogen protein.

[0068] Another aspect provides a method of improving skin conditions or preventing, ameliorating, or treating skin damage comprising administering to an individual in need thereof an effective amount of a nidogen protein.

[0069] Another aspect provides the use of a nidogen protein for use in the manufacture of a formulation for improving skin conditions or preventing, improving or treating skin damage.

[0070] Another aspect provides artificial skin comprising a nidogen protein.

[0071] The nidogen protein is as described above.

[0072] As used herein, the term "artificial skin" refers to three-dimensionally reconstructed skin using skin cells and skin constituents, and is used in the broadest sense to include all polymer composites that exhibit structural and functional properties similar to those of real skin and are accessible to ordinary technicians. Artificial skin can be composed of a dermal layer and an epidermal layer, similar to human skin. In this specification, the artificial skin may be an artificial skin consisting of a dermal layer containing fibroblasts and an epidermal layer containing keratinocytes and nidogenic proteins. Examples of artificial skin include, but are not limited to, the artificial skin described in Larouche et al. BioResearch Open Access (2016) 5(1):320-329.

[0073] Furthermore, the artificial skin may include a higher-level skin model that can replicate the complexity and function of in vivo skin tissue by further realizing not only the dermis and epidermis layers but also hair follicles, sweat glands, sebaceous glands, melanocytes, blood vessels, nerve cells, or subcutaneous fat. That is, in this specification, the term "artificial skin" can be used to collectively refer to "skin equivalents (or reconstructed skin)," "skin organoids," or "skin models."

[0074] The dermis layer may further include an extracellular matrix, such as collagen, laminin, elastin, chitosan, glycosaminoglycans (GAGs), hyaluronic acid (HA), and other components that constitute the dermis, in order to correlate with actual human skin.

[0075] The thickness of the dermis layer may generally be 0.05 cm or more, specifically 0.05 cm to 2 cm, but is not limited thereto, and may be increased or decreased as long as it does not impair the advantageous properties of the artificial skin according to the present invention.

[0076] The epidermal layer may be located above the dermal layer, and the nidogen protein may be present at the base of the epidermis in the artificial skin, or may be provided in the form of a coating between the dermal layer and the epidermal layer.

[0077] The keratinocytes may be any commonly used keratinocytes, including not only keratinocytes directly isolated from humans or cultured after isolation, but also any commonly used keratinocytes derived from other cells.

[0078] Another aspect provides a method for producing artificial skin, comprising the steps of culturing fibroblasts to produce a dermal layer, applying nidogen protein onto the dermal layer, and applying keratinocytes onto the dermal layer to which the nidogen protein has been applied to produce an epidermal layer.

[0079] The nidogen protein is as described above.

[0080] The concentration of the nidogen protein is 0.1 μg / ml to 1000 μg / ml, 0.1 μg / ml to 500 μg / ml, 0.1 μg / ml to 200 μg / ml, 0.1 μg / ml to 150 μg / ml, 0.1 μg / ml to 100 μg / ml, 1 μg / ml to 1000 μg / ml, 1 μg / ml to 500 μg / ml, 1 μg / ml to 20 0μg / ml, 10μg / ml~1000μg / ml, 10μg / ml~500μg / ml, 10μg / ml~200μg / ml, 20μg / ml~1000μg / m l, 30 μg / ml to 500 μg / ml, 40 μg / ml to 200 μg / ml, 45 μg / ml to 200 μg / ml, and 50 μg / ml to 200 μg / ml.

[0081] In the step of producing a dermal layer by culturing the fibroblasts, the fibroblasts can be cultured for about 1 to 8 weeks, about 3 to 5 weeks, or about 4 weeks after production.

[0082] The step of applying the nidogen protein may further include drying the nidogen protein after application.

[0083] The step of preparing the epidermal layer may be carried out by first preparing a dermal-epidermal structure by applying keratinocytes, and then culturing the dermal-epidermal structure on another dermal layer.

[0084] The epidermal layer may be cultured in an epidermal layer formation medium. Any medium known in the art may be used as the epidermal layer formation medium, for example, a medium containing bovine pituitary extract (BPE), human epidermal growth factor (hEGF), bovine insulin, hydrocortisone, gentamicin, and amphotericin-B (GA-1000). Alternatively, the medium may further contain epinephrine and transferrin in addition to the above ingredients.

[0085] The epidermal layer formation medium can be provided so as to be in contact with the lower surface of the dermal layer, and the surface opposite to the surface on which the epidermal layer formation medium is provided can be exposed to air.

[0086] In one embodiment, the dermal-epidermal structure may be obtained by culturing the keratinocytes for 1 to 14 days, 4 to 11 days, 6 to 8 days, or 7 days after application.

[0087] In one embodiment, the dermal layer may be obtained by culturing the dermal-epidermal structure on another dermal layer for 1 to 30 days, 10 to 20 days, 13 to 15 days, or 14 days.

[0088] When artificial skin is produced according to the above method, an artificial skin model that is substantially similar to real skin can be obtained, and the artificial skin can have improved characteristics due to the nidogen protein. [Effects of the Invention]

[0089] According to one embodiment, a composition containing Nidogen as an active ingredient strengthens the basement membrane-extracellular matrix bonds to improve the function of the basement membrane, maintains the proliferation of epidermal progenitor cells and the stem cell function of epidermal stem cells to improve keratinocyte turnover, and promotes collagen production and binding, thereby strengthening the basement membrane and influencing the epidermal and dermal layers. This can be useful for skin improvements such as strengthening the skin barrier, inhibiting or preventing skin pigmentation, moisturizing, improving skin elasticity, anti-aging, or tissue regeneration through wound treatment. [Brief explanation of the drawings]

[0090] FIG. 1 is a graph analyzing the cytotoxicity of nidogen to HEK and HaCaT cells. FIG. 2 shows images confirming the effect of nidogen coating concentration on cell adhesion ability. FIG. 3 shows images comparing the cell adhesion ability of coatings of nidogen with other extracellular matrix proteins (gelatin and collagen). FIG. 4 shows images comparing the cell proliferation ability of coatings of nidogen with other extracellular matrix proteins (gelatin and collagen). FIG. 5 is a graph comparing the cell proliferation ability of coatings of nidogen with other extracellular matrix proteins (gelatin and collagen). FIG. 6 shows cell proliferation images (top) and graphs (bottom) demonstrating the synergistic effect of nidogen and collagen dual coating on cell proliferation. FIG. 7 is an image showing the epidermal-dermal adhesive effect of nidogen in UVB-irradiated artificial skin (20X Scale bar: 100 μm, 40X Scale bar: 50 μm). FIG. 8 is an image showing the effect of nidogen in UVB-irradiated artificial skin on restoring basement membrane structure. FIG. 9 shows images showing the effect of nidogen in UVB-irradiated artificial skin on restoring epidermal layer differentiation (20X scale bar: 100 μm, 40X scale bar: 50 μm). FIG. 10 is an image showing the effect of nidogen in UVB-irradiated artificial skin on the restoration of epidermal basal layer proliferation (20X scale bar: 100 μm, 40X scale bar: 50 μm). FIG. 11 is an image showing the effect of nidogen in UVB-irradiated artificial skin on restoring collagen production in the dermis layer (20X scale bar: 100 μm, 40X scale bar: 50 μm). FIG. 12 shows an image of a Western blot (left) showing an increase in the expression of stem cell function-related proteins Ki67 and KRT14 in cells due to nidogen coating, and a graph (right) in which the intensity of each band was normalized and then quantified. FIG. 13 is a graph showing the synergistic effect of nidogen coating and epidermal progenitor cell culture medium on cell proliferation. FIG. 14 is a graph showing the effect of a formulation containing nidogen and an epidermal progenitor cell culture medium on cell proliferation. DETAILED DESCRIPTION OF THE INVENTION

[0091] Preferred embodiments are presented below to aid in understanding the present invention. However, the following embodiments are provided to facilitate understanding of the present invention, and the contents of the present invention are not limited to the following embodiments. Various modifications can be made to the embodiments, and the embodiments are not limited to the embodiments disclosed below, and can be implemented in various forms.

[0092] Example 1. Production of Nidogen-1 Recombinant Protein

[0093] An expression vector containing the nidogen-1 DNA sequence was constructed. After transforming Escherichia coli (E. coli) with the nidogen-1 expression vector, a strain containing this expression vector was isolated. After secondary screening for strains that successfully expressed nidogen-1 protein, a stock of nidogen-1 protein-expressing strains was isolated. This strain was cultured on a lab scale to confirm the expression and purification of nidogen-1 protein, followed by mass production of 200 L. The cultured strain was harvested by continuous centrifugation and disrupted to isolate inclusion bodies. After lysing the cells, the nidogen-1 protein was purified sequentially using various columns suited to the protein's properties. Finally, a highly concentrated nidogen-1 (hereinafter referred to as "nidogen") protein material was produced through concentration and buffer exchange processes.

[0094] Example 2. Preparation of epidermal progenitor cell-conditioned media (EPC-CM)

[0095] 2.1. Differentiation of stem cells into epidermal progenitor cells

[0096] Umbilical cords were isolated from placentas collected from healthy mothers who had given normal deliveries and provided informed consent. The isolated placenta and umbilical cord were washed 2–5 times with Ca / Mg-free Dulbecco's Phosphate-Buffered Saline (DPBS) to remove blood. The placenta tissue was then excised into pieces approximately 1–5 mm in size. The arteries and veins were then removed from the umbilical cord, and the umbilical cord was excised into pieces approximately 1–5 mm in size. The placenta and umbilical cord were then attached to a culture vessel and cultured for 10–15 days. After confirming cell growth from the cultured tissue, placenta-derived stem cells and umbilical cord-derived stem cells were isolated by adding 200 U / ml collagenase I for 5–6 hours.

[0097] To confirm that placenta-derived stem cells exhibit mesenchymal stem cell characteristics, flow cytometry was performed to analyze surface proteins. Cells were washed with DPBS, placed in DPBS containing 2% FBS, and incubated with CD44, CD73, CD90, CD105, CD45, CD34, CD31, CD29, CD49, CD9, HLA-ABC, and HLA-ER antibodies for approximately 20 minutes. Surface antigen characteristics were then analyzed using a flow cytometer (FACS Calibur, Becton Bickinson). High expression levels of CD44, CD73, CD90, CD105, CD29, CD49, CD9, and HLA-ABC in placenta-derived stem cells confirmed their mesenchymal stem cell characteristics.

[0098] The isolated cells are cultured at a density of 100 to 5,000 cells / cm. 2The cells were seeded into a multi-flask at a concentration of 0.01 mg / mL, and the cells were subcultured in MEM alpha GlutaMAX (PS-CM) medium containing 25 ng / mL fibroblast growth factor-4 (FGF-4), 1 μg / mL heparin, 50 μg / mL gentamicin, and 10% fetal bovine serum (FBS) at 37°C in 5% CO2 for 2 days for 5 passages.

[0099] The cultured cells were then inoculated into a multi-flask at 5 ml / cm with differentiation medium containing DMEM / F12 (Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12) medium, 0.3 μM ascorbic acid, 0.5 μg / ml hydrocortisone, and 10% fetal bovine serum (FBS). 2 The cells were added in a concentration of 0.01% and cultured for 11 days at 37°C in 5% CO2. As a result, both the placenta-derived stem cells and the umbilical cord-derived stem cells differentiated in the differentiation medium into small, uniformly sized, round cells similar in shape to epidermal progenitor cells.

[0100] 2.2. Preparation of epidermal progenitor cell culture medium

[0101] A culture medium was produced from the epidermal progenitor cells differentiated in Example 2.1. The differentiation medium was removed from the differentiated epidermal progenitor cells, and the cells were washed with DPBS to remove residual serum. Then, DMEM / F12 medium without choline chloride or phenol red was added to the culture plate at 2-3 ml / cm. 2 The cells were added so that the total volume of the cells was 1000 ml, and the mixture was cultured at 37°C in 5% CO for 3 days. The supernatant was then collected from the culture of differentiated epidermal progenitor cells mixed with the medium. The supernatant was then filtered through a 0.22 μm filter to obtain a stem cell-derived epidermal progenitor cell culture medium.

[0102] Experimental Example 1. Cytotoxicity evaluation after nidogen treatment

[0103] The cytotoxicity of nidogen-1 protein was evaluated in human epidermal keratinocytes (HEK) or HaCaT cells, a human keratinocyte cell line.

[0104] Specifically, HEK or HaCaT cells (3 × 10 4 Cells (1000 cells / well) were dispensed into 48-well plates and cultured in EpiLife (Gibco, USA) + HKGS supplement (Gibco, USA) or DMEM (WELGENE, Korea) + 10% FBS (Gibco, USA) for 24 hours. After replacing the medium with supplement-free or serum-free medium, the plates were cultured for 6 hours and 24 hours, respectively. Nidogen protein was then treated at various concentrations (1–100 mg / mL) and cultured for another 24 hours. Cytotoxicity was assessed using EZ-Cytox (DoGenBio, Korea). One-tenth the medium volume of EZ-Cytox was added per well and incubated in a 37°C CO2 incubator for 30 minutes. Absorbance was measured at 450 nm. The results are shown in Figure 1.

[0105] FIG. 1 is a graph analyzing the cytotoxicity of nidogen to HEK and HaCaT cells.

[0106] As shown in FIG. 1, nidogen protein did not show toxicity in HEK cells when treated at concentrations below 10 μg / mL, and did not show toxicity in HaCaT cells regardless of the nidogen treatment concentration.

[0107] Experimental Example 2. Evaluation of cell adhesion and viability after Nidogen coating

[0108] 2.1. Effect of nidogen protein coating on human keratinocyte adhesion Nidogen protein, quantified using the Pierce BCA protein assay kit (Thermo Scientific, USA), was diluted to various concentrations (0, 10, 20, 50, and 100 μg / ml) in DPBS (Dulbecco's Phosphate Buffered Saline). 500 μl of the diluted nidogen solution was dispensed into a 24-well plate, and the plate was coated at 4°C for 24 hours. The next day, 500 μl of 0.5% bovine serum albumin (BSA) solution was dispensed into each well, incubated at room temperature for 1 hour, and then washed with DPBS. HaCaT or HEK cells were suspended in serum-free DMEM or 1% supplement-containing EpiLife medium, respectively, at 5 × 10 per well. 4 The cells were cultured in a CO2 incubator at 37°C for 24 hours. Images of the cells were taken after 24 hours, and the results are shown in Figure 2.

[0109] FIG. 2 shows images confirming the effect of nidogen coating concentration on cell adhesion ability.

[0110] As shown in FIG. 2, it was confirmed that the adhesive ability of two types of keratinocytes (HEK and HaCaT cells) improved depending on the nidogen coating concentration.

[0111] 2.2. Comparison of the coating effects of nidogen protein and different extracellular matrix proteins on human keratinocyte adhesion

[0112] Nidogen-1 (10 μg / ml), gelatin (10 μg / ml), and 25% collagen solution (collagen concentration: 12.5 μg / ml) were dispensed into untreated cell culture plates (24-well plates) (NEST Scientific, China) and coated at 4°C for 24 hours. The next day, 500 μl of 0.5% BSA solution was dispensed per well, incubated at room temperature for 1 hour, and washed with DPBS. HaCaT cells were suspended in serum-free DMEM at 5 × 10 per well.4 The cells were cultured in a CO2 incubator at 37°C for 6 and 24 hours, respectively. Images of the cells were taken after 6 and 24 hours of culture, and the results are shown in Figure 3.

[0113] FIG. 3 shows images comparing the cell adhesion ability of coatings of nidogen with other extracellular matrix proteins (gelatin and collagen).

[0114] As shown in Figure 3, on the nidogen-coated plates, the cells adhered and proliferated by forming colonies in the form of undifferentiated keratinocytes, whereas on the gelatin- or collagen-coated plates, the cells spread, adhered, and proliferated over a wide area.

[0115] 2.3. Effect of nidogen protein coating on human keratinocyte proliferation

[0116] Nidogen protein quantified using the Pierce BCA assay kit was diluted to various concentrations (0, 5, 10, 20, 50 μg / ml) with Dulbecco's Phosphate Buffered Saline (DPBS) and dispensed at 500 μl per well into untreated cell culture plates (24-well plates). The plates were coated at 4°C for 24 hours. Gelatin (5, 10, 20 μg / ml) and collagen solutions (25%, 50%, 100%) were also diluted and coated in the same manner. The next day, 500 μl of 0.5% bovine serum albumin (BSA) solution was dispensed per well, incubated at room temperature for 1 hour, and washed with DPBS. HaCaT cells, a human keratinocyte cell line, were suspended in serum-free DMEM and plated at 5 × 10 cells per well. 4The cells were then plated in aliquots and allowed to adhere to the dish for 6 hours in a 37°C CO2 incubator. Cells that had not adhered after 6 hours were washed with DPBS, and the remaining cells were cultured in serum-free DMEM medium for 24 and 48 hours. Images of the cells were taken 6, 24, and 48 hours after seeding, and the results are shown in Figure 4. Cell viability and proliferation were measured by adding EZ-Cytox reagent (DoGenBio) in an amount equivalent to 1 / 10 of the medium volume per well after capturing images at each time point. The wells were then incubated in a 37°C CO2 incubator for 30 minutes, and the absorbance was measured at 450 nm. The results are shown in Figure 5. The cell proliferation rates at 24 and 48 hours were calculated using the following formula: [(absorbance after 24 or 48 hours - absorbance after 6 hours) / absorbance after 6 hours] × 100.

[0117] FIG. 4 shows images comparing the cell proliferation ability of coatings of nidogen with other extracellular matrix proteins (gelatin and collagen).

[0118] FIG. 5 is a graph comparing the cell proliferation ability of coatings of nidogen with other extracellular matrix proteins (gelatin and collagen).

[0119] As shown in Figures 4 and 5, cells on plates coated with nidogen protein showed higher adhesion ability in a nidogen concentration-dependent manner, and the proliferation rate of cells adhering to the nidogen coating over 24 and 48 hours was significantly higher than that of cells adhering to gelatin or collagen coatings.

[0120] 2.4. Effect of double coating of nidogen protein and other extracellular matrix proteins on human keratinocyte proliferation

[0121] It is well known that nidogen binds to the remaining components of the basement membrane and mediates its role. We analyzed the synergistic effect of double coating of nidogen protein with other extracellular matrix proteins on cell proliferation.

[0122] Nidogen-1 protein (10, 50 μg / ml) and collagen solution (12.5%, 25%, 50%, 100%) or nidogen-1 protein (1, 5, 10 μg / ml) and collagen solution (25%) were coated on a 24-well culture plate at 4°C for 24 hours. After blocking with 0.3% BSA solution for 1 hour, the plate was washed. HaCaT cells were suspended in serum-free DMEM at 1 × 10 per well. 4 The cells were then plated in aliquots and allowed to adhere to the dishes for 4 hours in a CO2 incubator at 37°C. After 4 hours, non-adherent cells were removed, and the adherent cells were further cultured for 8, 16, 24, and 48 hours. Images of the cells were taken at each time point and treated with EZ-Cytox to measure cell survival and proliferation. The results are shown in Figure 6.

[0123] FIG. 6 shows cell proliferation images (top) and graphs (bottom) demonstrating the synergistic effect of nidogen and collagen dual coating on cell proliferation.

[0124] As shown in Figure 6, coating with nidogen protein and collagen solution individually showed better cell adhesion and proliferation induction effects than uncoated culture plates, but the highest cell proliferation was induced in plates double-coated with a combination of 10 μg / mL nidogen protein and 25% collagen solution.

[0125] This confirmed that nidogen and collagen coating have a synergistic effect on cell proliferation.

[0126] Experimental Example 3. Effect of nidogen protein coating on UVB-treated full-thickness artificial skin model

[0127] 3.1. Epidermis-dermis adhesion effect of nidogen coating

[0128] The full-thickness artificial skin model was prepared based on the method disclosed in [Larouche et al. BioResearch Open Access (2016) 5(1):320-329]. Human dermal fibroblast (HDF) cells were cultured in DMEM medium supplemented with 10% FBS and 10 mg / mL ascorbic acid for 4 weeks to form a dermal sheet. Nidogen-1 protein (50 μg / mL or 100 μg / mL) was applied to the dermal sheet, which was then air-dried at room temperature for 45 minutes and washed twice with DPBS. HEK cells were planted on the dermal sheet and cultured for 1 week to form an epidermal-dermal structure. The formed epidermal-dermal structure was placed on another dermal sheet and exposed to air for 14 days to induce epidermal layer formation. The medium was changed every 2 days during the 14-day exposure period. UVB (60 mJ / cm2) was applied to the full-thickness artificial skin model. 2 ) treatment was performed once at the end of 14 days of differentiation, and the artificial skin was harvested 24 hours later. After the treatment, the artificial skin was fixed in 4% paraformaldehyde, then prepared into paraffin blocks and cut into 10 μm-thick sections to prepare slides for various histological staining. The fixed slides were placed in a staining jar and deparaffinized by immersion in xylene for 5 minutes. They were then immersed sequentially in 100%, 95%, 90%, 80%, and 70% ethanol, rinsed with water, and stained with Harris hematoxylin (Sigma-Germany) for 10 minutes. They were then rinsed with water, treated with 0.5% HCl, rinsed with water, treated with 0.5% ammonia, and rinsed again, before being stained with eosin solution for 2 minutes. After rinsing, the sections were dehydrated by sequential treatments in 70%, 80%, 95%, and 100% ethanol, immersed in xylene for 1 minute, and finally mounted in Canada balsam for observation under a light microscope. The results are shown in Figure 7.

[0129] FIG. 7 is an image showing the epidermal-dermal adhesive effect of nidogen in UVB-irradiated artificial skin (20X Scale bar: 100 μm, 40X Scale bar: 50 μm).

[0130] As shown in Figure 7, under normal conditions, no significant changes were observed with or without nidogen protein coating. However, under UVB irradiation, it was confirmed that the full-thickness model artificial skin coated with nidogen protein significantly reduced the epidermis-dermis separation phenomenon caused by UVB irradiation and the blistering phenomenon caused by UVB irradiation compared to the artificial skin that was not coated with nidogen protein.

[0131] 3.2. Nidogen coating restores basement membrane structure

[0132] The restoration effect of the disrupted basement membrane (BM) structure when UVB was irradiated after applying nidogen coating between the epidermis and dermis of a full-thickness artificial skin model was analyzed using transmission electron microscopy (TEM).

[0133] Specifically, full-thickness artificial skin models were prepared and irradiated with UVB in the same manner as in Experimental Example 3.1 to prepare transmission electron microscope specimens. The transmission electron microscope specimens were fixed in 2% glutaraldehyde-2% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) for 12 hours, washed with 0.1 M phosphate buffer, and then fixed in 1% OsO in 0.1 M phosphate buffer for 2 hours. They were then dehydrated in ascending ethanol series (50, 60, 70, 80, 90, 95, 100, and 100%) for 10 minutes each, and then infiltrated in propylene oxide for 10 minutes. The specimens were polymerized using the Poly / Bed 812 kit (Polysciences) in an electron microscope oven (TD-700, Dosaka, Japan) at 65°C for 12 hours. Then, 200-nm-thin sections were cut using an ultramicrotome (UC7, Leica Microsystems Ltd, Vienna, Austria) equipped with a diamond knife. They were then stained with toluidine blue and observed under an optical microscope. Regions of interest were then cut into 80-nm-thin cross sections using an ultramicrotome, placed on copper grids, and double-stained with 5% uranyl acetate for 20 minutes and 3% lead citrate for 7 minutes. Images were then taken using a transmission electron microscope (HT7800, Hitachi, Tokyo, Japan) equipped with an RC camera at an 80-kV accelerating voltage. The results are shown in Figure 8.

[0134] FIG. 8 is an image showing the effect of nidogen in UVB-irradiated artificial skin on restoring basement membrane structure.

[0135] As shown in Figure 8, in both the nidogen-untreated group (normal group) and the nidogen-treated (coated) group, the epidermal-dermal junction or basement membrane was clearly observed, and structures of hemidesmosomes, anchoring fibrils, and dermal collagen were clearly observed around the basement membrane. In contrast, in the artificial skin of the nidogen-untreated group exposed to UVB, the epidermal-dermal junction where the basement membrane is located was barely visible, and intradermal collagen with black dots was observed. However, in the nidogen-treated group, even after UVB exposure, the basement membrane and structures around the basement membrane were clearly observed, similar to those in the normal group, confirming the effect of nidogen in restoring the basement membrane structure damaged by UVB. Melasma skin exhibits a series of structural and functional changes in the epidermis, basement membrane, and upper dermis, which interact to induce and maintain a local hypermelanogenic phenotype, and basement membrane disruption or gaps are more frequently observed in melasma skin. Thus, damage to the basement membrane is associated with melanogenesis in melasma skin and photoaged skin.

[0136] Based on the above results, the nidogen of the present invention can maintain the structure of the basement membrane and induce extracellular matrix bonds within the basement membrane, thereby suppressing melanin production in skin with blemishes or skin with induced photoaging, improving pigmentation including blemishes and / or dullness, and / or inducing a skin whitening effect.

[0137] 3.3. Nidogen coating restores epidermal differentiation

[0138] Nidogen was applied to the epidermis-dermis interface of a full-thickness artificial skin model, and the recovery effect on the inhibition of epidermal differentiation when irradiated with UVB was analyzed by filaggrin (FLG) staining.

[0139] Specifically, full-thickness artificial skin models were prepared and irradiated with UVB using the same method as in Experimental Example 3.1. The fixed slide specimens were rinsed with tap water to remove paraffin and rehydrated. After rinsing for 3–5 minutes, they were washed in a recovery solution, 0.01 M citrate buffer (pH 6.0), and then microwaved three times for 5 minutes. They were then placed in phosphate-buffered saline (PBS) for 5 minutes, left in a 0.3% H2O2-methanol solution for 10 minutes, and then rinsed with PBS for 10 minutes. They were then treated with a blocking solution (1% BSA) for 1 hour and incubated overnight with primary antibodies (anti-filaggrin, anti-FLG antibodies) at 4°C. After rinsing with PBS for 10 minutes and incubation with secondary antibodies for 1 hour, they were stained with horseradish peroxidase-diaminobenzidine (HRP-DAB) and rinsed with PBS for 10 minutes. After approximately 1 minute of incubation with a DAB substrate kit (Abcam, England), the cells were washed with water and stained with Harris hematoxylin. After washing with water, the cells were dehydrated by sequential treatment with 70%, 80%, 90%, 95%, and 100% ethanol, and then immersed in xylene three times for 3 minutes each. Finally, the cells were mounted with Canada balsam and observed under an optical microscope. The filaggrin expression levels were analyzed in the nidogen-untreated group, nidogen (50 μg / ml)-treated group, and nidogen (100 μg / ml)-treated group. The results are shown in Figure 9.

[0140] FIG. 9 is an image showing the epidermal layer differentiation inhibitory effect of nidogen in artificial skin irradiated with UVB (20X Scale bar: 100 μm, 40X Scale bar: 50 μm).

[0141] As shown in Figure 9, when comparing the nidogen-untreated group with the UVB-irradiated group, it was observed that UVB treatment inhibited epidermal differentiation and weakened the filaggrin staining intensity from the granular layer. In the UVB-irradiated group, the inhibition of epidermal differentiation in the nidogen-treated group was overcome compared to the nidogen-untreated group, and the filaggrin staining intensity increased again from the granular layer. It was confirmed that the overcoming of the inhibition of epidermal differentiation and the effect of inducing filaggrin expression increased depending on the nidogen treatment concentration (0, 50, 100 μg / mL), regardless of the presence or absence of UVB irradiation.

[0142] This confirmed the effect of nidogen in restoring epidermal differentiation damaged by UVB.

[0143] 3.4. Nidogen coating's effect on the restoration of epidermal basal layer proliferation

[0144] The recovery effect on the inhibition of epidermal basal layer proliferation when nidogen was coated between the epidermis and dermis of a full-thickness artificial skin model and then irradiated with UVB was analyzed using Ki67 staining, a cell proliferation marker and stemness marker.

[0145] Specifically, full-thickness artificial skin models were prepared and irradiated with UVB in the same manner as in Experimental Example 3.1, and the expression levels of Ki67 were analyzed in the nidogen-untreated group, nidogen (50 μg / ml)-treated group, and nidogen (100 μg / ml)-treated group in the same manner as in Experimental Example 3.3, as shown in Figure 10.

[0146] FIG. 10 is an image showing the effect of nidogen in UVB-irradiated artificial skin on the restoration of epidermal basal layer proliferation (20X scale bar: 100 μm, 40X scale bar: 50 μm).

[0147] As shown in Figure 10, when comparing the nidogen-untreated group with the UVB-irradiated group and the unirradiated group, it was observed that UVB treatment reduced the number of Ki67-positive cells in the basal layer of the epidermis. In the UVB-irradiated group, it was observed that the number of Ki67-positive cells in the basal layer of the epidermis in the nidogen-treated group increased again in a nidogen treatment concentration-dependent manner compared to the nidogen-untreated group.

[0148] This confirmed the effect of nidogen in restoring proliferation of the epidermal basal layer damaged by UVB.

[0149] 3.5. Nidogen coating restores collagen production in the dermis

[0150] The recovery effect of nidogen coating on the epidermis-dermis interface of a full-thickness artificial skin model and UVB irradiation on the suppression of collagen production in the dermis layer was analyzed using Masson's trichrome (MT) staining method.

[0151] Specifically, full-thickness artificial skin models were prepared and irradiated with UVB light using the same method as in Experimental Example 3.1. The fixed slide specimens were placed in a staining jar and deparaffinized in xylene for 4 minutes. After hydration, they were immersed sequentially in 100%, 95%, 90%, 80%, and 70% ethanol, followed by immersion in Bouin's solution (Sigma-Aldrich, Germany) preheated at 60°C for 45 minutes. The slide specimens were rinsed with running tap water until they turned yellow. Nuclei were stained with Weigert's iron hematoxylin for 8 minutes and then washed. Next, the cytoplasm was stained with anionic dye (Biebrich scarlet), washed with water, treated with phosphomolybdic acid solution, treated with an acidic mordant solution for another 10 minutes, and immediately stained with aniline blue solution for 5 minutes. Finally, the specimens were treated with 1% acetic acid solution for 1 minute, rinsed with water, and dehydrated by sequential immersion in 70%, 80%, 95%, and 100% ethanol. The slides were immersed in xylene for 1 minute, mounted with Canada balsam, and observed under a light microscope. The level of collagen synthesis was analyzed by MT staining in the nidogen-untreated group, nidogen (50 μg / ml)-treated group, and nidogen (100 μg / ml)-treated group.

[0152] FIG. 11 is an image showing the effect of nidogen in UVB-irradiated artificial skin on restoring collagen production in the dermis layer (20X scale bar: 100 μm, 40X scale bar: 50 μm).

[0153] As shown in Figure 11, when comparing the nidogen-untreated group with the UVB-irradiated group and the unirradiated group, it was observed that UVB treatment significantly reduced collagen production in the dermis. In the UVB-irradiated group, collagen production in the dermis was reliably increased in the nidogen-treated group compared to the nidogen-untreated group, and a tendency for the increase to be nidogen treatment concentration-dependent was observed.

[0154] This confirmed that nidogen has the effect of restoring collagen production in the dermis layer damaged by UVB.

[0155] Experimental Example 4. Nidogen protein coating induces stem cell function marker expression

[0156] The protein expression of Ki67 and KRT14 (Keratin 14), stemness markers, in human keratinocytes (HEK) culture dishes coated with nidogen was measured by Western blotting.

[0157] Human keratinocytes (HEK) were cultured for 24 hours on nidogen-coated culture dishes and lysed. Protein content in the cell lysates was quantified, and 8 μg of protein from each test and control group was isolated by 7% polyacrylamide gel electrophoresis (SDS-PAGE). The isolated proteins were transferred to polyvinylidene fluoride (PVDF) membranes, semi-dried for 50 minutes, and then blocked with 5% nonfat dry milk for 1 hour. The PVDF membranes were then incubated overnight at 4°C with primary antibodies: anti-cytokeratin 14 (ab9220, 1:500), anti-Ki67 (MA5-14520, 1:500), and anti-GAPDH (sc47724, 1:2000). The next day, the PVDF membrane was washed with TBST solution containing Tween, Tris, and NaCl, and then incubated with anti-mouse IgG and anti-rabbit HRP-conjugated secondary antibodies at room temperature for 1 hour. Protein bands were analyzed using an Amersham Imager 680, and the results are shown in Figure 12.

[0158] FIG. 12 shows an image of a Western blot (left) showing an increase in the expression of stem cell function-related proteins Ki67 and KRT14 in cells due to nidogen coating, and a graph (right) in which the intensity of each band was normalized and then quantified.

[0159] As shown in Figure 12, GAPDH expression was clearly observed in both the nidogen-untreated group (Null) and the nidogen (50 μg / ml) group, but protein expression of Ki67 and KRT14, which are stem cell function markers of human keratinocytes (HEK), was more clearly observed in the nidogen (50 μg / ml) group than in the nidogen-untreated group. This confirmed that nidogen coating induces the expression of proteins related to the maintenance of stem cell function in keratinocytes.

[0160] Experimental Example 5. Synergistic effect of nidogen protein coating and EPC-CM treatment

[0161] The resistance effect of EPC-CM against oxidative stress has been well elucidated in human fibroblasts (Dermatol Ther (Heidelb) 2018 Jun;8(2):229-244). Therefore, we investigated the oxidative stress resistance effect of EPC-CM in human keratinocytes, HaCaT cells, and its synergistic effect with nidogen in keratinocytes.

[0162] HaCaT cells were suspended in DMEM + 10% FBS medium and then cultured at 3 × 10 cells per well. 4 The cells were then cultured in a CO2 incubator at 37°C for 1 day. The next day, after washing with DPBS, the medium was replaced with EPC-CM diluted with serum-free DMEM to final concentrations of 0, 1, 2.5, 5, 20, and 50% (v / v), and the cells were cultured in a CO2 incubator at 37°C for 24 hours. The same medium was treated with 600 μM H2O2 and cultured for an additional 24 hours. After treatment with EZ-Cytox, cell proliferation was measured, and the results are shown in Figure 13 (left).

[0163] In comparison, we investigated whether there was a difference between treatment with EPC-CM alone or EPC-CM after nidogen-1 protein coating under normal conditions. HaCaT cells were suspended in DMEM + 10% FBS medium and then cultured at 3 × 10 per well. 4 The cells were then cultured for 1 day in a CO2 incubator at 37°C. The next day, after washing with DPBS, the medium was replaced with EPC-CM diluted with serum-free DMEM to final concentrations of 0, 1, 5, 20, and 50% (v / v). The cells were then cultured for 24 hours in a CO2 incubator at 37°C. After that, the medium was replaced with serum-free DMEM, and the cells were treated with EZ-Cytox and cell proliferation was measured. The results are shown in Figure 13 (center).

[0164] Under normal conditions, we examined whether there was a difference in the effect of EPC-CM with or without nidogen-1 protein coating. Nidogen-1, measured by BCA assay, was diluted to a concentration of 10 μg / ml with DPBS and dispensed at 500 μl per well into untreated cell culture plates (24-well plates). The plates were coated at 4°C for 1 day (control group: 500 μl of DPBS). The next day, 500 μl of 0.5% BSA solution was dispensed per well, incubated at room temperature for 1 hour, and then washed with DPBS. The prepared HaCaT cells were suspended in serum-free DMEM medium and plated at 3 × 10 per well. 4 The cells were then cultured in a CO2 incubator at 37°C for one day. The next day, the medium was changed to EPC-CM diluted with serum-free DMEM to final concentrations of 0, 1, 5, or 50% (v / v), and the cells were cultured in a CO2 incubator at 37°C for 24 hours. After 24 hours, images of the cells were taken, the medium was changed to serum-free DMEM, and cell proliferation was measured after EZ-Cytox treatment. The results are shown in Figure 13 (right).

[0165] FIG. 13 is a graph showing the synergistic effect of nidogen coating and epidermal progenitor cell culture medium on cell proliferation.

[0166] As shown in Figure 13, under oxidative stress conditions induced by H2O2, treatment with EPC-CM at low concentrations (up to 5%) was effective in restoring cell proliferation. Unlike under oxidative stress conditions, under normal conditions, no significant effect on cell proliferation was observed regardless of the EPC-CM treatment concentration. However, nidogen coating treatment significantly increased cell proliferation in an EPC-CM concentration-dependent manner.

[0167] This confirmed that there is a synergistic effect between nidogen coating and EPC-CM on cell proliferation.

[0168] Experimental Example 6. Cellular and clinical effects of products containing nidogen protein and EPC-CM

[0169] A formulation containing 33% EPC-CM as the main ingredient and a formulation containing 10 ppm (10 μg / ml) of nidogen-1 protein in 33% EPC-CM were prepared, and their proliferation effects were evaluated in human fibroblast (HDF) cells.

[0170] A dilution concentration (25%) was established at which no toxicity was observed using a solvent control group containing excipients used during formulation manufacturing. HDFs were placed in a 48-well plate at 1 × 10 4 The cells were cultured for 24 hours after seeding, and the next day, serum-free DMEM was replaced with serum-free DMEM and cultured for another 24 hours. Each formulation was diluted to 25% with serum-free DMEM and treated with the cells for 24 hours. Cell survival and proliferation were then evaluated using EZ-Cytox. The results are shown in Figure 14.

[0171] FIG. 14 is a graph showing the effect of a formulation containing nidogen and an epidermal progenitor cell culture medium on cell proliferation.

[0172] As shown in Figure 14, the formulation containing EPC-CM significantly increased cell proliferation compared to the vehicle control group containing only the excipient. In comparison, the formulation containing EPC-CM and nidogen together demonstrated a greater effect.

[0173] Experimental Example 7. Nidogen coating and cell wound healing effects

[0174] The cell wound healing effect of wild-type nidogen-1 protein was evaluated by a cell scratch assay.

[0175] Specifically, nidogen (NID1) (10, 50 mg / mL), collagen (COL) (50 mg / mL), laminin (LAM) (10 mg / mL), and gelatin (GEL) (50 mg / mL) were coated onto a 6-well culture dish, which was then blocked with 0.3% BSA for 1 hour. HaCaT cells were then cultured at 1 × 10 per well. 5The cells were grown to 100% confluence and then wounded with a 200 mL tip. The medium was then replaced with serum-free medium or 10% FBS (positive control), and images were taken with an optical microscope 3, 6, and 24 hours later. The damaged area was measured using the Image J program, and the results are shown in Figure 15.

[0176] As shown in FIG. 15, it was confirmed that the wound healing effect of keratinocytes was greater when coated with nidogen than when coated with collagen, laminin, or gelatin.

[0177] These results suggest that nidogen has a concentration-dependent effect on cell adhesion and proliferation, and that it exerts a synergistic effect on cell proliferation when used in combination with epidermal progenitor cell culture medium or collagen. Nidogen therefore has an effect on the epidermis and dermis layers through strengthening the basement membrane, and can be useful for skin improvement, such as repairing skin damage, strengthening the skin barrier, inhibiting or preventing skin pigmentation, moisturizing, improving skin elasticity, and anti-aging.

[0178] The above description of the present invention is for illustrative purposes only, and those skilled in the art will understand that the present invention can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. Therefore, it should be understood that the above-described embodiments are illustrative in all respects and are not limiting.

Claims

1. A cosmetic composition for improving skin, comprising nidogen protein as an active ingredient.

2. The skin-improving cosmetic composition according to claim 1, further comprising an epidermal progenitor cell-conditioned media.

3. The cosmetic composition for improving skin according to claim 1 , wherein the protein comprises the amino acid sequence of SEQ ID NO:

1.

4. 2. The cosmetic composition for improving skin according to claim 1, wherein the skin improvement is one or more selected from the group consisting of skin barrier improvement, prevention or improvement of skin pigmentation, prevention or improvement of skin wrinkles, skin moisturizing, promotion of skin anti-aging, promotion of skin resistance, promotion of skin elasticity, skin soothing, skin regeneration, and prevention or improvement of skin diseases.

5. The cosmetic composition for improving skin according to claim 1, wherein the skin improvement comprises the following properties: - suppression of epidermal-dermal separation caused by UVB or ultraviolet light, - maintaining the alignment of basal keratinocytes or the structure of the basement membrane due to UVB or ultraviolet light, or - Alleviation of inhibition of differentiation of epidermal layers by UVB or ultraviolet rays.

6. The cosmetic composition for improving skin according to claim 1 , wherein the protein improves extracellular matrix junctions within the basement membrane or dermal density.

7. The cosmetic composition for improving skin according to claim 1, wherein the protein promotes the production of intracellular collagen, Ki67, or filaggrin.

8. A cosmetic composition for maintaining epidermal stem cell function (stemness), comprising nidogen protein as an active ingredient.

9. 9. The cosmetic composition for maintaining epidermal stem cell function (stemness) according to claim 8, wherein the protein upregulates the expression of a gene from the group consisting of Ki67, KRT14 (Keratin 14), ITGA6 (Integrin alpha-6), TP63, and SOX2 (SRY-Box Transcription Factor 2).

10. 9. The cosmetic composition for maintaining epidermal stem cell function (stemness) according to claim 8, wherein the maintenance of stem cell function is any one or more selected from the group consisting of inhibiting cellular senescence of stem cells, increasing cell proliferation ability, increasing stem cell functionality, increasing telomerase activity, increasing stem cell factor expression, increasing protein homeostasis, and increasing cell migration activity.

11. A pharmaceutical composition for preventing, improving, or treating skin damage, comprising a nidogen protein as an active ingredient.

12. The pharmaceutical composition of claim 11, wherein the skin injury is a skin wound, a skin scar, or a skin burn.

13. A skin external preparation composition for improving skin or preventing, improving or treating skin damage, comprising nidogen protein as an active ingredient.

14. A medium composition for maintaining epidermal stem cell function (stemness), comprising nidogen protein as an active ingredient.

15. Artificial skin consisting of a dermal layer containing fibroblasts and an epidermal layer containing keratinocytes and nidogen proteins.

16. The artificial skin of claim 15, wherein the nidogen protein is present in the basement membrane of the epidermal layer.

17. The artificial skin of claim 15, wherein the nidogen protein comprises the amino acid sequence of SEQ ID NO:

1.

18. Culturing fibroblasts to produce a dermis layer; applying a nidogen protein onto the dermis layer; and applying keratinocytes onto the dermis layer to which the nidogen protein has been applied to produce an epidermis layer.

19. The method according to claim 18, wherein the step of producing the epidermal layer comprises first producing a dermal-epidermal structure from applied keratinocytes, and then culturing the dermal-epidermal structure on another dermal layer.

20. A method for maintaining epidermal stem cell function in skin cells, comprising culturing the skin cells in a medium containing isolated nidogen protein.

21. A method for improving a skin condition or preventing, ameliorating or treating skin damage comprising the step of administering to an individual in need thereof an effective amount of a nidogen protein.

22. Use of a nidogen protein for use in the manufacture of a preparation for improving skin conditions or preventing, improving or treating skin damage.

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