Methods for evaluating skin sagging and wrinkles

Abstract

The objective is to provide a method for evaluating sagging and wrinkle conditions based on a novel mechanism. [Solution] The present inventors have discovered that frame-like collagen, formed by collagen bundles, exists in the dermis layer, and that frame-like collagen present in the dermis layer maintains the morphology of the skin and contributes to preventing sagging and wrinkles. Therefore, this disclosure relates to a method for evaluating the sagging and wrinkle state of skin by evaluating frame-like collagen formed by collagen bundles in the target skin.

Description

Technical Field

[0001] The present disclosure relates to a method for evaluating skin sagging and wrinkles.

Background Art

[0002] The occurrence of wrinkles and sagging in aging skin is a major visible sign of aging and a cosmetic concern for many women. One cause of wrinkles and sagging is the thinning of skin tissue with aging. In aging skin, there is a significant decrease in collagen fibers, which are the main matrix components of the dermis, and this is the main cause of the decrease in skin thickness. Therefore, promoting the production of collagen and maintaining the amount of collagen is considered effective in preventing and improving wrinkles and sagging. From the above perspective, cosmetics have been developed (Patent Document 1) to increase the amount of collagen and bring about prevention and improvement of wrinkles and sagging.

Prior Art Documents

Patent Documents

[0003]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0004] As described above, the development of cosmetics and beauty methods for increasing the amount of collagen and improving the sagging and wrinkle conditions has progressed, but research on improving the sagging and wrinkle conditions from other viewpoints has not progressed. Therefore, it is an object to provide a method for evaluating the sagging and wrinkle conditions based on a novel mechanism different from the causes of the skin sagging and wrinkle conditions so far.

Means for Solving the Problems

[0005] The inventors discovered that frame-like collagen, formed by collagen bundles, exists in the dermis. Surprisingly, they found that frame-like collagen in the dermis maintains skin morphology and contributes to anti-sagging and anti-wrinkle properties. Further research revealed that Wnt16b and biglycan are involved in the formation of frame-like collagen, leading to this disclosure.

[0006] Therefore, this disclosure relates to the following invention: [1] A method for evaluating skin sagging and wrinkles by evaluating frame-like collagen formed by collagen bundles in the target skin. [2] The evaluation method described in item 1, wherein the frame-like collagen is a mesh-like structure formed by collagen bundles of 50 to 500 μm. [3] The evaluation method described in item 1 or 2, wherein the frame-like collagen is evaluated by image analysis. [4] The evaluation of the frame-like collagen is as follows: A step of measuring the biglycan expression level and / or Wnt16b expression level in the dermis of the target skin, and The method according to item 1 or 2, which includes a step of determining the dermal collagen bundle level from the correspondence between the biglycan expression level and / or the Wnt16b expression level and the collagen bundle level. [5] Biglycan expression level is measured by the amount of biglycan protein or gene expression, and Wnt16b expression level is measured by the amount of Wnt16b protein or gene expression, as described in item 4 of the evaluation method. [6] The evaluation method described in item 4 or 5 for measuring the biglycan expression level and / or Wnt16b expression level in dermal fibroblasts. [7] The method according to any one of items 4 to 6, wherein the correspondence is a correspondence based on a correlation formula or a correspondence based on a threshold. [8] A method for inducing biglycan expression from dermal fibroblasts by applying Wnt16b to dermal fibroblasts. [9] A step of culturing a culture containing dermal fibroblasts in a culture medium containing a candidate drug; A step of measuring the expression level of biglycan or Wnt16b in the skin culture after culturing in a medium containing the candidate drug; and The process of comparing the measured expression levels with a control to determine which candidate drugs promote the formation of flame-shaped collagen. A method for screening agents that promote the formation of frame-like collagen, which is formed by collagen bundles in the skin.

[10] The screening method described in item 9, wherein the expression level of biglycan or Wnt16b is measured by the amount of biglycan or Wnt16b protein or the gene expression level.

[11] The method according to item 9 or 10, wherein the frame-like collagen formation promoter is a wrinkle or sagging improvement agent.

[12] The method according to any one of items 9 to 11, wherein the control is the expression level of biglycan or Wnt16b in a culture containing dermal fibroblasts cultured in a medium that does not contain the candidate drug, or a threshold determined from said expression level.

[13] A beauty treatment that promotes the formation of frame-like collagen by applying a temperature stimulus of 38°C to 45°C.

[14] The method of item 13, which promotes the formation of framing collagen, which is formed by collagen bundles in the skin, by increasing the expression of biglycans.

[15] The method of item 13, which promotes the formation of framing collagen, which is formed by collagen bundles in the skin, by increasing the expression of Wnt16b. [Effects of the Invention]

[0007] The evaluation method disclosed herein can be used to assess the condition of skin sagging and wrinkles. [Brief explanation of the drawing]

[0008] [Figure 1]Figure 1(A) shows frame-like collagen present in the dermis (indicated by black arrows). Figure 1(B) shows that sweat glands and blood vessels extend along bundles of collagen. [Figure 2-1] Figure 2(A) shows the function of frame-like collagen in the skin. It shows X-ray CT images taken before and after deformation of skin sections. Figure 2(B) is a graph showing the degree of deformation in each area after deformation treatment, comparing frame-like collagen identified in the X-ray CT images with non-frame-like collagen. [Figure 2-2] Figure 2(C) shows the appearance of the skin in young and elderly individuals, and the results of ultrasound measurements on cheek skin sections. Frame-like collagen is indicated by black arrows. Figure 2(D) shows the relationship between cheek sagging and frame-like collagen. [Figure 2-3] Figure 2(E) shows the relationship between frame-like collagen and dermal thickness and elasticity. [Figure 3] Figure 3 shows the results of immunostaining for biglycans in skin section samples. [Figure 4] Figure 4 shows a graph of biglycan expression when fibroblasts were cultured in Wnt16b-supplemented medium. It demonstrates that Wnt16b induces biglycan expression. [Figure 5-1] Figure 5(A) shows images of human skin sections from young and elderly individuals. Figure 5(B) shows that the amount of framing collagen decreases with age. [Figure 5-2] Figure 5(C) shows immunohistochemical staining images of biglycans in human skin sections from young and elderly individuals. Figure 5(D) shows that biglycans decrease with age. [Figure 5-3] Figure 5(E) shows immunohistochemical staining images of Wnt16b in blood vessels of human skin sections from young and elderly individuals. Wnt16b is indicated by white arrows. This shows that the amount of Wnt16b protein in blood vessels decreases with age. [Figure 5-4]FIG. 5(F) shows an immunostaining photograph of WNT16b in the sweat glands of human skin sections of the young and the elderly. Wnt16b is indicated by the white arrow. It shows that with aging, the protein amount of Wnt16b in the sweat glands decreases. [Figure 6-1] FIG. 6(A) is an immunostaining image of organ-cultured skin before and after temperature stimulation, and the graph shows that Wnt16 increased due to temperature stimulation. Blood vessels are indicated by the black arrow. FIG. 6(B) shows immunostaining of human skin sections before and after temperature stimulation, and the graph shows that biglycan increased due to temperature stimulation. FIG. 6(C) is an image of human skin sections before and after temperature stimulation, and the bar graph shows that the amount of collagen bundles increased due to temperature stimulation. FIG. 6(D) shows the height of the dermis before and after temperature stimulation, and the bar graph shows that the height of the dermis increased due to temperature stimulation. [Figure 6-2] FIG. 6(E) shows the width of the dermis before and after temperature stimulation, and the bar graph shows that the width of the skin decreased due to temperature stimulation. [Figure 7-1] FIG. 7(A) shows a photograph of human skin before and after temperature stimulation. Sagging is indicated by the white arrow. FIG. 7(B) shows the proportion of volunteers whose sagging was improved before and after temperature stimulation. [Figure 7-2] FIG. 7(C) shows the proportion of frame-shaped collagen before and after temperature stimulation.

MODE FOR CARRYING OUT THE INVENTION

[0009] One aspect of the present disclosure relates to a method for evaluating the sagging and wrinkling state of the skin by evaluating the frame-shaped collagen formed by collagen bundles in the target skin. Another aspect of the present disclosure relates to a method for screening a promoter for forming frame-shaped collagen formed by collagen bundles in the skin, using biglycan expression or Wnt16b expression as an index. Still another aspect of the present disclosure relates to a beauty method for promoting the formation of frame-shaped collagen by applying temperature stimulation.

[0010] The inventors discovered that frame-like collagen, formed by collagen bundles, exists in the dermis (Figure 1(A)), and found that frame-like collagen in the dermis maintains skin morphology and contributes to anti-sagging and anti-wrinkle effects (Figure 2). Furthermore, they discovered that biglycan is involved in the formation of frame-like collagen, which aggregates collagen bundles to form a reticular structure, that Wnt16b induces biglycan in dermal fibroblasts (Figure 4), and that thermal stimulation increases the expression of biglycan and Wnt16b (Figure 6(A)(B)).

[0011] In one aspect of this disclosure, this disclosure relates to a method for evaluating the sagging and wrinkle state of skin by evaluating frame-like collagen formed by collagen bundles in the target skin. Frame-like collagen is different from the amount of collagen, and it is possible to evaluate the sagging and wrinkle state from a different perspective, namely from a qualitative perspective, than conventional evaluations of sagging and wrinkle state based on the amount of collagen. By providing an evaluation method based on frame-like collagen in addition to the amount of collagen, it becomes possible to evaluate the skin condition from multiple perspectives.

[0012] Collagen fibers are formed when collagen proteins polymerize through intermolecular crosslinking. Collagen bundles are made up of clusters of collagen fibers. Collagen fibers do not exist randomly in the dermis, but rather have a certain directionality and aggregation. When collagen fibers aggregate, they form thicker collagen bundles, creating a matrix within the dermis.

[0013] Frame collagen refers to collagen with a reticular structure formed from bundles mainly composed of type I collagen fibers. Type I collagen is abundant in the dermis and constitutes the interstitial component of the reticular layer, a connective tissue with dense fibrous components. Frame collagen is present in the dermis and plays a role as a skeleton that maintains the skin's morphology. By functioning as a skeleton in the dermis, frame collagen increases the height of the skin, lifts excess skin that spreads out, and as a result improves sagging. Therefore, the level of frame collagen formed by collagen bundles in the skin can be used to evaluate the degree of skin sagging and wrinkles.

[0014] In this specification, a collagen bundle refers to a bundle with a diameter of approximately 50 to 500 μm, mostly around 100 μm, that is significantly aggregated compared to the surrounding collagen fibers. Frame collagen refers to a network-like structure formed from collagen bundles. Therefore, frame collagen may be determined based on the morphology of the collagen. When frame collagen is determined by the morphology of the collagen, the collagen bundle level may be used as the morphology of the collagen. The collagen bundle level is an index determined, for example, by the thickness of the collagen bundle. Since frame collagen plays a role as a skeleton that maintains the skin's morphology, it can also be called the dermal skeleton.

[0015] The evaluation of flame-like collagen may be performed by image analysis. The images used for image analysis are not particularly limited, and X-ray microscope images and fluorescence microscope images may be used for analysis. In fluorescence microscopy analysis, the determination may be based on the expression of proteins specifically expressed in flame-like collagen, such as type I collagen or biglycan. In addition to expression, flame-like collagen may also be determined by referring to its distribution and / or morphology.

[0016] The evaluation of framing collagen involves the following steps, for example: A step of measuring the biglycan expression level in the dermis of the target skin, and This may be performed by an evaluation method that includes a step of determining the dermal collagen bundle level from the correspondence between the biglycan expression level and the collagen bundle level. This allows for the evaluation of frame-like collagen formed by collagen bundles in the skin, and the evaluation of this frame-like collagen can also be used to assess the state of skin sagging and wrinkles.

[0017] The biglycan expression level in the dermis can be determined by measuring the amount of mRNA or protein in a dermal sample. mRNA levels can be measured using known methods in this art, such as quantitative PCR or Northern blotting. Protein levels can be measured using any known method in this art, such as Western blotting, immunohistochemistry, or FACS. Antibodies that specifically bind to biglycans are used in these methods. For dermal samples, samples obtained from the face can be used to evaluate sagging and wrinkles.

[0018] Biglycans are small proteoglycans present in various tissues that interact with many molecules to control multiple physical reactions and are involved in collagen fiber formation. The inventors have discovered that biglycans assemble collagen bundles. Therefore, there is a correlation between increased biglycan expression and the promotion of collagen bundle formation. It is thought that when collagen bundle formation is promoted, the amount of framing collagen also increases.

[0019] The process for determining the dermal collagen bundle level in this disclosure can uniquely determine the dermal collagen bundle level from the biglycan expression level by using a predetermined correspondence. Such a correspondence may be a correlation formula or a threshold-based correspondence. For multiple subjects, the skin sagging / wrinkling state and the dermal collagen bundle level can be measured and a correspondence can be created, and such a correspondence, as well as its threshold and correlation formula, can be created by methods well known in the art.

[0020] Wnt proteins are highly conserved secreted signaling molecules that form a family of signaling molecules that bind to cell surface receptors encoded by Frizzled and low-density lipoprotein receptor-associated proteins (LRPs). These proteins are involved in multiple developmental processes, including tumorigenesis, as well as the regulation of cell fate and pattern formation during embryonic development.

[0021] Wnt16b secreted from sweat glands and blood vessels induces dermal fibroblasts into a subpopulation of fibroblasts that highly express biglycan. Therefore, biglycan expression levels may be measured by the biglycan expression level in the subpopulation of dermal fibroblasts. Another aspect of this disclosure relates to a method for inducing dermal fibroblasts into a subpopulation of dermal fibroblasts that highly express biglycan by applying Wnt16b to dermal fibroblasts.

[0022] The evaluation of framing collagen involves the following steps, for example: A step of measuring the expression of Wnt16b in the dermis of the target skin, and This may be performed by an evaluation method that includes a step of determining the dermal collagen bundle level from the correspondence between the Wnt16b expression level and the collagen bundle level. This allows for the evaluation of frame-like collagen formed by collagen bundles in the skin, and the evaluation of this frame-like collagen can also be used to assess the state of skin sagging and wrinkles.

[0023] The Wnt16b expression level can be determined by measuring either mRNA or protein levels. mRNA levels can be measured using known techniques in this art, such as quantitative PCR or Northern blotting. Protein levels can be measured using any known technique in this art, such as Western blotting, immunohistochemistry, or FACS. In these methods, antibodies that specifically bind to Wnt16b are used.

[0024] The process for determining the dermal collagen bundle level in this disclosure can uniquely determine the dermal collagen bundle level from the Wnt16b expression level by using a predetermined correspondence. Such a correspondence may be a correlation formula or a threshold-based correspondence.

[0025] By applying Wnt16b to dermal fibroblasts, the expression of biglycans from these dermal fibroblasts can be induced. Biglycans cause the formation of frame collagen, which aggregates collagen bundles to form a reticular structure. Therefore, the level of frame collagen, determined using Wnt16b expression as an indicator, can be used to evaluate the state of skin sagging and wrinkles.

[0026] In one aspect of this disclosure, this disclosure relates to a method for screening flame collagen formation promoters using the expression level of biglycan or Wnt16b as an indicator, for example, the following steps: A step of culturing a culture containing dermal fibroblasts in a culture medium containing a candidate drug; A step of measuring the expression level of biglycan or Wnt16b in the skin culture after culturing in a medium containing the candidate drug; and The process of comparing the measured expression levels with a control to determine which candidate drugs promote the formation of flame-shaped collagen. Includes. A catalyst for promoting the formation of frame-like collagen can improve wrinkles or sagging of the skin.

[0027] The candidate components used in the screening method of this disclosure can be any library, such as cosmetic materials, food materials, or pharmaceutical materials. Such libraries may include compound libraries, extract libraries, etc. The compounds and extracts contained in each library may be commercially available compounds and extracts, or synthesized compounds and prepared extracts.

[0028] The screening method of this disclosure may include a pre-culture step in which dermal fibroblasts are cultured in a medium containing the candidate drug, before the step of culturing dermal fibroblasts in a medium containing the candidate drug. Alternatively, it may include a post-culture step in which dermal fibroblasts are further cultured in a medium that does not contain the candidate drug after the step of culturing dermal fibroblasts in a medium containing the candidate drug. In the step of culturing dermal fibroblasts in a medium containing the candidate drug, the candidate drug or a diluted solution thereof may be added directly to the culture obtained in the pre-culture step, or the culture may be performed by replacing the medium with one containing the candidate drug.

[0029] The expression level of biglycan or Wnt16b can be determined by measuring mRNA or protein levels. mRNA levels can be measured using known methods in the art, such as quantitative PCR or Northern blotting. Protein levels can be measured using any known method in the art, such as Western blotting, immunohistochemistry, or FACS. These methods utilize antibodies that specifically bind to biglycan or Wnt16b.

[0030] The control may be the expression level of biglycan or Wnt16b, differing in that the cells are cultured in a medium that does not contain the candidate drug. Experiments with the control may be conducted in parallel with or prior to the screening method of this disclosure. The control may be a threshold determined from the expression level of biglycan or Wnt16b in a culture containing dermal fibroblasts cultured in a medium that does not contain the candidate drug.

[0031] One aspect of this disclosure relates to a cosmetic method that promotes the formation of frame-like collagen by applying temperature stimulation. By increasing the expression of biglycans and / or Wnt16b through temperature stimulation, skin wrinkles or sagging can be improved by promoting the formation of frame collagen.

[0032] The inventors found that applying thermal stimulation to cultured skin increased Wnt16b in blood vessels and sweat glands, increased biglycans, and increased flamed collagen. While not intended to be theoretical, thermal stimulation increases biglycan expression and promotes the formation of flamed collagen through increased Wnt16b expression. This can improve wrinkles or sagging. Thermal stimulation may preferably be applied to facial skin.

[0033] The preferred temperature stimulus is 38°C to 45°C. Preferably, it is 38°C to 43°C, and more preferably, 40°C.

[0034] The devices that provide temperature stimulation are not particularly limited as long as they can deliver a predetermined temperature as stimulation, and examples include hot masks, gels and creams, other bases, devices, and heated beauty tools.

[0035] In this specification, methods for improving wrinkles or sagging skin refer to cosmetic methods for aesthetic purposes and can be distinguished from treatments performed by doctors or medical professionals; they can be considered non-therapeutic methods. Such cosmetic methods may be performed individually or in places such as beauty salons, cosmetics stores, or esthetic salons.

[0036] All references made herein are incorporated herein by reference in their entirety.

[0037] The embodiments of the Disclosure described below are for illustrative purposes only and do not limit the technical scope of the Disclosure. The technical scope of the Disclosure is limited solely by the claims. Modifications to the Disclosure, such as adding, deleting, or replacing constituent elements of the Disclosure, may be made without departing from the spirit of the Disclosure. [Examples]

[0038] Example 1. Discovery of frame-shaped collagen Preparation of skin samples: This study used 30 surplus facial skin specimens from plastic surgery departments (subject age range: 20–92 years). 20 abdominal skin specimens (subject age range: 22–73 years) obtained from Biopredic International (Rennes, France) were used as standard samples. Skin specimens for observation under physiological conditions were immediately placed in Dulbecco's Modified Eagle Medium (DMEM) containing 10% FBS. All studies were approved by the ethics committees of all participating institutions.

[0039] X-ray Micro-CT : According to the following paper, approximately 3 mm was measured using X-ray micro-CT (Xradia; Zeiss, Oberkochen, Germany). 3 We observed the skin specimens. During the testing period, the physical condition of the skin specimens was maintained in good condition and confirmed to have recovered to its original state. Skin structures in all CT images were automatically identified by the artificial intelligence (AI) deep learning system, Dragonfly (Object Research Systems, Montreal, Canada). Paper: Ezure T (2023) Subcutaneous fat infiltration into the dermal layer induces wrinkle formation. Skin Res Technol 29:e13296-e13300.

[0040] X-ray images of human skin tissue were used to focus on collagen fibers present in the dermis. It was found that the dermis contains super-aggregated collagen bundles, each composed of several thick type I collagen fibers, forming a reticular structure (Figure 1(A)). The collagen fibers previously observed are located within this frame-like collagen. Furthermore, sweat glands and blood vessels extend along these bundles (Figure 1(B)). Collagen fibers aggregate to form collagen bundles. Further aggregation of these bundles creates frame-like collagen.

[0041] Example 2. Functional analysis of flame-shaped collagen The function of frame-like collagen was analyzed using dynamics analysis by X-ray micro-CT as described in Example 1. Specifically, X-ray analysis was performed while pressure was applied to deform a skin sample, and the state of frame-like collagen and non-frame-like collagen before and after deformation was confirmed. Frame-like collagen resisted skin deformation, while non-frame-like collagen was deformed (Figure 2(A)).

[0042] The relationship between sagging and frame-like collagen was confirmed non-invasively in actual facial skin using the method described below. Sagging skin evaluation: The evaluation was conducted using photograph-based evaluation criteria, as described in the following paper. Paper: Ezure T, Yagi E, Kunizawa N, Hirao T, Amano S (2011) Comparison of sagging at the cheek and lower eyelid between male and female faces. Skin Res Technol 17:510-515. Determining the state of frame-like collagen: The state of the frame-like collagen was determined using the Prosound alpha 5 ultrasound diagnostic device (Aloka Co., Ltd., Tokyo, Japan) according to the following paper. Paper: Ezure T, Amano S (2010) Influence of subcutaneous adipose tissue mass on dermal elasticity and sagging severity in lower cheek. Skin Res Technol 16:332- 338. In the ultrasound image shown in Figure 2(C), the white brightness areas (indicated by arrows) in the dermal region were identified as framed collagen, and the black areas as non-framed collagen. The density of framed collagen was then measured. The statistical analysis will be performed using the following methods. statistical analysis Differences between groups were evaluated using Student's t-test. However, for efficacy studies in humans, the Wilcoxon rank test was used (*: p<0.05 and **: p<0.01). Correlations were evaluated using Pearson's correlation coefficient or Spearman's correlation coefficient. A p-value of less than 0.05 was considered statistically significant.

[0043] Subjects with less sagging had abundant framing collagen, while subjects with severe sagging had very little framing collagen (Figure 2(C)). Loss of framing collagen was statistically significantly associated with increased sagging. Framing collagen increased skin thickness and elasticity (Figure 2(E)).

[0044] Example 3. Functional analysis of biglycans In Example 1, skin section samples were obtained and immunostained using biglycan antibody (Abcam).

[0045] Immunohistofluorescence staining: Paraffin-embedded skin sections were treated with biglycan antibody (Abcam), then immunofluorescence staining was performed using a fluorescently labeled secondary antibody (Cell Signaling), and the samples were observed under a fluorescence microscope (Keyence).

[0046] Gene expression analysis revealed that the expression of the biglycan gene coincided with the location of bundles of highly aggregated collagen. This confirmed that the bundles of highly aggregated collagen were surrounded by biglycan (Figure 3).

[0047] Example 4. Induction of biglycan expression from dermal fibroblasts Human dermal fibroblasts (Lonza; Basel, Switzerland) were cultured in DMEM containing 10% FBS at 37°C under a 5% CO2 humidified atmosphere. Secretory factors were obtained from R&D Systems (MN, USA). Fibroblasts highly expressing biglycan were obtained by in vitro cloning. Compared to dermal fibroblasts, biglycan-expressing fibroblasts promoted the formation of highly aggregated collagen bundles.

[0048] Example 5. Promotion of biglycan expression Human dermal fibroblasts (Lonza; Basel, Switzerland) were cultured in DMEM containing 10% FBS at 37°C under a 5% CO2 humidified atmosphere. When the cultured human fibroblasts reached 10-20% confluence, the medium was replaced with 10 ug / mL Wnt16 supplemented medium (Wnt16b: R&D systems) and cultured for 48 hours. As a control, the same experiment was performed using an experimental system that differed only in that it did not contain Wnt16b. After 48 hours of culture, the cells were harvested, RNA was recovered using RNeasy (Qiagen), and biglycan expression was measured by real-time PCR. Real-time PCR was performed using the following primer set and a Light-cycler (Roche, IN, USA). [Table 1]

[0049] Example 6. Analysis of frame-like collagen due to aging Human skin imaging revealed that frame-like collagen decreases with age, and dissociated collagen fibers become dominant (Figure 5(A)). Fluorescent immunostaining using an antibody against biglycan (Abcam), performed using the method described in Example 3, revealed that biglycan also decreases with age (Figure 5(C), (D)). Skin samples prepared in Example 1 were immunostained using Wnt16 antibody (Abcam). Visualization was performed using a fluorescently labeled secondary antibody (Cell Signaling), and observation was performed with a fluorescence microscope (Keyence). It was found that Wnt16b decreases with age in both blood vessels and sweat glands (Figure 5(E), (F)).

[0050] Example 7. Effects of temperature stimulation on cultured skin tissue Human skin specimens were cultured at 37°C in DMEM containing 10% FBS under a 5% CO2 humidified atmosphere. The cultured skin prepared in Example 1 was subjected to thermal stimulation at 40°C for 1 hour. Cultured skin tissue was obtained after thermal stimulation, and immunostaining for Wnt16b and biglycan was performed as in Examples 3 and 6 (Figure 6A, B). Collagen bundles were also evaluated by X-ray CT (Figure 6C). The height and width of the skin sections were also evaluated (Figure 6D, E). Increases in Wnt16b, biglycan, and flamed collagen were observed with thermal stimulation (Figure 6). Furthermore, thermal stimulation at 40°C increased the height of the skin and decreased the width of the skin (Figure 6D, E).

[0051] Example 8. Effects of temperature stimulation on human skin. Ten female volunteers in their 40s were subjected to a 15-minute temperature stimulation at 40°C once a day for four weeks. Sagging, frame-like collagen, and skin properties were evaluated using the methods described in Example 2 and below (Figure 7). Measurement of skin properties: Skin properties were measured using a Cutometer 580® (Courage & Khazaka, Cologne, Germany) according to the following paper. Paper: Ryu HS, Joo YH, Kim SO, Park KC, Youn SW (2008) Influence of age and regional differences on skin elasticity as measured by the Cutometer. Skin Res Technol 14:354-358.

Claims

1. A method for evaluating skin sagging and wrinkles by assessing the frame-like collagen formed by collagen bundles in the target skin.

2. The evaluation method according to claim 1, wherein the frame-like collagen is a mesh-like structure formed by collagen bundles of 50 to 500 μm.

3. The evaluation method according to claim 1 or 2, wherein the evaluation of the frame-like collagen is performed by image analysis.

4. The evaluation of the aforementioned frame-like collagen is as follows: A step of measuring the biglycan expression level and / or Wnt16b expression level in the dermis of the target skin, and The method according to claim 1 or 2, wherein the evaluation method includes the step of determining the dermal collagen bundle level from the correspondence between the biglycan expression level and / or the Wnt16b expression level and the collagen bundle level.

5. The evaluation method according to claim 4, wherein the biglycan expression level is measured by the amount of biglycan protein or the gene expression level, and the Wnt16b expression level is measured by the amount of Wnt16b protein or the gene expression level.

6. The evaluation method according to claim 4, which measures the biglycan expression level and / or Wnt16b expression level in dermal fibroblasts.

7. The method according to claim 4, wherein the correspondence is a correspondence based on a correlation formula or a correspondence based on a threshold.

8. A method for inducing biglycan expression from dermal fibroblasts by applying Wnt16b to dermal fibroblasts.

9. A step of culturing a culture containing dermal fibroblasts in a culture medium containing a candidate drug; A step of measuring the expression level of biglycan or Wnt16b in the skin culture after culturing in a culture medium containing the candidate drug; and The process of comparing the measured expression levels with a control to determine which candidate drugs promote the formation of flame-shaped collagen. A method for screening agents that promote the formation of frame-like collagen, which is formed by collagen bundles in the skin.

10. The screening method according to claim 9, wherein the expression level of biglycan or Wnt16b is measured by the amount of biglycan or Wnt16b protein or the gene expression level.

11. The method according to claim 9, wherein the frame-like collagen formation promoter is a wrinkle or sagging improvement agent.

12. The method according to any one of claims 9 to 11, wherein the control is the expression level of biglycan or Wnt16b in a culture containing dermal fibroblasts cultured in a medium that does not contain the candidate drug, or a threshold determined from said expression level.

13. This beauty treatment promotes the formation of frame-like collagen by applying a temperature stimulus of 38°C to 45°C.

14. The method according to claim 13, which promotes the formation of frame collagen, which is formed by collagen bundles in the skin, by increasing the expression of biglycans.

15. The method according to claim 13, wherein the formation of frame-like collagen, which is formed by collagen bundles in the skin, is promoted by increasing the expression of Wnt16b.

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