Method for evaluating sagging and wrinkles of skin

Abstract

The present invention addresses the problem of providing a method for evaluating a sagging / wrinkle state on the basis of a novel mechanism. The present inventors have found that frame-like collagen formed by collagen bundles is present in the dermal layer, and discovered that the frame-like collagen present in the dermal layer maintains skin shape and contributes to anti-sagging and anti-wrinkle of the skin. Therefore, the present disclosure relates to a method for evaluating a sagging / wrinkle state of skin by evaluating the frame-shaped collagen formed by collagen bundles in a target skin.

Description

Method for Evaluating Skin Sagging and Wrinkles

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

[0002] The occurrence of wrinkles and sagging seen in aging skin is a major external sign of aging and is a cosmetic concern for many women. One cause of wrinkles and sagging is the thinning of skin tissue due to 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, it is considered effective to promote collagen production and maintain the amount of collagen for the prevention and improvement of wrinkles and sagging. From the above perspective, cosmetics have been developed (Patent Document 1) to increase the amount of collagen and bring about the prevention and improvement of wrinkles and sagging.

[0003] Japanese Patent Application Laid-Open No. 2013-35776

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

[0005] The present inventors have found that frame-shaped collagen formed by collagen bundles exists in the dermis layer. Surprisingly, it has been discovered that the frame-shaped collagen present in the dermis layer maintains the shape of the skin and contributes to anti-sagging and anti-wrinkling of the skin. Furthermore, through further research, it has been found that Wnt16b and biglycan are involved in the formation of frame-shaped collagen, leading to the present disclosure.

[0006] Therefore, this disclosure relates to the following inventions: [1] A method for evaluating the sagging and wrinkle state of skin by evaluating frame-like collagen formed by collagen bundles in the target skin. [2] The evaluation method according to 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 according to item 1 or 2, wherein the evaluation of the frame-like collagen is performed by image analysis. [4] The method according to item 1 or 2, wherein the evaluation of the frame-like collagen is performed by an evaluation method that includes the steps of measuring the biglycan expression level and / or Wnt16b expression level in the dermis of the target skin, and determining the dermal collagen bundle level from the correspondence between the biglycan expression level and / or Wnt16b expression level and the collagen bundle level. [5] The evaluation method described in item 4, wherein the biglycan expression level is measured by the amount of biglycan protein or gene expression, and the Wnt16b expression level is measured by the amount of Wnt16b protein or gene expression. [6] The evaluation method described in item 4 or 5 for measuring the biglycan expression level and / or the Wnt16b expression level in dermal fibroblasts. [7] The method according to any one of items 4 to 6, wherein the correspondence is a correspondence by 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 method for screening for a frame collagen formation promoter in skin, comprising the steps of: culturing a culture containing dermal fibroblasts in a medium containing a candidate drug; measuring the expression level of biglycan or Wnt16b in the skin culture after culturing in a medium containing a candidate drug; and comparing the measured expression levels with a control to determine the candidate drug as a frame collagen formation promoter.

[10] The screening method according to 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 cosmetic method for promoting the formation of frame collagen by applying a temperature stimulus of 38°C to 45°C.

[14] The method according to item 13, which promotes the formation of frame collagen formed by collagen bundles in the skin by increasing the expression of biglycan.

[15] The method according to item 13, which promotes the formation of frame collagen formed by collagen bundles in the skin by increasing the expression of Wnt16b.

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

[0008] 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(A) shows the function of frame-like collagen in the skin. X-ray CT images taken before and after deformation of skin sections are shown. Figure 2(B) shows a graph of the degree of deformation in each area due to deformation treatment, between frame-like collagen identified in the X-ray CT images and non-frame-like collagen. Figure 2(C) shows the appearance of skin in young and elderly individuals and the results of measurements taken with an ultrasound diagnostic device 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(E) shows the relationship between frame-like collagen and dermal thickness and elasticity. Figure 3 shows the results of immunostaining of biglycan in skin section samples. Figure 4 shows a graph of biglycan expression when fibroblasts were cultured in Wnt16b-supplemented medium. It shows that Wnt16b induces biglycan expression. Figure 5(A) shows images of human skin sections from young and elderly individuals. Figure 5(B) shows that the amount of frame collagen decreases with age. Figure 5(C) shows immunohistochemical images of biglycan in human skin sections from young and elderly individuals. Figure 5(D) shows that biglycan decreases with age. Figure 5(E) shows immunohistochemical images of WNT16b in blood vessels of human skin sections from young and elderly individuals. Wnt16b is indicated by a white arrow. It shows that the amount of Wnt16b protein in blood vessels decreases with age. Figure 5(F) shows immunohistochemical images of WNT16b in sweat glands of human skin sections from young and elderly individuals. Wnt16b is indicated by a white arrow. This shows that the amount of Wnt16b protein in sweat glands decreases with age. Figure 6(A) shows immunohistochemical staining images of organ cultured skin before and after temperature stimulation, and the graph shows that Wnt16 increased with temperature stimulation. Blood vessels are indicated by black arrows. Figure 6(B) shows immunohistochemical staining of human skin sections before and after temperature stimulation, and the graph shows that biglycan increased with temperature stimulation.Figure 6(C) shows images of human skin sections before and after temperature stimulation, with the bar graph indicating an increase in collagen bundles due to temperature stimulation. Figure 6(D) shows the height of the dermis before and after temperature stimulation, with the bar graph indicating an increase in dermal height due to temperature stimulation. Figure 6(E) shows the width of the dermis before and after temperature stimulation, with the bar graph indicating a decrease in skin width due to temperature stimulation. Figure 7(A) shows photographs of human skin before and after temperature stimulation. Sagging is indicated by white arrows. Figure 7(B) shows the percentage of volunteers whose sagging improved before and after temperature stimulation. Figure 7(C) shows the percentage of frame-like collagen before and after temperature stimulation. Embodiments for carrying out the invention.

[0009] One aspect of this disclosure relates to a method for evaluating the state of skin sagging and wrinkles by evaluating frame-like collagen formed by collagen bundles in the target skin. Another aspect of this disclosure relates to a screening method for promoting the formation of frame-like collagen formed by collagen bundles in the skin, using biglycan expression or Wnt16b expression as indicators. Yet another aspect of this disclosure relates to a cosmetic method for promoting the formation of frame-like 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 (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-shaped 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-shaped collagen, such as type I collagen or biglycan. In addition to expression, flame-shaped collagen may also be determined by referring to its distribution and / or morphology.

[0016] The evaluation of frame-like collagen may be performed by an evaluation method that includes, for example, the following steps: measuring the biglycan expression level in the dermis of the target skin, and 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 frame-like collagen can also be used to evaluate 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 relationship. Such a correspondence relationship may be a correlation formula or a threshold-based correspondence relationship. For multiple subjects, the skin sagging / wrinkling state and the dermal collagen bundle level can be measured and a correspondence relationship can be created. Such a correspondence relationship, as well as its threshold and correlation formula, can be created using 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, the biglycan expression level 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 frame-like collagen may be performed by an evaluation method that includes, for example, the following steps: measuring the expression of Wnt16b in the dermis of the target skin, and 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 frame-like collagen can also be used to evaluate the state of skin sagging and wrinkles.

[0023] The Wnt16b expression level can be determined by measuring the amount of mRNA or protein. 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. 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 the present disclosure, the present disclosure relates to a method for screening flame collagen formation promoters using the expression level of biglycan or Wnt16b as an indicator, comprising, for example, the following steps: culturing a culture containing dermal fibroblasts in a medium containing a candidate drug; measuring the expression level of biglycan or Wnt16b in the skin culture after culturing in the medium containing the candidate drug; and determining the candidate drug as a flame collagen formation promoter by comparing the measured expression level with a control. Flame collagen formation promoters 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 on 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 for promoting the formation of frame-like collagen by applying temperature stimulation. By increasing the expression of biglycan and / or Wnt16b through temperature stimulation, it is possible to improve wrinkles or sagging of the skin by promoting the formation of frame-like collagen.

[0032] The inventors have found that applying thermal stimulation to cultured skin increases Wnt16b in blood vessels and sweat glands, increases biglycans, and increases flamed collagen. Although 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 temperature stimulus is preferably 38°C to 45°C. Preferably, it is 38°C to 43°C, 39°C to 42°C, 40°C to 41°C, and more preferably 40°C. For example, the temperature stimulus can be applied within any continuous range of temperatures from 38°C to 39°C, 39°C to 40°C, 40°C to 41°C, 41°C to 42°C, and 42°C to 43°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, 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 citation 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.

[0038] Example 1. Discovery of Frame-like Collagen Preparation of Skin Samples: This study used 30 surplus facial skin specimens from plastic surgery (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). ３Skin specimens were observed. During the study period, the physical condition of the skin specimens was maintained in good condition and confirmed by their recovery to their 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 inflammation into the dermal layer induces winkle 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 forms the frame-like collagen.

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

[0042] The relationship between sagging and frame-shaped collagen in actual facial skin was non-invasively confirmed in the following manner. Evaluation of sagging: Based on the following paper, evaluation was performed using evaluation criteria based on photographs. 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.Judgment of the state of frame-shaped collagen: The state of frame-shaped collagen was determined using the ultrasonic diagnostic device Prosound alpha 5 (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 ultrasonic image shown in Fig. 2(C), the white luminance (indicated by an arrow) in the dermis region was determined as frame-shaped collagen, and the black part was determined as non-frame-shaped collagen, and the frame-shaped collagen density was measured. Statistical analysis was performed by the following analysis method. The differences between statistical analysis groups were evaluated by Student's t-test. However, for the efficacy test in humans, the Wilcoxon rank sum test was used (*: p<0.05 and **: p<0.01). Correlation was evaluated by Pearson's correlation coefficient or Spearman's correlation coefficient. A p-value less than 0.05 was considered significant.

[0043] Subjects with less sagging had abundant frame-shaped collagen, while subjects with severe sagging had little frame-shaped collagen (Fig. 2(C)). The loss of frame-shaped collagen was statistically significantly associated with an increase in sagging. Frame-shaped collagen increased skin thickness and elasticity (Fig. 2(E)).

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

[0045] Fluorescent immunostaining: Paraffin-embedded skin section samples were treated with a biglycan antibody (Abcam), followed by fluorescent immunostaining using a fluorescent-labeled secondary antibody (Cell Signaling), and observed under a fluorescence microscope (Keyence).

[0046] Gene expression analysis revealed that the expression of the biglycan gene coincides with the position of the highly aggregated collagen bundles. It was confirmed that the highly aggregated collagen bundles are wrapped by biglycan (Figure 3).

[0047] Example 4. Induction of biglycan expression from dermal fibroblasts Human skin fibroblasts (Lonza; Basel, Switzerland) were cultured in DMEM containing 10% FBS at 37°C under a 5% CO ２ humid atmosphere. Secretion factors were obtained from R&D Systems (MN, USA). Fibroblasts highly expressing biglycan were obtained by in vitro cloning. In fibroblasts highly expressing biglycan, the formation of highly aggregated collagen bundles was promoted compared to dermal fibroblasts.

[0048] Example 5. Promotion of biglycan expression Human skin fibroblasts (Lonza; Basel, Switzerland) were cultured in DMEM containing 10% FBS at 37°C under a 5% CO ２ humid atmosphere. When the cultured human fibroblasts reached 10 - 20% confluence, they were replaced with a medium supplemented with 10 μg / mL Wnt16 (Wnt16b: R&D systems) and cultured for 48 hours. As a control, the same experiment was conducted in an experimental system that differed only in the absence of Wnt16b. After 48 hours of culture, the cells were harvested, and RNA was recovered using RNeasy (Qiagen), and the expression of biglycan was measured by real-time PCR. Real-time PCR was performed using the following primer set with a Light-cycler (Roche, IN, USA).

[0049] Example 6. Analysis of frame-like collagen due to aging. Human skin images 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 each treated in DMEM containing 10% FBS and 5% CO2. ２ The cultured skin was incubated at 37°C in a humidified atmosphere. The cultured skin prepared in Example 1 was subjected to a thermal stimulus of 40°C for 1 hour. Cultured skin tissue was obtained after the thermal stimulus and immunostaining was performed for Wnt16b and biglycan as in Examples 3 and 6 (Figures 6A, B). X-ray CT was also performed to evaluate collagen bundles (Figure 6C). The height and width of the skin sections were also evaluated (Figures 6D, E). Increases in Wnt16b, biglycan, and flamed collagen were observed with thermal stimulation (Figure 6). Furthermore, the height of the skin increased and the width of the skin decreased with the 40°C thermal stimulus (Figures 6D, E).

[0051] Example 8. Effects of temperature stimulation on human skin Ten female volunteers in their 40s were subjected to temperature stimulation at 40°C for 15 minutes 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 (registered trademark) (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 wrinkle conditions by evaluating 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-shaped collagen is performed by image analysis.

4. The method according to claim 1 or 2, wherein the evaluation of the frame-like collagen is performed by an evaluation method comprising the steps of measuring the biglycan expression level and / or Wnt16b expression level in the dermis of the target skin, and determining the dermal collagen bundle level from the correspondence between the biglycan expression level and / or 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 the 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 method for screening for a catalyst for the formation of framing collagen, which is formed by collagen bundles in the skin, comprising the steps of: culturing a culture containing dermal fibroblasts in a medium containing a candidate drug; measuring the expression level of biglycan or Wnt16b in the skin culture after culturing in a medium containing a candidate drug; and comparing the measured expression level with a control to determine the candidate drug as a catalyst for the formation of framing collagen.

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. 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 according to claim 13, wherein the formation of frame collagen, which is formed by collagen bundles in the skin, is promoted by increasing the expression of biglycans.

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

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