WO2018089047A1

WO2018089047A1 - Three-dimensional aged skin model and method of creating the same

Info

Publication number: WO2018089047A1
Application number: PCT/US2017/031904
Authority: WO
Inventors: Suhyoun CHON (Su); Lucia PICCOTTI; Debbie Y. NGAI; Karien J. RODRIGUEZ
Original assignee: Kimberly-Clark Worldwide, Inc.
Priority date: 2016-11-10
Filing date: 2017-05-10
Publication date: 2018-05-17

Abstract

A three-dimensional aged skin model and methods of creating the same are disclosed. A method of creating a three-dimensional aged skin model can include providing a three-dimensional skin model and treating the three-dimensional skin model with a treatment solution comprising hydrogen peroxide for a treatment period. The hydrogen peroxide can be at various concentrations. The method also includes removing the treatment solution from the three-dimensional skin model after the treatment period to provide a three-dimensional aged skin model.

Description

THREE-DIMENSIONAL AGED SKIN MODEL AND METHOD OF CREATING THE SAME

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent Application Serial No. 62/420191 , filed November 10, 2016, the contents of which are hereby incorporated by reference in their entirety, for all purposes.

BACKGROUND

The present disclosure relates to an aged skin model and methods for creating the same. More specifically, the present disclosure relates to a three-dimensional aged skin model and methods for creating the same.

Compromised skin health has been well recognized in aging skin, especially in the elderly. As skin ages, it changes and the epidermal and dermal layers thin, and in turn, skin becomes more fragile. Dermal strength and elasticity are conferred through the organization of extracellular (ECM) proteins such as elastin, collagens I and III, and collagen chaperone protein Hsp47. In addition, dermal epidermal junction (DEJ) complexes serve to anchor cytoskeletal keratins of the epidermis with connective tissue of the dermis. As individuals age, these ECM proteins are down-regulated and resident proteins are broken down by proteases, which result in a disorganized and weakened dermis. Expression of DEJ anchoring proteins can also decline, thereby, weakening the bond between the epidermis and dermis. These structural changes increase the potential for the epidermis to detach from the dermis, or more severely, both layers detaching from the subcutaneous fat layer. Skin of elderly individuals is therefore more susceptible to injuries such as pressure ulcers and skin tears when the skin comes into contact with friction, shear stress, blunt trauma, or applied pressure.

Primary skin cells or explants from young and old human subjects have been used in in-vitro studies for skin aging research. Reconstructed three-dimensional skin models have also been prepared to study aged skin and products interacting with the skin. Such reconstructed skin models can be useful tools for in-vitro bench testing in active screening and conducting research on a variety of products and formulations, and can provide helpful information prior to any clinical testing. UV irradiation has traditionally been the medium used to create such a reconstructed skin model. Another approach that has been used to create an aged skin equivalent is to use Mitomycin-C (MMC). MMC has been reported to reduce expression of filaggrin, collagen 1 and elastin in skin equivalents. However, there are some challenges and limitations in preparing aged skin models via UV irradiation or MMC. A number of studies have investigated how solar UV irradiation results in premature skin aging, also called photo-aging and described the different characteristics between photo-aging and normal skin aging. The traditional method of UV irradiation has a drawback in that it is difficult to apply in a consistent manner such that the dermal matrix is aged, but does not trigger a detrimental toxic effect on the epidermis. Additionally, variability may be created by the choice or ratio of UVA (long wave ultraviolet rays) and UVB (shortwave ultraviolet rays) in trying to produce an aged skin model. Furthermore, MMC is not a preferred method for creating an aged skin model as the chemical is highly toxic and may potentially be carcinogenic.

Thus, there is a desire to create a three-dimensional aged skin model by a method that is more consistent and more closely replicates aged skin.

SUMMARY

In one aspect of the disclosure, a method of creating a three-dimensional aged skin model is provided. The method can include providing a three-dimensional skin model. The method can also include treating the three-dimensional skin model with a treatment solution comprising hydrogen peroxide for a treatment period. The method can additionally include removing the treatment solution from the three- dimensional skin model after the treatment period to provide the three-dimensional aged skin model.

In another aspect of the disclosure, an additional method of creating a three-dimensional aged skin model is provided. The method can include providing a three-dimensional skin model. The method can also include treating the three-dimensional skin model with a treatment solution comprising hydrogen peroxide at a concentration of about 1.0 mM to about 4.0 mM for a treatment period. The method can additionally include removing the treatment solution from the three-dimensional skin model after the treatment period to provide the three-dimensional aged skin model.

DESCRIPTION OF THE DRAWINGS

FIGS. 1 A-1 D illustrate the cytotoxicity results of various embodiments of three-dimensional skin models exposed to various concentrations of hydrogen peroxide or UV rays as quantified by lactate dehydrogenase assay.

FIGS. 2A-2D illustrate histology images of an embodiment of a three-dimensional skin model that was treated with 2.5 J/cm² of UV irradiation over time compared to a sham control three-dimensional skin model. FIGS. 3A-3C illustrate histology images of an embodiment of a three-dimensional skin model that was treated with 0.5 J/cm² of UV irradiation over time compared to a sham control three-dimensional skin model.

FIGS. 4A-4D illustrate histology images of embodiments of three-dimensional skin models that were treated with various concentrations of hydrogen peroxide over time compared to an untreated control three-dimensional skin model.

FIGS. 5A-5F illustrate gene expression of ECM and DEJ components in a three-dimensional skin model treated with 0.5 J/cm² of UV irradiation over time compared to a sham control three- dimensional skin model.

FIGS. 6A-6D illustrate protein expression of ECM and DEJ components in a three-dimensional skin model treated with 0.5 J/cm² of UV irradiation over time compared to a sham control three- dimensional skin model.

FIGS. 7A-7F illustrate gene expression of ECM and DEJ components in a three-dimensional skin model treated with 2.0 mM hydrogen peroxide over time compared to an untreated control three- dimensional skin model.

FIGS. 8A-8D illustrate protein expression of ECM and DEJ components in a three-dimensional skin model treated with 2.0 mM hydrogen peroxide over time compared to an untreated control three- dimensional skin model.

FIGS. 9A and 9B illustrate the cytotoxicity results of various embodiments of three-dimensional skin models exposed to various concentrations of hydrogen peroxide.

FIGS. 10A-10F illustrate histology images of embodiments of three-dimensional skin models that were treated with various concentrations of hydrogen peroxide over time compared to an untreated control three-dimensional skin model.

FIGS. 1 1A-1 - 1 1 F-2 illustrate gene expression of ECM and DEJ components in embodiments of three-dimensional skin models treated with various ranges of concentrations of hydrogen peroxide over various treatment periods compared to an untreated control three-dimensional skin model.

FIGS. 12A-1 - 12D-2 illustrate protein expression of ECM and DEJ components in embodiments of three-dimensional skin models treated with various ranges of concentrations of hydrogen peroxide over various treatment periods compared to an untreated control three-dimensional skin model. DETAILED DESCRIPTION

The present disclosure generally relates to a three-dimensional aged skin model that includes structural and molecular changes to skin equivalents (i.e., not human skin) that resemble features found in aged skin, especially targeting weakening of dermis.

A commercially available three-dimensional skin model was used as the subject of treatment in producing a three-dimensional aged skin model 10. The three-dimensional skin model that was used in the experiments discussed herein was a skin equivalent produced by MatTek Corporation (Ashland, Massachusetts), model number EFT400-K2F2 (tissue cultured with adult donor matched keratinocytes and dermal fibroblasts). However, it is believed that the techniques and procedures discussed herein would be as effective at producing a physiologically relevant three-dimensional aged skin model 10 with other available three-dimensional skin equivalents.

In the studies described herein, the use of hydrogen peroxide in a tissue culture media (referred to as a treatment solution), at specified concentrations and lengths of treatment (referred to as treatment periods), was found to be effective at producing a physiologically relevant three-dimensional aged skin model 10 verified by the down-regulation in expression of specific ECM and DEJ components that are present in aged skin. As will be described further herein, tissue cytotoxicity, tissue histology, and expression of ECM and DEJ components were analyzed following hydrogen peroxide treatment and compared to measures from non-treated control and a UV irradiated three-dimensional skin model 10 to determine effectiveness and accuracy of creating a three-dimensional aged skin model 10 with treatment of hydrogen peroxide.

As a primary measure, cytotoxicity was evaluated after acute and chronic exposure of hydrogen peroxide and solar UV irradiation on a three-dimensional skin equivalent. Cytotoxicity was evaluated by lactate dehydrogenase (LDH) assay. Damaged cells release LDH, an enzyme, into the cell culture media. LDH is quantified by a coupled enzymatic reaction. LDH converts lactate to pyruvate via reduction of oxidized nicotinamide adenine dinucleotide (NAD+) to reduced nicotinamide adenine dinucleotide (NADH). In parallel, an enzyme diaphorase uses NADH to reduce (convert) tetrazolium salt (INT) to formazan. Therefore, formazan production and its quantification at absorbance 490nm is a measurement of LDH in the cell culture media and serves as an indicator of cytotoxicity. For this experimentation, media samples were collected at multiple time points (day 1 -2, 1-3, 3-5, or 5-7) to evaluate acute and chronic effects.

FIGS. 1A-1 D illustrate the results of a cytotoxicity evaluation, depicting various three- dimensional aged skin models 10 on the 'x' axis and absorbance of formazan (at wavelength of 490nm) as an indicator of cytotoxicity on the 'y' axis. Data in FIGS. 1A-1 D is shown as mean ± SEM. * p<0.05, ** p<0.01 , *** p<0.001 , and **** pO.0001 compared to respective controls; One-Way ANOVA; Posthoc: Dunnett's Test. The sham control sample 12A was the same three-dimensional skin equivalent that was subjected to the same conditions as the skin equivalents subjected to UV irradiation to provide an aged skin model 10, except the sham control sample 12A was not subjected to any UV irradiation. The control sample 12B for the 2 and 10 mM H2O2 treatment samples 10 was a skin equivalent treated only with a water vehicle (labeled as UNT) that was exposed to the same amount of water as the 2 and 10 mM treatment samples 10.

FIG. 1A depicts cytotoxicity in skin models 10 following 24 hour acute exposure of 2 and 10 mM H2O2 treatment and 2.5 J/cm² and 5 J/cm² UV treatment. The hydrogen peroxide was dissolved in water to provide the treatment solution. Although the treatment solutions described herein included hydrogen peroxide and water, it is contemplated that the treatment solution could include alternative vehicles other than water and/or additional components as well. Single exposure of 2.5 J/cm² and 5 J/cm² UV on tissues resulted in marked increase in LDH values compared to sham control 12A within the first 24 hours (FIG. 1A). As also depicted in FIG. 1A, a one day exposure of 2 mM hydrogen peroxide and 10 mM hydrogen peroxide (H2O2) also increased LDH values significantly compared to untreated control sample 12B (* p<0.05, ** p<0.01 vs. untreated control), but the magnitude was modest compared to UV exposure.

FIG. 1 B depicts results of chronic exposure of 2 mM and 10 mM H2O2 treatment and 2.5 J/cm² and 5 J/cm² UV (day 1 , 3, and 6) treatment on skin equivalents in a 7 day culture. In contrast to FIG. 1 A, FIG. 1 B depicts that the LDH data from the final 48 hours (culture media collected on day 7) showed the opposite trend of FIG. 1A in that the three-dimensional aged skin models 10 treated with hydrogen peroxide demonstrated increased values of LDH as compared to the three-dimensional aged skin models 10 treated with UV, in regard to their respective control skin models 12B, 12A. However, the relatively lower LDH values from UV irradiation at this time frame doesn't mean UV was less toxic compared to the hydrogen peroxide; rather, these lower LDH values for the UV irradiated skin models 10 in FIG. 1 B is believed to be due to the fact that there were not enough viable cells left in the UV irradiated skin models 10 at that time frame to produce LDH at day 5 due to the substantial amount of cells that were dead already following 24 hours of UV irradiation.

Histological analysis for the three-dimensional skin samples 10 treated with 2.5 J/cm² UV was conducted to examine any morphological changes that occurred due to this insult, and is depicted in FIGS. 2A-2D. Unless stated otherwise, histology images of skin models 10 and sham and untreated models 12A, 12B, such as those shown in FIGS. 2A-2D, were captured at 20x magnification and are representative images presented from triplicate samples. The horizontal scale bar 14 shown in FIGS. 2A-2D represents 200μιη. As depicted in FIG. 2B, 2.5 J/cm² UV exposure even at day 2 caused detrimental toxicity to the epidermal layer 16 (or epidermis), which can be noticed by comparing to the sham model 12A at day 2 depicted in FIG. 2A. Furthermore, the day seven skin model 10 that was exposed to 2.5 J/cm² depicted in FIG. 2D shows similar results with the day 2 sample in FIG. 2A, with no viable epidermal layer 16 being left and the epidermis 16 and dermis 18 (or dermal layer) were detached from each other post-UV exposure. This finding is consistent with LDH data in FIG. 1 A.

Based on histological evaluation and cytotoxicity measurements, 2.5 J/cm² and 5.0 J/cm² UV treatments were too aggressive on the three-dimensional skin equivalents to provide a viable three- dimensional aged skin model 10, as the LDH values were more than double their respective controls and the cytotoxicity even at the level of 2.5 J/cm² was noticeable in the histological analysis. Additionally, based on the LDH values in FIGS. 1A and 1 B, it was determined that the treatment using 10 mM hydrogen peroxide was also too aggressive of a treatment on the three-dimensional skin equivalent for chronic exposure to provide a viable three-dimensional aged skin model 10.

A reduced dose of UV exposure (0.5 J/cm²) and H2O2 treatment at 2 mM were further explored. FIG. 1 C depicts the LDH values for samples 10 treated with 2 mM H2O2 treatment and 0.5 J/cm² UV treatment after day 1-3. FIG. 1 D depicts the 7 day chronic exposure of 2 mM H2O2 treatment and 0.5 J/cm² UV treatment in skin model samples 10, with the media from days 5-7 being tested for the assay. As depicted in FIGS. 1 C and 1 D, at any time point, there was no significant increase in LDH by either 0.5 J/cm² UV or 2 mM H2O2 treatment on skin samples 10 in either acute exposure (FIG. 1 C) or in chronic exposure (FIG. 1 D).

Histological analysis was also conducted for the treatment of the skin equivalent with 0.5 J/cm² UV and the 2 mM H2O2 treatment and is depicted in FIGS. 3A-4D. Similar to the scale in FIGS. 2A-2D, the horizontal scale bar shown in FIGS. 3A-4D represents 200μιη.

The histological analysis for 0.5 J/cm² UV treatment on the skin model 10 is illustrated in FIGS. 3A-3C. FIGS. 3A and 3B provide a comparison by showing the day 7 results of the untreated (UNT) skin equivalent control sample 12B (FIG. 3A) and the sham control sample 12A (FIG. 3B). As shown in FIGS. 3A-3C, unexpected thinning in the epidermal layer 16 was observed in the sham control sample 12A compared to the untreated control sample 12B. However, multiple doses of the 0.5 J/cm² UV treatment did not cause any detrimental effect on tissue structure of the skin model 10 illustrated in FIG. 3C as was noticed in the 2.5 J/cm² UV treatment of the skin model 10 discussed above and depicted in FIGS. 2B and 2D.

FIGS. 4A-4D display the histological analysis for 2 mM H2O2 treatment on skin model samples 10. As illustrated in FIGS. 4B and 4D, the structure of the dermal compartment 18 of the samples 10 treated with 2 mM H202 were more disorganized and less dense when compared to respective controls 12B at day 4 (FIG. 4A) and at day 7 (FIG. 4C). In fact, a clear difference was observed due to 2 mM H2O2 treatment on skin models 10 at day 4 collection compared to the untreated skin control 12B at day 4 (comparing FIGS. 4A and 4B). No drastic change was observed in the epidermal layers 16 treated with 2 mM H2O2 at day 4 compared to the untreated skin control 12B at day 4 (comparing FIGS. 4A and 4B), except the thickness of epidermis 16 in the skin model 10 appeared to be increased due to 2 mM H2O2 treatment. However, this was not consistently observed in all experiments (data not shown). As illustrated in FIG. 4D, several intercellular vacuoles 20 were found in the epidermis 16 of the skin model 10 treated with 2 mM H2O2 at day 7 (one vacuole 20 labeled in FIG. 4D), which were not observed in the untreated control sample 12B at day 7 (FIG. 4C).

Since there was no detrimental effect on tissue morphology and cytotoxicity with skin models 10 treated with 0.5 J/cm2 UV or 2 mM HteCtet, gene and protein expression analysis of various biomarkers for skin aging was conducted following these treatments in the skin models 10. Based on the gene expression and protein expression analysis, it was unexpectedly found that the 2 mM H2O2 treatment provided a more physiologically relevant three-dimensional aged skin model 10 than the use of UV irradiation at 0.5 J/cm², as will be further discussed below.

Gene Expression Study

For each respective gene marker (discussed further below), three rounds of independent experiments were performed and tissues were harvested for evaluation of gene expression in skin models 10 treated with UV or hydrogen peroxide. At harvest, each tissue was cut into halves and one- half of the tissue was submerged in 200 uL of RIPA buffer for a subsequent protein assay. For RNA isolation, triplicates of one-half tissues were pooled into one tube and placed in 500 ul RLT buffer. Tissues were vigorously vortexed in buffer and homogenized using TissueRuptor (Qiagen Cat# 9001271 ). Samples were centrifuged at maximum speed for 10 minutes and supernatants were collected. Lysates were transferred to the Qiacube instrument and RNA was isolated using Qiagen RNeasy protocol with DNase I digestion (Cat#79254). Reverse transcription was performed using a High Capacity cDN A kit (Life Technologies, Cat#4368814). For each sample, a total of 5-10 ng of cDNA was used for qPCR analysis. TaqMan gene expression assays were purchased from Life Technologies. Data were normalized by the reference gene GAPDH. Relative gene expression was calculated by using the comparative CT method. Selected biomarkers are described in Table 1 .

A total of nine markers for ECM/DEJ components were analyzed for the gene expression analysis, as shown in Table 1 below. The markers tested and the methodology used was consistent between the UV samples and the hydrogen peroxide samples. Gene expression was evaluated by qPCR based analysis, as described above.

Table 1 : Selected Markers for gene expression analysis for skin models treated by UV and H2O2

Results from six representative markers for the 0.5 J/cm² UV treated skin samples 10 are presented in FIGS. 5A-5F. Multiple exposures of 0.5 J/cm² solar UV increased MMP1 gene expression by 2-3 fold at day 7 (FIG. 5A). COL1A1, elastin {ELN) and TNC were down-regulated by UV exposure (FIGS. 5C, 5D and 5F). However, down-regulation of TIMP1 and COL4A1 was not consistently observed in the skin models 10 treated with 0.5 J/cm² UV (Figure 5B and 5E).

Protein Expression Study

A total of four markers for ECM/DEJ components were analyzed for protein expression, as shown in Table 2 below. For each protein marker tested, three rounds of independent experiments were conducted and one half of the tissue was harvested for evaluation of protein expression in skin models 10 treated with UV or hydrogen peroxide. After harvest, protein expression was evaluated according to the enzyme-linked immunosorbent multiplex bead based assays as noted below. The markers tested and the methodology used was consistent between the UV samples and the hydrogen peroxide samples in the protein expression analysis.

Table 2: Selected Markers for protein expression analysis for skin models treated by UV and H2O2 Multiplex Bead Based Assay - MMP1 and TIMP1 Measurements

Expression of MMP1 and TIMP1 was measured using a custom magnetic bead-based assay kit (R&D Systems, Catalog #LXSAHM-02). Supernatant samples were diluted 1 :25 (first round samples) or 1 :35 using Calibrator Diluent provided by the kit prior to being analyzed in the assay. Supernatant samples that exceeded the standard curve due to tissue treatment conditions were re-evaluated at a dilution factor of 1 :100 with Calibrator Diluent. Fluorescent intensity was measured using a MagPix System (Luminex Corp., Austin TX).

Multiplex Bead Based Assay - MMP1 and TIMP1 Sample Optimization

Supernatant samples collected from the first round of experiments with MatTek tissue were diluted at 1 :5, 1 :25, 1 :100, 1 :250, 1 :500, 1 :1000, and 1 :2000 using Calibrator Diluent provided by the kit prior to being analyzed in the assay. Supernatant samples were determined to be within the range of both analytes at 1 :25 and 1 :100 dilutions.

Multiplex Bead Based Assay - TNC and COL4A1 Measurements

Expression of TNC and COL4A1 was measured using a custom magnetic bead-based assay kit (R&D Systems, Catalog #LXSAHM-02). Tissue lysate samples were diluted 1 :100 using Calibrator Diluent provided by kit prior to being analyzed in the assay. All values were normalized to total protein, measured using a BCA protein assay (Thermo Fisher Scientific, Catalog #23227). Fluorescent intensity was measured using a MagPix System (Luminex Corp.).

Multiplex Bead Based Assay - TNC and COL4A1 Sample Optimization

Tissue lysates collected from the second and third round of experiments were diluted at 1 :5, 1 :100, 1 :500, 1 :1000, and 1 :2000 using Calibrator Diluent provided by the kit prior to being analyzed in the assay. Tissue lysate samples were determined to be within the range of both analytes at 1 :100 and 1 :500 dilutions.

The results for the protein expression analysis for the 0.5 J/cm² solar UV samples are illustrated in FIGS. 6A-6D. The MMP1 and TIMPI protein levels were measured in the supernatant samples while TNC and COL4A1 protein levels were measured in the tissue lysates. MMP1 and TIMP1 levels in supernatant samples were measured at two time points from the second set of skin models 10 irradiated with 0.5 J/cm² solar UV on days 1 , 3, and 6, respectively. As illustrated in FIG. 6A, the MMP1 levels significantly increased with 0.5 J/cm² solar UV treatment compared to the sham control sample 12A at day 7. There was no decrease in TIMP1 levels, and instead, the TIMP1 levels significantly increased on day 7 (FIG. 6B). The TNC and COL4A1 levels in tissue lysates were processed on day 7. TNC and COL4A1 levels did not change in the skin models 10 after multiple 0.5 J/cm² solar UV exposures (FIGS. 6C and 6D). Tissue lysate samples were pooled into one sample per condition. All data shown in FIGS. 6A and 6B is mean ± SEM. * p<0.05 and ** pO.01 ; one-Way ANOVA; Post-hoc: Dunnett's Test.

As mentioned above, gene expression analysis and protein expression analysis was also conducted for the skin models 10 treated with 2 mM H2O2 using the same markers and methods as noted above for the respective gene expression analysis and protein expression analysis for the skin models treated with 0.5 J/cm² UV discussed above.

Results from six representative markers for gene expression analysis of the 2 mM H2O2 treatment samples 10 are presented in FIGS. 7A-7F. The 2 mM H2O2 treatment increased MMP1 gene expression by 2-3 fold in both day 4 and day 7 (FIG. 7A). TIMP1 expression showed the trend to be decreased by treatment, though the magnitude of change was minor compared to other markers (FIG. 7B). All other markers for ECM/DEJ components in the 2 mM H2O2 treatment samples 10 including COL1A1, ELN, COL4A1 and TNC were down-regulated by 2 mM H2O2 at both time points (FIGS. 7C- 7F). The data shown in FIGS. 7A-7F was normalized by GAPDH expression and the value from day 7 untreated control was set as 1 . Triplicate samples were pooled for this gene expression analysis. The results shown are representative of three experiments.

The results of the protein expression analysis for the 2 mM H2O2 treatment samples is illustrated in FIGS. 8A-8D. As illustrated in FIG. 8A, MMP1 levels increased significantly with 2 mM H2O2 treatment compared to untreated tissue in supernatant collected between days 5-7. TIMP1 levels decreased significantly with 2 mM H2O2 treatment after day 3 and a significant reduction was also observed on day 7 (FIG. 8B). TNC levels did not change in the tissue samples 10 after 2 mM H2O2 treatment (FIG. 8C) although a slight decrease in COL4A1 was observed (FIG. 8D). Overall, the change in protein expression for the skin model 10 treated with 2 mM H2O2 was consistent with the gene expression for the skin model 10 treated with 2 mM H2O2 described above and illustrated in FIGS. 7A-7F. Data shown in FIGS. 8A and 8B is shown as mean ± SEM . ** p<0.01 and *** pO.001 versus untreated tissue control at each time point; one-Way ANOVA; Post-hoc: Dunnett's Test.

The 2mM H2O2 treatment on the skin models 10 resulted in a more significant magnitude of change in protein and gene expression of various ECM and DEJ markers compared to the 0.5 J/cm² UV treatment on the skin models 10. In addition, the effect of the 2 mM H2O2 treatment was consistent across tissues prepared in different batch or from different donors. In contrast, UV treatment was not able to deliver as consistent of an effect across different sets of tissues.

Overall from histological analysis and from gene and protein expression results, it was discovered that 2 mM H2O2 treatment for 4-7 days significantly reduced expression of multiple ECM and DEJ components in a skin equivalent model 10, successfully mimicking key features of aged skin. As described earlier, UV dosing proved to be difficult in effectively and consistently inducing a change in ECM/DEJ components such that the dosage was not toxic on the skin equivalent model 10. On the other hand, H2O2 treatment effectively and consistently down-regulated dermal matrix components without severe morphological changes as seen with a high dose of UV exposure, suggesting better efficacy of H2O2 treatment to provide a skin equivalent model 10 mimicking dermal weakening in aged skin. The H2O2 treatment substantially reduced gene expression of COL1A1, COL4A1, ELN, TIMP1 and TNC, accompanied by a drastic increase in MMP1 gene expression, characteristic of aged skin. Similar changes were observed in protein expression of these markers, confirming results from gene expression analysis. While skin equivalent models 10 exposed multiple times to 0.5 J/cm² of solar UV also exhibited changes in some ECM and DEJ markers, the UV treatment was less effective than 2 mM H2O2 treatment and the results were not as consistent across multiple experiments. Therefore, it was discovered that 2 mM H2O2 treatment was more effective than solar UV irradiation to alter the expression of dermal ECM and DEJ components.

Additional testing was conducted for treatment solutions including a range of concentrations of hydrogen peroxide treatments on a skin model 10, and over various treatment periods. Specifically, the concentrations of hydrogen peroxide that were used to perform additional testing were 0.25 mM, 0.5 mM, 1 mM, 2 mM, 4 mM, and 8 mM .

FIGS. 9A and 9B illustrate the cytotoxicity results of various embodiments of three-dimensional skin models 10 exposed to treatment solutions including various concentrations of hydrogen peroxide. One skin model 10 was used for treating with various concentrations of hydrogen peroxide for FIG. 9A and a separate skin model 10 was used for treating with the same concentrations of hydrogen peroxide for FIG. 9B, providing two separate experiments. Each experiment also utilized an untreated control skin model 12B, which was only exposed to water treatment solution (as previously discussed), that was analyzed in each of FIG. 9A and 9B. Supernatant samples were taken at each time point of days 3, 5, and 7 from each tissue to generate the cytotoxicity data for FIGS. 9A and 9B. Tissue culture supernatant in each condition were collected every two days (D3, D5, and D7) and LDH release of each time point were measured using the same cytotoxicity analysis as described above for the cytotoxicity data generated for FIGS. 1A-1 D, except background absorbance correction at 680nm. Cytotoxicity data shown in FIGS. 9A and 9B is mean ± SEM . (n=7 for day 3, and n=4 for day 5 and 7) * p<0.05, ** p<0.01 , *** pO.001 , and **** pO.0001 versus untreated control. One-Way ANOVA. Post-hoc: Dunnett's Test.

As depicted in FIGS. 9A and 9B, hydrogen peroxide at concentration ranges of 0.25 mM, 0.5 mM. and 1 mM exhibited low levels of LDH release comparable to the untreated control skin model 12B, upon testing at day 3, day 5, and day 7. The skin models 10 treated with 2 mM hydrogen peroxide exhibited higher levels of cytotoxicity compared to untreated control skin model 12B, with cytotoxicity levels generally increasing as the treatment period extended from day 3, to day 5, and to day 7. The cytotoxicity levels of the skin models 10 treated with the 2 mM hydrogen peroxide treatment solution and depicted in FIGS. 9A and 9B generally exhibited higher levels of cytotoxicity than the skin models 10 treated with 2 mM hydrogen peroxide treatment solution discussed above and illustrated in FIGS. 1 A - 1 D. This discrepancy is believed to be due to the variability from skin equivalents from batch to batch or donor to donor difference. The skin models 10 treated with 4 mM and 8 mM hydrogen peroxide provided potentially feasible options if treated in shorter treatment periods. For example, FIGS. 9A and 9B demonstrate that 4 mM and 8 mM hydrogen peroxide treatment provided for a treatment period of three days exhibited from about 0.5 to about 0.7 fold change. However, longer treatment periods of five and seven days for 4 mM and 8 mM hydrogen peroxide treatment solutions exhibited much higher levels of cytotoxicity. Thus, if higher concentration ranges of hydrogen peroxide treatment solutions are desired to be employed in generating a three-dimensional aged skin model 10, it is beneficial to use shorter treatment periods.

FIGS. 10A-10F illustrate histology images of embodiments of three-dimensional skin models 10 that were treated with various concentrations of hydrogen peroxide treatment solutions over a treatment period of seven days compared to an untreated control three-dimensional skin model 12B. The images in FIGS. 10A-10F provide representative images out of two experiments. As illustrated in FIGS. 10B- 10E, treatment solutions including 0.25 mM hydrogen peroxide to 2.0 mM hydrogen peroxide did not exhibit significant tissue damage in the skin models 10 after a treatment period of seven days. However, FIG. 10F depicts that a skin model 10 treated with 8.0 mM hydrogen peroxide treatment solution for a treatment period of seven days exhibited higher levels of tissue damage, with the epidermal layer 16 separating from the dermal layer 18 and fewer viable cells in epidermis 16 (viable cells are depicted as black dots) further demonstrating higher levels of cytotoxicity in the skin models 10 as seen in FIGS. 9A and 9B that were treated with a higher concentration of hydrogen peroxide treatment solution for a seven day treatment period. No representative skin model 10 was available for the treatment solution including hydrogen peroxide at a concentration of 4 mM .

FIGS. 1 1A-1 - 11 F-2 illustrate further gene expression analysis of ECM and DEJ components in embodiments of three-dimensional skin models 10 treated with treatment solutions including various ranges of concentrations of hydrogen peroxide over treatment periods of three days and six days (respectively D4 and D7) compared to an untreated control three-dimensional skin model 12B. Two separate experiments were conducted, each on a separate skin model 10, and gene expression was measured. As an example, FIG. 1 1 A-1 demonstrates COL4A1 gene expression for the first skin model 10 and FIG. 1 1 A-2 demonstrates COL4A1 gene expression for the second skin model 10. For each of the two experiments, the relative gene expression was evaluated by qPCR analysis in the same fashion as the Gene Expression Study discussed above, with the exception that the three samples for each experiment were tested independently of one another instead of being pooled together. The error bars in each figure demonstrate the range of fold change exhibited by the three samples for each experiment conducted for the six different gene expression markers. Data was normalized by GAPDH expression and the value from untreated control of each day was set as 1 . Data presented as mean ± SEM. *p<0.05, **p<0.01 , ***p<0.001 , ****p<0.0001 versus untreated control. One-way ANOVA, Dunnett's post hoc analysis.

As depicted in FIGS. 1 1A-1 - 11 F-2, treatment solutions having a hydrogen peroxide concentration of less than 2.0 mM did not appear to consistently down-regulate the gene markers of COL1A1, ELN, TIMP1, COL4A1, and TNC when the treatment period was limited to three days (i.e., the results at day 4), however, treatment solutions having a hydrogen peroxide concentration of 2.0 mM and above did consistently down-regulate such gene markers in such a treatment period. Thus, based on a treatment period of three days, it is preferable to have a treatment solution having hydrogen peroxide at a concentration of about 1 .0 mM to about 8.0 mM, more preferably from about 2.0 mM to about 8.0 mM, more preferably from about 2.0 mM to about 4.0 mM . Additionally, when the treatment period was extended to six days (i.e., the results at day 7), treatment solutions having a hydrogen peroxide concentration of 0.25 mM and greater had at least some down-regulation of at least some of the gene markers of COL 1 A 1, ELN, TIMP1, COL4A1, and TNC, but treatment solutions having hydrogen peroxide at a concentration of about 1 .0 mM to about 4.0 mM were more consistent, and treatment solutions having hydrogen peroxide at a concentration of about 2.0 mM to about 4.0 mM being even more preferable. As illustrated in FIGS. 1 1 C-1 and 1 1 C-2, MMP1 was up-regulated for each treatment solution tested having concentration ranges of 0.25 mM H2O2 to 8.0 mM H2O2.

FIGS. 12A-1 - 12D-2 illustrate additional protein expression analysis of ECM and DEJ components in embodiments of three-dimensional skin models 10 treated with various treatment solutions having ranges of concentrations of hydrogen peroxide over treatment periods of three days and six days (respectively D4 and D7) compared to an untreated control three-dimensional skin model 12B. As with the gene expression analysis of FIGS. 1 1A-1 - 11 F-2 discussed above, two separate experiments were conducted, each on separate skin models 10. Three skin samples were treated per concentration and harvested in each experiment to measure COL4A1 and TNC, with the error bars depicting the variability in the protein expression in each experiment. Otherwise, the protein expression analysis was optimized and sampled in the same fashion as noted above with respect to the protein expression analysis depicted in FIGS. 8A-8D. In FIGS. 12A-1 - 12D-2, data is presented as mean ± SEM. (n=7 for day 3, and n=4 for day 5 and 7) *p<0.05, **p<0.01 , ***p<0.001 , ****p<0.0001 versus untreated control. One-way ANOVA, Dunnett's post hoc analysis.

As depicted in FIGS. 12A-1 - 12D-2, treatment solutions having a hydrogen peroxide concentration of less than 2.0 mM did not appear to consistently down-regulate the gene markers of TIMP-1, COL4A1, and TNC when the treatment period was limited to three days (i.e., the results at day 4), however, treatment solutions having a hydrogen peroxide concentration of 2.0 mM and above did down-regulate such protein markers in such a treatment period. From this testing, it was discovered that based on a treatment period of three days, it is preferable to have a treatment solution having hydrogen peroxide at a concentration of about 1 .0 mM to about 8.0 mM, more preferably from about 2.0 mM to about 8.0 mM. When the treatment period was extended to six days (i.e., the results at day 7), treatment solutions having a hydrogen peroxide concentration of 0.25 mM and greater down-regulated the protein markers of TIMP1 and COL4A1 , with treatment solutions having hydrogen peroxide at a concentration of about 1.0 mM to about 4.0 mM being more consistent in down-regulating such protein markers, and treatment solutions having hydrogen peroxide at a concentration of about 2.0 mM to about 4.0 mM being even more preferable. As illustrated in FIGS. 12A-1 and 12A-2, the gene o MMP1 was consistently up-regulated for treatment solutions having concentration ranges of 1.0 mM H2O2 to 8.0 mM H2O2 for treatment periods of three days and six days. Significant up-regulation of MMP1 was exhibited by treatment solutions having concentration ranges of 2. O mM H2O2 to 4.0 mM H2O2 for treatment periods of both three days and six days.

From the additional testing completed on treatment solutions including various concentration ranges of hydrogen peroxide and for various treatment periods, preferable ranges for the concentration of the hydrogen peroxide with a respective treatment period were discovered to provide consistent regulation of gene and protein markers indicative of aged skin without providing a cytotoxic effect to the skin model 10. As noted above, treatment solutions including higher concentration ranges of hydrogen peroxide, such as 8.0 mM, are preferably limited in the treatment period in which they are applied (such as a treatment period of three days or less) to avoid cytotoxic effects on the skin model 10. Additionally, while treatment solutions including concentration ranges of hydrogen peroxide less than 1 .0 mM did provide some down-regulation of gene and protein markers, especially for treatment periods of four to six days, such lower concentration ranges of hydrogen peroxide were not as consistent at regulating the gene and protein markers as treatment solutions including the higher concentration ranges of hydrogen peroxide. In one preferred embodiment for a method for creating a three-dimensional aged skin model, the treatment solution can include hydrogen peroxide at a concentration of about 1 .0 mM to about 4.0 mM when the treatment period is between three to six days. In a further preferred embodiment, the treatment solution can include hydrogen peroxide at a concentration of about 2.0 mM to about 4.0 mM when the treatment period is between three to six days. This additional testing further confirmed the effectiveness of utilizing a treatment solution including hydrogen peroxide over a treatment period on a skin model to provide a three-dimensional aged skin model 10 that mimics aged skin.

PROCEDURES

Tissue Culture

Full thickness skin equivalents (EFT 400-K2F2) were purchased from MatTek Corporation. Tissues were constructed from donor matched keratinocytes and dermal fibroblasts (31Y, Caucasian female and 43Y Caucasian female). In total, three independent experiments were conducted to identify culture conditions which compromise expression of ECM/DEJ components. MatTek tissues were challenged with various doses of solar UV (UVA/UVB) irradiation on the top of the tissue while H2O2 treatment were added via culture medium (n=3 per condition). Culture medium with or without 2 mM H2O2 was replenished every other day. Dose and time of exposure in each experiment are described in Table 3, below. Supernatant samples were collected at two time points (days 1 -3 and days 5-7) and tissue samples were lysed on days 2, 4, and 7.

Table 3: Experimental conditions to develop three-dimensional aged skin model in-vitro

Histology

General tissue morphology was examined by H&E staining. After designated treatments, tissues were fixed in 10% neutral buffered formalin solution for one day, and then transferred to 70% ethanol solution. Samples were shipped to American Histology Lab (Gaithersburg, MA) for staining. Histology images were captured by EVOS AL Auto Cell Imaging Systems (Life Technologies) at 20X magnification.

While the invention has been described in detail with respect to the specific embodiments thereof, it will be appreciated that those skilled in the art, upon attaining an understanding of the foregoing, may readily conceive of alterations to, variations of, and equivalents to these embodiments. Accordingly, the scope of the present disclosure should be assessed as that of the appended claims and any equivalents thereto.

Embodiments

Embodiment 1 : A method of creating a three-dimensional aged skin model, the method comprising: providing a three-dimensional skin model; treating the three-dimensional skin model with a treatment solution comprising hydrogen peroxide for a treatment period; and removing the treatment solution from the three-dimensional skin model after the treatment period to provide the three-dimensional aged skin model.

Embodiment 2: The method of embodiment 1 , wherein the treatment solution includes hydrogen peroxide at a concentration of about 1 .OmM to about 4.0mM.

Embodiment 3: The method of embodiment 1 or 2, wherein the treatment period is between three to six days.

Embodiment s The method of embodiment 1 , wherein the treatment solution includes hydrogen peroxide at a concentration of about 0.25mM to about 8.0mM and the treatment period is about three days or less.

Embodiment 5: The method of embodiment 1 , wherein the treatment solution includes hydrogen peroxide at a concentration of about 0.50mM to about 8.0mM and the treatment period is about three days or less.

Embodiment 6: The method of embodiment 1 , wherein the treatment solution includes hydrogen peroxide at a concentration of about 1 .OmM to about 8.0mM and the treatment period is about three days or less.

Embodiment 7: The method of embodiment 1 , wherein the treatment solution includes hydrogen peroxide at a concentration of about 2.0mM to about 4.0mM and the treatment period is between three days to about six days.

Embodiment 8: The method of embodiment 1 , wherein the treatment solution includes hydrogen peroxide at a concentration of about 0.25mM to about 4.0mM and the treatment period is about six days.

Embodiment 9: The method of embodiment 1 , wherein the treatment solution includes hydrogen peroxide at a concentration of about 0.50mM to about 4.0mM and the treatment period is about six days.

Embodiment 10: The method of claim 1 , wherein the treatment solution includes hydrogen peroxide at a concentration of about 1 .OmM to about 4.0mM and the treatment period is about six days.

Embodiment 11 : The method of any one of the preceding embodiments, wherein the three-dimensional skin model is a three-dimensional skin equivalent. Embodiment 12: The method of any one of the preceding embodiments, wherein treating the three- dimensional skin model comprises exposing the three-dimensional skin model directly to the treatment solution substantially constantly over the treatment period.

Embodiment 13: A three-dimensional aged skin model, wherein the three-dimensional aged skin model is prepared according to the method of any one of the preceding embodiments.

Embodiment 14: A method of creating a three-dimensional aged skin model, the method comprising: providing a three-dimensional skin model; treating the three-dimensional skin model with a treatment solution comprising hydrogen peroxide at a concentration of about 1 .0 mM to about 4.0 mM for a treatment period; and removing the treatment solution from the three-dimensional skin model after the treatment period to provide the three-dimensional aged skin model.

Embodiment 15: The method of embodiment 14, wherein the treatment period is between three to six days.

Embodiment 16: The method of embodiment 14 or 15, wherein the treatment solution includes hydrogen peroxide at a concentration of about 2.0 mM to about 4.0 mM.

Embodiment 17: A three-dimensional aged skin model, wherein the three-dimensional aged skin model is prepared according to the method of any one of embodiments 14-16.

Claims

CLAIMS What is claimed is:

1. A method of creating a three-dimensional aged skin model, the method comprising:

providing a three-dimensional skin model;

treating the three-dimensional skin model with a treatment solution comprising hydrogen peroxide for a treatment period; and

removing the treatment solution from the three-dimensional skin model after the treatment period to provide the three-dimensional aged skin model.

2. The method of claim 1 , wherein the treatment solution includes hydrogen peroxide at a concentration of about 1 .OmM to about 4.0m M.

3. The method of claim 2, wherein the treatment period is between three to six days.

4. The method of claim 1 , wherein the treatment solution includes hydrogen peroxide at a concentration of about 0.25mM to about 8.0mM and the treatment period is about three days or less.

5. The method of claim 1 , wherein the treatment solution includes hydrogen peroxide at a concentration of about 0.50mM to about 8.0mM and the treatment period is about three days or less.

6. The method of claim 1 , wherein the treatment solution includes hydrogen peroxide at a concentration of about 1 .OmM to about 8.0mM and the treatment period is about three days or less.

7. The method of claim 1 , wherein the treatment solution includes hydrogen peroxide at a concentration of about 2.0mM to about 4.0mM and the treatment period is between three days to about six days.

8. The method of claim 1 , wherein the treatment solution includes hydrogen peroxide at a concentration of about 0.25mM to about 4.0mM and the treatment period is about six days.

9. The method of claim 1 , wherein the treatment solution includes hydrogen peroxide at a concentration of about 0.50mM to about 4.0mM and the treatment period is about six days.

10. The method of claim 1 , wherein the treatment solution includes hydrogen peroxide at a concentration of about 1 .OmM to about 4.0mM and the treatment period is about six days.

11 . The method of claim 1 , wherein the three-dimensional skin model is a three-dimensional skin

equivalent.

12. The method of claim 1 , wherein treating the three-dimensional skin model comprises exposing the three-dimensional skin model directly to the treatment solution substantially constantly over the treatment period.

13. A three-dimensional aged skin model, wherein the three-dimensional aged skin model is prepared according to the method of claim 1 .

14. A method of creating a three-dimensional aged skin model, the method comprising:

providing a three-dimensional skin model;

treating the three-dimensional skin model with a treatment solution comprising hydrogen peroxide at a concentration of about 1 .0 mM to about 4.0 mM for a treatment period; and

15. The method of claim 14, wherein the treatment period is between three to six days.

16. The method of claim 15, wherein the treatment solution includes hydrogen peroxide at a

concentration of about 2.0 mM to about 4.0 mM.

17. A three-dimensional aged skin model, wherein the three-dimensional aged skin model is prepared according to the method of claim 15.