Study of the Repair Action and Mechanisms of a Moisturizing Cream on an SLS-Damaged Skin Model Using Two-Photon Microscopy

Abstract

This study evaluates the efficacy of a novel moisturizing cream using a sodium lauryl sulfate (SLS)-induced skin damage model, supported by advanced imaging with two-photon microscopy (TPM). TPM’s capabilities allow for in-depth, non-invasive visualization of skin repair processes, surpassing traditional imaging methods. The innovative formulation of the cream includes ceramide NP, ceramide NS, ceramide AP, lactobacillus/soybean ferment extract, and bacillus ferment, targeting the enhancement of skin hydration, barrier function, and structural integrity. In SLS-stimulated 3D skin models and clinical settings, the cream significantly improved the expression of key barrier proteins such as filaggrin (FLG), loricrin (LOR), and transglutaminase 1 (TGM1), while reducing inflammatory markers like IL-1α, TNF-α, and PGE2. Notably, the cream facilitated a significant increase in epidermal thickness and improved the dermal–epidermal junction index (DEJI), as observed through TPM, indicating profound skin repair and enhanced barrier functionality. Clinical trials further demonstrated the cream’s reparative effects, significantly reducing symptoms in participants with sensitive skin and post-intense pulsed light (IPL) treatment scenarios. This study highlights the utility of TPM as a groundbreaking tool in cosmetic dermatology, offering real-time analysis of the effects of skincare products on skin structure and function.

1. Introduction

Skin acts as the body’s first line of defense against environmental stressors, and maintaining its barrier function is crucial for preventing dehydration and protecting against harmful agents [1,2]. Compromised barrier function can lead to conditions such as dryness, irritation, and inflammation, often resulting in visible skin damage [3,4,5]. Sodium lauryl sulfate (SLS) is a widely used irritant that disrupts the skin’s barrier function, making it an effective in vivo model to study barrier dysfunction and assess the efficacy of potential skincare formulations aimed at restoring skin health [6,7]. Traditional methods for evaluating skin barrier repair have focused on clinical outcomes such as skin hydration levels and transepidermal water loss (TEWL) [8,9,10]. However, these measurements have limitations in providing insights into the underlying cellular and molecular mechanisms. This study utilizes two-photon microscopy (TPM), an advanced imaging technique, to offer high-resolution, real-time images of skin structure and function during the repair process of SLS-induced skin damage with a novel moisturizing cream.

TPM is a powerful technique for obtaining high-resolution in vivo imaging without the need for invasive biopsies [11]. It detects autofluorescence signals from keratin, NAD(P)H, FAD, elastic fibers, collagen fibers, melanin, and other skin components [12,13,14]. By combining two-photon absorption, excitation principles, and laser scanning confocal microscopy, TPM enables the reconstruction of the skin’s 3D structure through layer-by-layer scanning and imaging. TPM’s advantages in visualizing three-dimensional morphological features, such as epidermal thickness and the dermal–epidermal junction (DEJ), with exceptional clarity, have led to its broad application in clinical medicine [15,16], cosmetics [17,18], and various other fields. In this study, TPM was used as an advanced imaging tool to explore the tissue repair mechanisms of the moisturizing cream, particularly through the quantitative analysis of epidermal thickness and DEJ undulation. A key innovation of this study is the introduction of the Dermal–Epidermal Junction Index (DEJI), a novel 3D metric that quantifies DEJ undulation. TPM’s ability to capture depth-resolved two-photon excited fluorescence (TPEF) and second harmonic generation (SHG) signals enabled us to compute DEJI.

The moisturizing cream evaluated in this study was specifically formulated to enhance skin hydration, restore barrier integrity, and promote skin regeneration. The formulation includes ceramide NP, ceramide NS, ceramide AP, Lactobacillus/soybean ferment extract, and bacillus ferment. Ceramides are crucial components of the stratum corneum’s intercellular lipids, playing an essential role in maintaining the skin’s moisturizing and barrier functions, alongside fatty acids and cholesterol. A reduction in ceramide levels is closely associated with compromised barrier function [19]. Lactobacillus/soybean ferment extract provides nerve-soothing effects, alleviating skin discomfort caused by SLS-stimulated damage [20]. Bacillus ferment has been shown to enhance type IV collagen production, improving DEJ integrity and increasing laminin-332, which in turn enhances the undulation of the DEJ, promoting better skin cohesion and overall barrier function [21]. The benefit of the cream is mainly attributed to the essential ingredients of ceramide NP, ceramide NS, ceramide AP, lactobacillus/soybean ferment extract, and bacillus ferment, as well as the synergistic interplay of these components with other ingredients in the formulation.

Integrating findings from our prior in vitro and clinical studies, this investigation explores the cream’s effects on key skin biomarkers. In SLS-stimulated 3D skin models and in vivo tests on subjects, the cream significantly increased the expression of barrier proteins such as filaggrin (FLG), loricrin (LOR), and transglutaminase 1 (TGM1), while reducing inflammatory markers such as IL-1α, TNF-α, and PGE2. Histological examinations further demonstrated improved tissue structure and skin barrier integrity following cream application, consistent with findings from recent studies on ceramide-enriched formulations [22,23]. Clinical trials also confirmed the cream’s reparative properties, as evidenced by a reduction in lactic acid sting test (LAST) scores and facial redness in participants with sensitive skin, as well as increased moisturization, reduced TEWL and decreased facial pigmentation after intense pulsed light (IPL) treatment.

This study aims to demonstrate the potential of TPM in cosmetic dermatology by revealing subtle, structural changes in the skin during the repair process and assessing the cream’s effectiveness in enhancing skin repair and barrier function. The use of TPM complements the growing trend of utilizing advanced imaging techniques to track the performance of skincare formulations, offering a deeper understanding of their effects at the structural and morphological levels, as seen in recent advancements in skin imaging [24,25].

2. Materials and Methods

2.1. Research Product

The test product was a cream formulated with ceramides (ceramide NP, ceramide NS, ceramide AP), lactobacillus/soybean ferment extract, and bacillus ferment, provided by Proya Cosmetics Co., Ltd. under the name Proya Advanced Original Repair Concentrating Essence Cream (ORC). The sample group using this cream in the experiment was the ORC group. A comprehensive list of the cream’s formula ingredients is detailed in Data S1.

2.2. Prior In Vitro and Clinical Studies

2.2.1. The SLS-Stimulated 3D Epidermal Skin Model

In vitro analysis was conducted using a 3D epidermal skin model (EpiKutis^®, BioCell Biotechnology Co., Ltd., Xian, China, Batch No.: ES220106), stimulated with 0.2% SLS (10% in H₂O, Sigma, St. Louis, MO, USA, Batch No.: 220119) to induce barrier damage [26]. This study included four groups: a blank control group (BC), which received no SLS stimulation or treatment; a negative control group (NC), which was stimulated with SLS but received no treatment; a positive control group (PC), treated with 0.01% dexamethasone solution (C₂₂H₂₉FO₅, MW: 392.46, purity > 98%, Sigma, St. Louis, MO, USA, Batch No.: 220119) following SLS stimulation; and a cream treatment group (ORC). Barrier-associated proteins FLG (Abcam, Cambridge, UK, Batch No.: ab218397), LOR (Abcam, Cambridge, UK, Batch No.: ab198994), and TGM1 (Abcam, Cambridge, UK, Batch No.: 12912-3-AP) were evaluated using immunofluorescence [27]. Tissue morphology was assessed via H&E staining. Inflammatory cytokines including IL-1α (Abcam, Cambridge, UK, Batch No.: ab100560), IL-6 (Abcam, Cambridge, UK, Batch No.: ab46027), TNF-α (Abcam, Cambridge, UK, Batch No.: ab46087), and PGE2 (Abcam, Cambridge, UK, Batch No.: ab133021) were quantified using ELISA [28]. More details of the in vitro methods are provided in Data S2.

2.2.2. Clinical Studies

A 4-week clinical study involving 34 participants with sensitive skin is detailed in Data S3.

This split-face controlled study enrolled 12 healthy Chinese women (aged 26–44). Following full-face IPL treatment, participants’ facial halves were randomly assigned to either the cream-treated experimental group (ORC group) or the untreated control group (NC group). Skin parameters were measured, and subjective assessments were made at baseline, 30 min after IPL treatment, and on days 3, 7, 14, and 28. Participants adhered to prescribed skincare regimens (cream, medical dressing, cleanser, sunscreen) during the 4-week study. Adverse events were monitored throughout. More details are provided in Data S4.

2.3. Evaluation of the Cream by TPM

2.3.1. Subjects

This study enrolled 40 healthy Chinese participants, with 22 of them screened using the SLS stimulation model experiment. The participants, aged 24–34 years, exhibited skin intolerance to 0.2% SLS stimulation on their inner forearms. All participants were free from skin or systemic diseases and had not applied any topical treatments to the test areas for 30 days. Exclusion criteria included pregnant or lactating women, individuals planning to become pregnant during the study period, those with a history of significant cosmetic allergies, or those with severe skin conditions. Participants provided informed written consent after being fully briefed on the benefits, risks, and potential complications.

2.3.2. Methods

Evaluations were conducted in a controlled environment, maintaining a temperature of 19–23 °C and a relative humidity of 40–60%. Participants were acclimatized for at least 30 min prior to testing. Three skin sites were randomly marked on the inner left and right forearms of subjects for the blank control (BC), negative control (NC), and experimental groups. Next, 200 μL of 0.2% SLS solution was applied using an 18 mm diameter patch device to the test areas of the experimental and NC groups. A blank patch device was applied to the BC group. The sequence of application was randomized to ensure blinding for both researchers and participants. Baseline instrumental measurements were taken prior to the application of 0.2% SLS. The induction time for the SLS was 24 h, during which the test area was kept dry. After induction, the patch was removed, and subjects rested for 30 min. Clear and obvious erythema responses were assessed to confirm successful skin damage modeling. Data acquisition and image capture were then performed.

Subjects were instructed to apply the cream to the experimental test area once in the morning and evening, while the NC group and the BC group were not instructed to apply any product. Assessments were performed at the following time points: post-modeling, 24 h, 72 h, and 168 h. The images were captured using the TPM. For each test site, three non-overlapping fields of view (140 × 140 μm each) were randomly selected for imaging. The DEJI was calculated for each field, and the mean of the three measurements was used for analysis to minimize variability and avoid area-specific bias.

2.3.3. Instrumental Measurement

Instrumental assessments included stratum corneum moisture content, measured by a Corneometer CM825 (Courage & Khazaka, Cologne, Germany), and TEWL, determined by a Tewameter TM HEX (Courage & Khazaka, Cologne, Germany). Layer-by-layer skin images were captured using a TPM (Transcend Vivoscope, Beijing, China) with a laser wavelength of 780 ± 10 nm, an imaging field of view of 140 × 140 μm, and a scanning depth of 120 μm. The DEJ can be precisely delineated based on the transition from the appearance of collagen to the disappearance of columnar or cubic basal cells. Epidermal thickness is defined as the mean distance from the skin’s upper surface to the DEJ. The DEJI is defined as the ratio of the actual 3D surface area of the DEJ to its 2D projected area along the skin surface normal. A DEJI value ≥ 1 is expected: a completely flat DEJ yields a DEJI of 1, whereas greater undulation results in higher DEJI values. A higher DEJI thus indicates a larger contact area between the epidermis and dermis. The DEJI was computed using a dedicated computer software algorithm, based on the SHG signal from collagen fibers, which allowed clear 3D delineation of the DEJ surface. Our algorithm was adapted from previously published studies [29,30]. More details of the algorithm are provided in the Supplementary Data.

DEJI = Actual Surface Area of the DEJ/Projected Flat Surface Area

(1)

2.3.4. Statistical Analysis

Statistical analyses were conducted using SPSS 25.0 software. The Shapiro–Wilk test was used to assess data distribution normality. Paired t-tests were performed for comparisons between two groups if the data followed a normal distribution; otherwise, the rank sum test was used. For comparisons involving more than two groups, one-way ANOVA was employed for data with normal distribution and equal variance, while the Welch t-test was used for data with normal distribution and unequal variance. The Kruskal–Wallis test was applied if the data did not follow a normal distribution. A p value of less than 0.05 was considered statistically significant in all tests.

3. Results

3.1. Prior In Vitro and Clinical Study Results

3.1.1. SLS-Stimulated 3D Epidermal Skin Model: Barrier Protein Expression, Tissue Morphology Analysis, and Regulation of Inflammatory Factors

In the SLS-stimulated 3D epidermal skin model, the focus was on barrier protein expression, tissue morphology analysis, and the regulation of inflammatory factors. Barrier proteins, particularly LOR, FLG, and TGM1, are essential for maintaining skin barrier integrity and function. The reduction of these proteins is a significant factor contributing to compromised skin barrier function. LOR and FLG, in particular, are key components of keratinocyte differentiation, strengthening the skin barrier and enhancing moisturization. After SLS stimulation, the immunofluorescence intensity of FLG, LOR, and TGM1 was significantly lower in the NC group compared to the BC group (Figure 1B), indicating skin barrier damage. However, the application of the cream significantly increased the levels of these proteins, with improvement rates of 69%, 300%, and 775% for FLG, LOR, and TGM1, respectively (Figure 1A).

Tissue morphology analysis, conducted using hematoxylin and eosin (H&E) staining, revealed the physiological structure of the tissues. Compared to the BC group, the skin barrier in the NC group became lax after SLS stimulation. However, the cream application alleviated these structural changes induced by SLS, indicating a positive effect on skin barrier integrity (Figure 1C). IL-1α, an inflammatory factor, is released into the extracellular space following cell membrane damage. IL-6 and TNF-α are inflammatory cytokines released during the activation of inflammation-related pathways. PGE2, a prostaglandin metabolite of arachidonic acid, primarily induces inflammatory responses. In this study, the levels of IL-1α, IL-6, TNF-α, and PGE2 were significantly elevated in the NC group compared to the BC group. However, the application of the cream notably reduced the secretion of IL-1α, TNF-α, PGE2, and IL-6, with inhibition rates of 58%, 40%, 65%, and 47%, respectively (Figure 1D).

The significant improvements in the levels of barrier proteins and the reduction in inflammatory factors underscore the cream’s efficacy in counteracting the damaging effects of SLS-induced irritation. These findings are in line with recent studies that highlight the role of barrier proteins in skin resilience and their potential for repair through specific skincare formulations [5,6].

3.1.2. Assessment of Soothing and Repairing Efficacy in the 4-Week Clinical Study

A total of 34 healthy female participants with sensitive skin completed this 4-week study. The results demonstrated that the cream effectively soothes skin irritation and reduces skin redness, further emphasizing its role in skin comfort and barrier restoration. The observed improvement in skin tolerance was consistent with findings from similar clinical trials, which have demonstrated that this cream can significantly improve skin hydration and reduce inflammation, particularly in sensitive skin types [22,23]. More details are provided in Data S5.

3.1.3. Evaluation of Skin Improvement After IPL Treatment

Following IPL treatment, the cream was found to increase skin hydration, reduce TEWL, and minimize pigmentation compared to the control group. These findings are consistent with previous studies that show the positive effects of ceramides and probiotic extracts in promoting skin regeneration and hydration [19,21]. More details are provided in Data S6.

3.2. Evaluation of the Cream Under SLS-Damaged Skin Model by TPM

3.2.1. Subject Skin Condition

Differences in skin condition among various test groups at the initial stage, as well as the stability of skin condition throughout the test period, are presented in

Table 1. Variability of indicators in the BC group at each time point and comparison of initial indicators among groups (ns, p > 0.05).

Indicator	Comparison of BC Group at Each Time Point	Comparison Among Groups Before Modeling
Moisture content	0.056 ns	0.969 ns
TEWL value	0.995 ns	0.615 ns
T/C Value	0.689 ns	0.591 ns
DEJI	0.995 ns	0.188 ns
Epidermal thickness	0.364 ns	0.905 ns

. In the BC group, the test indicators showed fluctuations around the baseline values at different testing times (pre-modeling, post-modeling, 24 h, 72 h, 168 h). However, these fluctuations were not statistically significant. This finding suggests that the skin barrier function of the subjects remained stable and consistent throughout the testing process, exerting a negligible impact on the results. Furthermore, no significant differences were observed in the initial values of the test indicators among the BC group, the NC group, and the cream application group prior to modeling.

This consistency in the baseline values ensures that the observed effects are attributed to the intervention (the cream) rather than baseline variations—an important aspect of validating the study’s methodology and results. Additionally, this consistency further supports the reliability of TPM as an imaging technique for assessing skin condition over time [16,18].

3.2.2. SLS Damage Model

After 0.2% SLS stimulation, the subjects exhibited significant erythema on the inner forearm. The use of the TEWL value is an effective approach for measuring skin barrier function [8,9,10]. The ratio of the TEWL value to the stratum corneum moisture content (T/C value) represents the rate of transdermal water loss per unit of stratum corneum moisture content per unit area. This ratio provides a comprehensive reflection of the skin’s water barrier function: the lower the T/C value, the better the skin’s water barrier performance [31]. The changes in stratum corneum moisture content, TEWL value, and T/C value before and after modeling are shown in Figure 2. Following modeling, there was a decrease in stratum corneum moisture content and an increase in both TEWL and T/C values, all of which showed significant differences compared to the pre-modeling period. These results confirm the success of the skin damage model.

3.2.3. The Repair Efficacy of the Cream

Skin Stratum Corneum Moisture Content

The effect of the cream on increasing the moisture content of the skin’s stratum corneum is shown in Figure 3. In the BC group, the stratum corneum moisture content fluctuated around the baseline value at each test time point. In the NC group, the moisture content increased 24 h after SLS modeling due to edema infiltration in the tested area and then gradually decreased back to lower levels after 72 and 168 h (Figure 3). After cream application, the moisture content followed the same initial trend as in the NC group, but the mean values remained higher at all subsequent time points. Notably, compared to the NC group, the stratum corneum moisture content was significantly increased at various test time points.

The TEWL Value

In both the NC and ORC groups, TEWL values gradually declined over time following modeling (Figure 4). After 168 h of cream application, the TEWL value in the ORC group was significantly reduced by 9% compared to the NC group, indicating the cream’s beneficial effect on skin repair.

The T/C Value

The variation in T/C value over time is presented in Figure 5. The data indicate a stable skin condition in the BC group, as the T/C value fluctuated around baseline values throughout the testing period. In the NC group, the T/C value decreased significantly 24 h after SLS damage modeling, as the TEWL value decreased and the moisture content of the stratum corneum increased. After the application of the cream, the T/C value continued to decrease, with a significant reduction of 36% compared to the NC group after 168 h. The T/C value was significantly lower than that of the NC group at 24, 72, and 168 h post-cream application, indicating effective skin repair by the cream.

Skin Epidermal Thickness

TPM provided non-invasive imaging at various skin depths, revealing detailed skin structure through TPEF and SHG images. The different layers of the skin, from the stratum corneum to the stratum basale, were clearly visible (Figure 6). Post SLS modeling, epidermal thickness peaked at 24 h in the NC group and then tapered off. Similarly, in the ORC group, the epidermal thickness substantially increased at 24 h and continued to rise over time. After 168 h, the increase in the ORC group was 8.5 μm, a significant difference compared to the 2.9 μm increase in the NC group (Figure 7).

The Morphology of the DEJ

The DEJ boundary was determined by identifying the transition zone between SHG signals (red) and TPEF signals (green). The interface between strong SHG and dominant TPEF signals was designated as the DEJ. The DEJI value indirectly reflects the contact area between the dermis and epidermis. Post SLS modeling, changes in DEJI value are shown in Figure 8. In the NC group, DEJI values fluctuated slightly over 168 h period. After cream application, the DEJI value improved significantly by 7% after 168 h compared to the NC group. This improvement signifies an increase in the contact area between the dermis and epidermis. Notably, longitudinal skin section images show that the DEJ changed from a flatter to a more undulating wavy shape after the cream application (Figure 9).

4. Discussion

This study confirms the efficacy of a novel moisturizing cream in repairing SLS-induced skin damage, with improvements observed in skin hydration, barrier function, and structural integrity. Using TPM, we gained detailed insight into the skin repair process—specifically, changes in epidermal thickness and DEJ undulation—that are difficult to capture using traditional imaging techniques.

The observed increase in epidermal thickness and DEJI, as indicated by TPM, suggests that the cream not only restores the skin’s structural integrity but also enhances its functionality. The substantial decrease in the T/C value within the treatment group further supports the cream’s role in enhancing skin barrier function. These findings are consistent with recent studies emphasizing the importance of a well-maintained DEJ in promoting skin health, as DEJ plays a crucial role in cellular communication, metabolic exchange, and skin barrier integrity [32,33].

Our histological and biochemical analyses align with TPM findings, revealing the cream’s repair mechanisms. In SLS-stimulated 3D skin models, the cream markedly increased the expression of barrier proteins such as FLG, LOR, and TGM1, while reducing inflammatory markers like IL-1α, TNF-α, and PGE2. The results from H&E staining also supported the TPM findings, showing that the cream helped mitigate the structural damage induced by SLS. The change from loose to dense tissue structure shown by H&E and the increase in epidermal thickness shown by TPM both reflect the cream’s effect on repairing the epidermal barrier. This was particularly evident in the restoration of the skin barrier, which is known to be crucial for maintaining skin hydration and preventing irritants from penetrating the skin [5,6]. The findings confirm that the cream’s impact is multifaceted, addressing both the immediate effects of hydration and long-term barrier repair, similar to previous research on ceramide-based formulations and their role in maintaining epidermal integrity and moisture retention [19,22].

The moisturizing cream is formulated with ceramide NP, ceramide NS, ceramide AP, lactobacillus/soybean ferment extract, and bacillus ferment, all of which are crucial for maintaining skin hydration, enhancing barrier function, and promoting skin regeneration. Ceramides, particularly ceramide NP, which is the most abundant ceramide in the human stratum corneum, have been extensively studied for their ability to repair the skin barrier and restore skin moisture in individuals with compromised skin [34]. Lactobacillus/soybean ferment extract, a biotechnological ingredient, is known to help reduce irritation and enhance skin recovery by supporting epidermal thickness and rejuvenating the skin [35]. Bacillus ferment, on the other hand, has been shown to stimulate the production of type IV collagen and laminin-332, which are essential for improving DEJ integrity and promoting better cohesion between the epidermis and dermis [36]. These ingredients work synergistically to enhance the overall efficacy of the cream, offering a comprehensive solution for skin repair and barrier restoration.

TPM offers the advantage of visualizing structural and morphological details within the skin without requiring invasive biopsies, enabling detailed, real-time analysis of skin recovery [24,25]. TPEF signals reveal the distribution of endogenous fluorophores in the epidermis and dermis, while SHG signals highlight collagen fibers within the dermis. Since most collagen fibers only appear in the dermis, the contour of the DEJ can be distinguished well from a 3D perspective [29]. The ability to distinctly visualize the DEJ in three dimensions underscores its critical role in skin homeostasis and function as a dynamic signal transduction barrier between fibroblasts and keratinocytes [32]. The flattened appearance of DEJ in aged skin [33], which typically exhibits reduced surface area, was notably improved in our study, suggesting an increase in metabolic exchange within epidermal cells, thereby stabilizing skin condition.

Despite its strengths, this study faced limitations such as the small field of view provided by TPM, which restricted our ability to fully observe the DEJ. Moreover, the absence of pre-treatment and post-treatment histopathological images limited our ability to directly compare TPM results with biopsy samples. However, the non-invasive nature of TPM provides a significant advantage by reducing patient discomfort and risk, a crucial consideration in dermatological research.

5. Conclusions

The findings underscore the potential of the novel moisturizing cream in effectively repairing skin damage induced by SLS exposure. This was evidenced by enhanced skin hydration, reduced TEWL value, increased epidermal thickness and improved DEJI, as corroborated by both clinical assessments and detailed TPM analysis. The use of TPM not only enabled real-time, high-resolution imaging of the skin’s repair process but also provided insights into the cream’s reparative mechanisms, confirming its benefits in epidermal regeneration and DEJ repair. This study highlights the utility of TPM as an essential tool in the cosmetic dermatology field, providing critical insights into the efficacy of skincare products and guiding the formulation of treatments for compromised skin.