The skin, the largest organ of the human body, serves as a vital and multifaceted interface between our internal environment and the external world. The skin serves as a multifunctional barrier, encompassing physical, biochemical, microbiome, and immunological defenses (1–4).

In its role as a physical barrier, the skin’s outermost layer, the stratum corneum, is composed of densely arranged dead skin cells called the corneocytes, which are surrounded by lipids and bound together by corneodesmosomes. This well-structured arrangement creates a robust defense against external pressures, including abrasion, cuts, and shocks. The appropriate thickness of the stratum corneum is therefore crucial in the barrier function. Moreover, keratinocytes forming tight junctions add an additional layer of defense to the skin’s physical barrier, further enhancing its protective capabilities. The skin’s structural design also plays a crucial role in preventing excessive water loss from the body, thereby averting dehydration and maintaining optimal hydration levels (1, 5). Indeed, in the stratified epithelium of the skin, filaggrin, loricrin, involucrin, and corneodesmosin, are recognized as essential proteins for a functional epidermal barrier. Filaggrin contributes to the structural integrity of the stratum corneum. Deficiencies in filaggrin are associated with increased skin pH, potentially hindering repair mechanisms and promoting dehydration. Loricrin constitutes a vital component of the cornified envelope. Reduced loricrin expression compromises the structural integrity of this crucial barrier. Involucrin plays a critical role in the initial stages of keratinocyte differentiation, influencing the formation of the cornified envelope. Corneodesmosin (CDSN) is an intercellular protein that plays a critical role in maintaining skin barrier function (6, 7). CDSN deficiency resulted in lethal-skin barrier disruption in a mouse model (7) and enhanced viral penetration in an organotypic skin model (8). These essential proteins for epidermal barrier function act in concert to ensure a robust epidermal barrier, safeguarding the underlying tissues.

The skin’s immune barrier is composed of two primary types of immune cells: innate immune cells and adaptive immune cells. Among them, keratinocytes and Langerhans cells play crucial roles as key components of the skin’s innate immune system. They actively participate in protecting the skin from potential infections and contribute to its overall immune surveillance. In response to skin barrier damage resulting from injury or exposure to pathogens, keratinocytes and Langerhans cells trigger an innate immune response. They release various antimicrobial peptides and cytokines that further strengthen the skin’s defense mechanisms against infections, providing an essential line of protection for the body (4, 9, 10).

Recently, the recommended hygiene measures to prevent the spread of SARS-CoV-2, such as wearing masks, frequent handwashing and the use of sanitizers have been blamed for altering the skin barrier integrity (11). Repeated exposure to soaps, surfactants, detergents, and high-concentration ethanol disrupts the skin’s natural barrier, leading to loss of the lipid barrier of the stratum corneum, protein denaturation, and changes in keratinocyte cell membranes (12). This weakens the skin barrier, causing increased transepidermal water loss (TEWL) (13), greater irritant and allergen penetration, and an elevated risk of skin lesions and inflammation, potentially increasing the risk of bacterial and viral transmission through the skin (14–16). In the context of the COVID-19 pandemic, understanding the role of the skin barrier in human health has gained paramount importance.

Previous studies already showed the impact of various stressors on skin barrier function, such as the tape stripping technique (17–19), sodium dodecyl sulfate (SDS) treatment (20–23), microneedles (24–27), and abrasion (19, 28, 29). But so far, no studies have investigated the impact of mask wearing and frequent handwashing on human skin barrier. In addition, previous studies have primarily examined isolated aspects of skin health and function.

Here we aim to develop ex vivo models of human skin damaged by different treatments. We used both chemical treatments (SDS and ethanol solution) and mechanical treatments (tape stripping, dermabrasion using skin preparation pad, and application of surgical mask). Then we aim to systematically characterize the barrier integrity via an array of relevant parameters, such as the permeability, dermal absorption capacity, stratum corneum thickness and gene expression of barrier function markers. As inflammation is linked to skin barrier integrity, inflammatory markers were also analyzed. Having a range of well characterized degrees of skin damage models could be instrumental to assess the efficacy of topical products targeted at skin repair or to evaluate the effects of damaged skin barrier on viral penetration.

The skin barrier plays a critical role in safeguarding the body from infections and dermatological disorders by acting as a defense against external agents. The emergence of the COVID-19 pandemic has emphasized the importance of maintaining a healthy skin barrier, as a compromised barrier could potentially facilitate viral entry (30–35). With the widespread adoption of hygiene practices during the pandemic to prevent SARS-CoV-2 transmission, concerns regarding potential alterations to the skin barrier have arisen. Therefore, developing models to assess and investigate damaged skin barriers becomes even more crucial.

The primary goal of this research was to establish reliable ex vivo models for compromised human skin, offering more ethical alternatives to animal models and aligning with the three Rs principles of replacement, reduction, and refinement in experimental research. Due to substantial physiological differences between animal and human skin, translating animal research findings to humans can be unreliable. Human-based ex vivo models developed in this study provide a more accurate representation of human skin, thereby increasing research relevance. These models offer a more accurate representation of human skin function and response to various stimuli compared to animal models.

Transepidermal electrical resistance (TEER) and transepidermal water loss (TEWL) provide complementary insights into skin barrier function. TEER measures the electrical resistance across the skin, reflecting its overall integrity. Conversely, TEWL quantifies water loss through the skin, increasing as the barrier’s integrity weakens. A variety of techniques were used to compromise the skin barrier, including tape stripping for sunburn replication, dermabrasion for procedure-related damage, SDS for induction of itch-like irritation, and simulations of excessive handwashing and mask use.

All treatments significantly compromised skin barrier integrity, as evidenced by changes in TEER and TEWL. SDS, ethanol, and surgical mask reduced TEER, while the skin preparation pad decreased TEER and increased TEWL. Tape stripping primarily increased TEWL without significantly affecting TEER.

The difference between the effects of tape stripping on TEER and TEWL can be attributed to the different properties being measured and the specific mechanisms involved. TEER is a measure of the electrical resistance across a biological barrier, traditionally reflecting the tightness of cell junctions. While tight junctions are not typically found throughout the entire stratum corneum, some of their components can be detected in the lower portions of the stratum corneum, and occasionally even in the outer stratum corneum under certain circumstances. Therefore, when the stratum corneum is removed through tape stripping, the electrical resistance may not be significantly affected because TEER primarily depends on the integrity of these tight junction components within the stratum corneum. It’s worth noting that tape stripping might not directly disrupt these tight junctions. On the other hand, TEWL measures the rate of water evaporation from the skin surface. When the stratum corneum is partially removed, the skin’s barrier function is compromised, allowing more water to evaporate from the skin surface. As a result, TEWL increases after tape stripping, indicating an increase in water loss from the skin.

Our findings are consistent with published data demonstrating the detrimental effects of various treatments on skin barrier integrity. Studies investigating the impact of SDS on the skin have consistently shown its disruptive effects on the stratum corneum and barrier function. The significant decrease in TEER observed in our ex vivo model following SDS treatment is in line with previous research that highlights the weakening of the skin barrier upon exposure to SDS (36, 37). Likewise, the use of the skin preparation pad in our study resulted in a substantial reduction in TEER and a significant increase in TEWL, confirming its damaging effects on the skin barrier. These results are in agreement with previous investigations that have reported compromised barrier integrity following dermabrasion or similar mechanical interventions (38, 39). In the case of tape stripping treatment, our findings are also supported by previous studies (17, 18, 40).

Our findings suggest a potential for temporary skin barrier disruption with mask wear. Therefore, it is important to consider how mask use compares to clothing in terms of their impact on the skin barrier. Compared to most clothing, masks generally have closer and more continuous contact with the skin. This can lead to increased friction and occlusion, potentially impacting barrier function. This, along with the warm and humid microclimate created by masks around the mouth and nose, could influence the skin microbiome. Clothing generally has a less dramatic effect on microclimate. Moreover, some mask materials may contain chemicals or dyes that could potentially irritate sensitive skin, further impacting the barrier. In contrast, clothing fabrics are typically chosen for comfort and breathability, minimizing these concerns. These factors suggest that mask wear might have a more significant impact on the skin barrier compared to clothing.

Microscopic analysis revealed a significant reduction in stratum corneum thickness following SDS, skin preparation pad, tape stripping, ethanol and surgical mask treatments, providing further evidence of compromised skin barrier integrity. Reduction in stratum corneum thickness is a well-known feature of barrier impairment, and numerous studies investigating the effects of chemical and mechanical treatments on the skin have extensively documented this phenomenon (19, 20). Moreover, the reduced thickness or the stratum corneum also leads to altered tissue integrity. As the outermost layer exposed to the external environment, the stratum corneum serves as the body’s primary protective barrier. When this layer is damaged, the skin becomes more permeable (22, 38, 39, 41, 42). Consequently, all methods of damage often lead to increased dermal absorption of molecules, particularly hydrophilic molecules, while lipophilic molecules may show less pronounced increase.

In conclusion, the study highlights that, alongside revealing an increase in dermal absorption when the skin barrier is compromised, the findings emphasize the correlation between the magnitude of this increase and the severity of skin damage. For example, in cases of SDS-induced damage, the level of dermal absorption is comparatively lower than that arising from tape stripping, which in turn is less than the dermal absorption resulting from dermabrasion. Altogether, our study provides robust support for the idea of increased dermal absorption in the presence of a compromised skin barrier.

However, it is important to acknowledge the inherent limitations of our ex vivo skin models. One key limitation is the absence of systemic circulation, which distinguishes it from in vivo conditions where blood vessels and systemic circulation are present. This absence can influence the distribution of substances and potentially affect skin responses and also cannot recapitulate the recruitment of immune cells from the blood circulation. Furthermore, our models may not fully replicate the complex interactions that occur between skin and the body’s internal systems, which are inherent to in vivo conditions.

Importantly, our ex vivo skin models have been instrumental in assessing the permeation of substances through the skin, shedding light on dermal absorption mechanisms. This recognition has led regulatory agencies to integrate ex vivo skin models into specific regulatory processes, including considerations of bioequivalence waivers. One of the key advantages related to dermal absorption is that our models closely resemble the conditions of diseased skin, where dermal absorption is known to be increased. This aspect holds great relevance in terms of risk assessment when considering systemic exposure to substances applied topically. Understanding how substances permeate damaged skin barriers in these situations can have significant implications for assessing potential systemic exposure and safety profiles.

To further explore the molecular impacts of the damaging treatments, we assessed the expression of markers of barrier function and inflammation. Our results reveal that SDS, dermabrasion, ethanol and surgical mask treatments elicited a significant decrease in filaggrin and loricrin expression. These findings are in line with the observed effects on TEER, TEWL, and stratum corneum thickness, further confirming damage to the skin barrier. In contrast, tape stripping had no discernible effect on these markers.

Filaggrin, loricrin, and involucrin represent pivotal proteins with important roles in the formation of the epidermal skin barrier (23, 24). Filaggrin’s critical contribution to stratum corneum construction underscores the widespread consequences of its decreased expression, directly impacting the resilience of the skin barrier. Moreover, diminished filaggrin levels could lead to a rise of skin pH, obstructing repair mechanisms and potentially causing dehydration. Loricrin constitutes a crucial element of the cornified envelope. Diminishing the expression of loricrin compromises the structural integrity of this protective barrier. The reduction in the expression of these barrier-related molecules, filaggrin and loricrin, closely mirrors the barrier dysfunction triggered by SDS, dermabrasion, ethanol and surgical mask treatments. Notably, the roles of these molecules extend beyond the results of our study, as they have been implicated in dermatological diseases such as atopic dermatitis and psoriasis, where their dysregulation contributes to compromised skin barriers (23–29). Involucrin acts as a significant biomarker indicating the initial stages of keratinocyte differentiation. Its expression is triggered subsequently to the migration of mature keratinocytes from the basal layer but before the onset of cross-linking in the upper epidermal layer’s envelope. It also plays a critical role in shaping the cornified envelope formation. The marked increase of involucrin expression may provide insight into a compensatory mechanism adopted by keratinocytes in response to the reduced levels of loricrin and filaggrin arising from the different treatments. On a different note, the lack of an apparent impact from tape stripping on these markers could potentially be attributed to the insufficiency of 10 tape strips to induce significant molecular alterations, particularly considering the potentially superficial nature of tape stripping.

Cytokines play a pivotal role in various inflammatory skin conditions. The observed modulation of cytokine expression resulting from the diverse damaging treatments explored in our study underscores the intricate interplay between disruptions in the skin barrier and the innate immune defense mechanisms of the skin.

Of particular significance is the substantial increase in IL-8 expression following the SDS, tape stripping, and dermabrasion treatments. This increase in IL-8 levels signifies that the damaging treatments elicited an inflammatory reaction. IL-8 is a cytokine that plays a central role in skin inflammation. The observed rise in IL-8 levels in our study correlates with prior research that demonstrated elevated IL-8 levels following SDS treatment in human subjects (29).

The effect of the different treatments on Thymic Stromal Lymphopoietin (TSLP) expression presents interesting perspectives. TSLP is a cytokine that plays a pivotal role in the initiation and maintenance of immune responses, particularly within the context of skin inflammation and allergic reactions. The increase in TSLP expression following SDS treatment is of particular interest. SDS, well known for its ability to induce skin irritation as a surfactant, disrupts the skin protective barrier and triggers inflammation. The increased TSLP expression we observed after SDS treatment could potentially serve as an indicator of the immune system’s reaction to the irritation resulting from the compromised integrity of the skin barrier (43). Conversely, the decrease in TSLP expression subsequent to tape stripping and dermabrasion could indicate a potential mechanism through which the skin seeks to regulate excessive immune activation caused by these treatments. This could potentially be a safeguard mechanism to avoid an over-aggressive immune response that might lead to heightened inflammation.

The consistent expression of human beta-defensins (hBD) in suprabasal keratinocytes is also worth mentioning. Beyond their antimicrobial function, these defensins contribute to wound healing, immune cell attraction, and regulation of inflammation (44). The maintenance of adequate regulation of hBD closely correlates with skin barrier integrity. The observed increase in DEFB1 expression in the aftermath of the damaging treatments could indicate the skin’s adaptive responsiveness to the challenges encountered. Collectively, these findings underscore the intricate and multifaceted nature of immune responses triggered by various damaging treatments.

The impact of various treatments on key barrier parameters in ex vivo human skin is summarized in Table 2.

Finally, we evaluated the efficacy of a commercially available skin barrier repair product in restoring skin integrity following damage induced by SDS or dermabrasion. While the product demonstrated some anti-inflammatory effects, as evidenced by the modulation of the expression of IL-8 and TSLP, it was less effective in repairing the physical barrier. Notably, the product did not significantly improve TEER values or stratum corneum thickness, indicating a limited ability to restore the epidermal barrier. Furthermore, the product exacerbated the detrimental effects of SDS-induced damage, as seen by the further reduction in filaggrin and involucrin expression. These findings suggest that some components of the product may interact with SDS and that the product may not be suitable for repairing surfactant-induced damage and could potentially worsen the condition.

While the product exhibited anti-inflammatory properties in both SDS- and dermabrasion-damaged skin, its impact on the physical barrier was less pronounced. This discrepancy highlights the need for further investigation into the product’s mechanism of action and its optimal application for different types of skin damage. To gain a more comprehensive understanding of the product’s efficacy and limitations, future studies should focus on investigating the product’s impact on lipid barrier components, evaluating the product’s long-term effects to determine its sustained efficacy in repairing damaged skin, and exploring its interaction with different types of skin damage.

In conclusion, this study describes the characterization of ex vivo models of damaged skin upon different treatment modeling different degrees of skin damage. The ex vivo models offer a valuable platform for investigating various aspects of human skin barriers. They are based on well-preserved human skin explants, closely mimicking human skin physiology by preserving the natural structure encompassing both the dermal and epidermal layers, as well as other key aspects of skin structure and function. The approach used in this study highlights the complexity of the skin’s response to damage and the importance of considering both physical and inflammatory aspects when evaluating skin barrier integrity. However, further work is necessary to standardize such ex vivo models to ensure they comply with current regulations and facilitate wider adoption. In addition to refining the model, future research should investigate the role of the skin microbiome in maintaining barrier function and its potential influence on susceptibility to microbial infections and other skin diseases.