Development of a novel in vitro strategy to understand the impact of shaving on skin health: combining tape strip exfoliation and human skin equivalent technology

Abstract

Introduction: The removal of unwanted hair is a widespread grooming practice adopted by both males and females. Although many depilatory techniques are now available, shaving remains the most common, despite its propensity to irritate skin. Current techniques to investigate the impact of shaving regimes on skin health rely on costly and lengthy clinical trials, which hinge on recruitment of human volunteers and can require invasive biopsies to elucidate cellular and molecular-level changes.

Methods: Well-characterised human skin equivalent technology was combined with a commonplace dermatological technique of tape stripping, to remove cellular material from the uppermost layer of the skin (stratum corneum). This method of exfoliation recapitulated aspects of razor-based shaving in vitro, offering a robust and standardised in vitro method to study inflammatory processes such as those invoked by grooming practices.

Results: Tape strip insult induced inflammatory changes in the skin equivalent such as: increased epidermal proliferation, epidermal thickening, increased cytokine production and impaired barrier function. These changes paralleled effects seen with a single dry razor pass, correlated with the number of tape strips removed, and were attenuated by pre-application of shaving foam, or post-application of moisturisation.

Discussion: Tape strip removal is a common dermatological technique, in this study we demonstrate a novel application of tape stripping, to mimic barrier damage and inflammation associated with a dry shave. We validate this method, comparing it to razor-based shaving in vitro and demonstrate the propensity of suitable shave- and skin-care formulations to mitigate damage. This provides a novel methodology to examine grooming associated damage and a platform for screening potential skin care formulations.

Keywords: exfoliation; inflammation; shaving; skin equivalent; stratum corneum; tape strip.

Figure 1

Number of tape strips removed correlates with level of barrier damage. Schematic protocol depicting the adhesion and removal of tape strips from FT-HSEs in vitro (A). Tape strips were applied with controlled pressure using a spring-loaded applicator and removed from the surface of HSEs using forceps. Cellular material is visible on tape strips upon removal and models were either analysed immediately or returned to culture for 4 days prior to analysis. H&E staining (B) of HSEs immediately following insult (Ba–c) ranging from 0–10 tape strip removal, or following a 4 days recovery period (Be–g). Immunofluorescence of terminal differentiation marker Filaggrin (C) stained in green and nuclei are highlighted by DAPI in blue. Demonstrates changes in barrier biomarker expression immediately following insult (Ca–d) and following 4 days recovery (Ce–h). Transepidermal water loss (TEWL) increased with increasing number of tape strips immediately following insult (D) (data represent mean ± SEM, n = 9, one-way ANOVA with Tukey’s multiple comparison test). TEWL recovered over a 4 days period (E) (data represent ± SEM, n = 9, two-way ANOVA with Tukey’s multiple comparison test) to a level comparable to 0 tape strip controls (dashed line). Scale bar: 100 μm. ** = p < 0.01 and **** = p < 0.0001.

Figure 2

Increased keratinocyte proliferation drives epidermal thickening following tape strip insult. Representative immunofluorescence images (A) of FT-HSEs 4 days after removal of 0–10 tape strips. ki67 is stained in green and DAPI highlights nuclei in blue. Epidermal thickness (B) (data represent mean ± SEM, n = 9, one-way ANOVA with Tukey’s multiple comparison test) significantly increases 4 days following tape strip insult, as does keratinocyte ki67 expression (C) (data represent mean ± SEM, n = 9, one-way ANOVA with Tukey’s multiple comparison test), suggesting an increase in proliferation is responsible for epidermal thickening. Scale bar: 50 μm. * = p < 0.1 and **** = p < 0.0001.

Figure 3

Proinflammatory cytokines are released in response to tape strip insult. An increase in a number of pro-inflammatory cytokines in the culture medium of tape stripped FT-HSEs was detected using a commercially available array. FT-HSEs were tape stripped, returned to culture for 4 days and culture medium harvested for analysis. Pro-inflammatory cytokines including: TNFα (A), IL-12p40 (B), IL-8 (C), IL-1Ra (D), IL-1β (E) and GM-CSF (F) were all increased in tape stripped samples (data represent mean ± SEM, n = 27, one-way ANOVA with Tukey’s multiple comparison test). * = p < 0.1, ** = p < 0.01, *** = p < 0.001, and **** = p < 0.0001.

Figure 4

Tape strip induced epidermal barrier damage is comparable to single dry razor pass. Schematic protocol for the dry shaving of FT-HSEs in vitro using a disposable razor (A). HSEs were unclipped from culture inserts and a razor was passed over the in the absence of lubrication, cellular material is visible on the blades and models were returned to culture for 4 days prior to analysis. H&E staining (Ba–c) of HSEs immediately following a dry razor pass or 4 days post-insult. Immunofluorescence images (Bd–f) reveal ki67 positive cells (green) and nuclei (blue), and barrier marker filaggrin (green) with nuclei (blue) (Bg–i). Transepidermal water loss (TEWL) (C) (data represent mean ± SEM, n = 9, two-way ANOVA with Tukey’s multiple comparison test) increased following razor pass compared with control samples (black dashed line) to a level comparable with severe tape stripping (dotted line), and recovered over a 4 days period. Both epidermal thickness (D) (data represent mean ± SEM, n = 9, one-way ANOVA with Tukey’s multiple comparison test) and ki67 positivity (E) (data represent mean ± SEM, n = 12, one-way ANOVA with Tukey’s multiple comparison test) increased following a dry razor pass to a level comparable to mild-moderate tape strip insult (dotted line). Scale bar: 50 μm. * = p < 0.1, ** = p < 0.01, and **** = p < 0.0001.

Figure 5

Pre-application of shaving foam reduces razor-mediated skin barrier damage. Schematic protocol for both dry razor shaving and pre-application of shaving foam to FT-HSEs in vitro (A). Gillette classic sensitive shaving foam was administered with 10 μL of formulation spread evenly across the surface of HSEs with a glass rod prior to razor-pass. HSEs were either harvested immediately or returned to culture for 4 days post-insult and H&E staining (Ba–d) reveals their morphology. Representative immunofluorescence staining of filaggrin, a biomarker of an intact barrier in green and nuclei are highlighted in blue (Be–h). Transepidermal water loss (TEWL) (C) (data represent mean ± SEM, n = 9, unpaired two-tailed student’s t-test) was reduced by pre-application of foam, as was the concentration of protein removed by a single razor pass (D) (data represent mean ± SEM, n = 9, unpaired two-tailed student’s t-test). Epidermal thickness (E) (data represent mean ± SEM, n = 9, unpaired two-tailed student’s t-test) was reduced after a 4 days recovery period when pre-application of shaving foam was incorporated into the regimen. Scale bar: 50 μm. ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001.

Figure 6

Post-insult moisturisation attenuates tape strip-induced damage. Schematic protocol depicting tape strip application and subsequent topical application (A). Two tape strips were removed from HSEs, a moisturising formulation was added topically to the surface and spread evenly using a glass rod, HSEs were then returned to culture for 4 days. H&E staining of HSEs immediately following removal of two strips (Ba) and 4 days post-insult treated with (Bc) and without moisturisation (Bb) or with no tape strips removed (Bd). Representative images of ki67 staining (Be–h) (ki67 green, nuclei blue), demonstrating changes in proliferation marker expression with treatment conditions. Transepidermal water loss (TEWL) (C) (data represent mean ± SEM, n = 3, one-way ANOVA with Tukey’s multiple comparison test) as a percentage of day 0 measurements. TEWL increases with time in culture without tape stripping (dashed line, 0 TS) but to a greater degree with tape stripping, which is reduced with post-tape strip moisturisation. Epidermal thickness (D) (data represent mean ± SEM, n = 3, one-way ANOVA with Tukey’s multiple comparison test) and percentage ki67 positive cells (E) (data represent mean ± SEM, n = 3, one-way ANOVA with Tukey’s multiple comparison test) are both increased with tape strip insult and reduced by post-insult moisturisation. Concentration of pro-inflammatory cytokines: TNFα (F), IL-1β (G), IL-Ra (H) and IL-12p40 (I) were increased in the culture medium following tape strip insult, and subsequently reduced by moisturisation (data represent mean ± SEM, n = 6, one-way ANOVA with Tukey’s multiple comparison test). Scale bar: 50 μm. * = p < 0.1, ** = p < 0.01, *** = p < 0.001, and **** = p < 0.0001.