Applying a vernix caseosa based formulation accelerates skin barrier repair by modulating lipid biosynthesis

Abstract

Restoring the lipid homeostasis of the stratum corneum (SC) is a common strategy to enhance skin barrier function. Here, we used a ceramide containing vernix caseosa (VC)-based formulation and were able to accelerate barrier recovery in healthy volunteers. The recovery was examined over 16 days by monitoring trans-epidermal water loss (TEWL) after barrier disruption by tape-stripping. Four skin sites were used to examine the effects of both treatment and barrier recovery. After 16 days, samples were harvested at these sites to examine the SC ceramide composition and lipid organization. Changes in ceramide profiles were identified using principal component analysis. After barrier recovery, the untreated sites showed increased levels of ceramide subclass AS and ceramides with a 34 total carbon-atom chain length, while the mean ceramide chain length was reduced. These changes were diminished by treatment with the studied formulation, which concurrently increased the formulated ceramides. Correlations were observed between SC lipid composition, lipid organization, and TEWL, and changes in the ceramide subclass composition suggest changes in the ceramide biosynthesis. These results suggest that VC-based formulations enhance skin barrier recovery and are attractive candidates to treat skin disorders with impaired barrier properties.

Keywords: barrier recovery; clinical study; epidermis; lipid organization; lipidomics; mass spectrometry; sphingolipids; stratum corneum; treatment.

Fig. 1.

Study design. A: Timeline of the clinical study (Time points are indicated as t with days as subscripts). Volunteers were first screened by a dermatologist (t_D-7), followed by a 1-week washout period. At t_D0 the SC was removed by tape-stripping. The formulation was applied two times daily for 2 weeks followed by 2 days washout. The activities and measurements are indicated at each time point (t_D-7 to t_D16). At all indicated time points, TEWL was measured, except for t_D-7. B: The studied sites on the ventral forearms. On both arms, one site was tape-stripped; on one arm, formulation was applied. C: The time points at which TEWL was measured to monitor barrier recovery of the stripped sites.

Fig. 2.

The effect of treatment on barrier recovery. A: The typical elapse of barrier recovery over time for untreated and treated stripped sites for a single volunteer. Points are means (±SD) of three measurements within one site. Lines are point-to-point connections. The dotted line indicated the difference in time (Δt) between the untreated and treated curve at a designated recovery percentage. B: The AUCs/1000 for all volunteers. Lines connect data points of the same volunteer. A paired t-test showed significant increase at the treated sites. C: The Δt for all volunteers at recovery percentages between 5 and 90%. The data were nonnormally distributed and are shown as median and interquartile range. Deviations from zero were tested with a Wilcoxon signed-rank test.

Fig. 3.

PCA of all SC ceramides and the amount of ceramides NS C40 and EOS C66. A: The PCA loading plot. Inertia per PC is indicted as percent variation. Variables are all ceramides quantified in samples obtained from tape-strips 5–8 and 17–20; the latter are indicated with a 2 after the subclass name. B: A plot of the PCA samples (the four sites of all volunteers). The samples were clustered per site.

Fig. 4.

The effect of treatment on ceramide groups correlating to barrier function. A, B: Correlations between TEWL and the amount of subclass AS (ng/SQ). The Pearson r and P are indicated (A: Samples of tape-strips 5–8; B: Samples of tape-strips 17–20). C: The amount subclass AS (ng/SQ) at all sites at depths of both 5–8 and 17–20 tape-strips (mean ± SD). Effect size was determined with an LMM. D, E: Correlations between TEWL and the amount of ceramides with a chain length of 34 carbon atoms (ng/SQ). The Pearson r and P are indicated (D: Samples of tape-strips 5–8; E: Samples of tape-strips 17–20). F: The amount of ceramides with a chain length of 34 carbon atoms (ng/SQ) at all sites at depths of both 5–8 and 17–20 tape-strips (mean ±SD). Effect size was determined with a linear mixed model. G: Pearson r of the correlation between TEWL and the total amount of each subclass at all sites at depths of both 5–8 and 17–20 tape-strips. H: The correlation between TEWL and ratio of subclasses $\frac{\text{NdS}\mathrm{+}\text{NP}\mathrm{+}\text{NH}\mathrm{+}\text{AdS}\mathrm{+}\text{AP}\mathrm{+}\text{AH}}{\text{NS}\mathrm{+}\text{AS}}$ of both depths, excluding NS C40. Pearson r per site were: control, −0.648; stripped, −0.808; treated,−0.729; and stripped+treated, −0.694. All statistical outputs of the LMM are found in Table 1.

Fig. 5.

Mean chain length and lamellar spacing. A: The mean total chain length (carbon atoms) for the four different sites at two depths excluding the ceramides of the formulation in the calculation. Data were compared using an LMM. B: The correlation between changes in fraction EO ceramides and changes in the mean chain lengths (carbon atoms) at the stripped site due to treatment (amount at the stripped site subtracting the stripped+treated). C: The EO fraction of the samples at the four different sites at two depths. Data were compared using an LMM. D: The difference between the SAXD peak 2 position in the stripped and stripped+treated sites. The differences were tested with a Wilcoxon matched pairs signed rank test. E: The correlation between the SAXD peak 2 (nm) and TEWL (g/m²/h) at the end of the study. Pearson r and P are provided. F: The correlation between the SAXD peak 2 (nm) and the mean chain length (carbon atoms) of all ceramides. Pearson r and P are provided. G. The wavenumber in cm⁻¹ of the stretching peak position. Data were compared using an LMM.

Fig. 6.

An overview of the effects of barrier disruption and treatment. Negative effects are indicated with a − sign and positive effects with a + sign. Barrier disruption induces barrier recovery and treatment accelerates this process. Changes in the EO fraction, mean chain length, and lamellar spacing showed correlations (cor) indicated by the arrows. The changes in lipid composition, organization, and subsequent effects are indicated. The ratio $\frac{\text{dS}\mathrm{+}\text{P}\mathrm{+}\text{H}}{\text{S}}$ is mainly determined by the enzymes human-dihydro-Δ4-desaturate (DES1) and human-sphingolipid-C4-hydroxylase (DES2), responsible for the production of sphingosine and phytosphingosine ceramides from dihydrosphingosine ceramides, respectively. The arrows indicate that barrier disruption changed the lipid synthesis decreasing this ratio and treatment had the opposite effect. The correlation of the TEWL at day 16(d16) to the subclass ratio is indicated; however, the amount of C34 and the SAXD Peak 2 position correlated to TEWL as well.