Human Stratum Corneum Research Articles

Uptake and desorption of hydrophilic compounds from human stratum corneum

Small, polar compounds, both ionic and uncharged, partition into human stratum corneum immersed in aqueous solutions to an extent comparable to the water volume fraction of the tissue, then desorb in two phases. The fast phase has a time constant on the order of a few minutes, whereas the slow phase occurs over many hours. A physical model for this behavior involving a combination of tranverse diffusion through the tissue and lateral diffusion and exchange with skin appendages is presented. This concept is probed using excised human stratum corneum exposed to aqueous solutions of radiolabeled sodium chloride, tetraethyl ammonium bromide and mannitol, plus previously published data on six other compounds of varying molecular size and polarity. The fast phase desorption process becomes unimportant for lipophilic compounds. Slow phase desorption rates are size-selective, with larger species desorbing much more slowly than smaller ones. Interpreting the size-selectivity in terms of smooth cylindrical pores using the centerline approximation leads to an optimum pore radius of about 8–12Å, depending on the model chosen.

Control of human skin wettability using the pH of anionic surfactant solution treatments

The outermost layer of human skin, or stratum corneum, acts as a protective barrier between underlying living tissue and the external environment. The wettability of this tissue layer can influence spreading of chemicals and the adhesion of pathogenic microorganisms. We show in this article that the wettability of isolated human stratum corneum can be controlled through treatment with solutions of the anionic surfactant, sodium lauryl sulfate, buffered to different pH values. Relative to control treatments with the buffer solution alone, surfactant solution treatments under acidic conditions cause delipidated stratum corneum to become more hydrophobic. In contrast, alkaline conditions cause the stratum corneum to become more hydrophilic; irrespective of lipid composition. This transition is consistent with a reorientation of bound surfactants at the tissue interface. Under acidic conditions, electrostatic binding of negatively charged surfactant head groups with positively charged keratin in the stratum corneum would increase tissue hydrophobicity due to the exposed hydrophobic tails. However, a hydrophobic based attraction of the apolar surfactant tails to the stratum corneum surface under alkaline conditions would leave the hydrophilic surfactant head groups exposed, causing increased tissue hydrophilicity. Changes in wettability with pH become diminished when lipids ordinarily found in stratum corneum are present, suggesting the lipids partially inhibit surfactant binding. Profilometry studies of the tissue topography highlight that surfactant induced changes in stratum corneum surface roughness cannot account for the observed changes in wettability.

Quaternized carboxymethyl chitosan/organic montmorillonite nanocomposite as a novel cosmetic ingredient against skin aging

It is of technical and economic importance to develop an effective cosmetic ingredient against skin aging through low-cost and facile production. This study developed a polymer/layered silicate nanocomposite as cosmetic ingredient against skin aging. Firstly, quaternized carboxymethyl chitosan (QCMC)/organic montmorillonite (OMMT) nanocomposite (QCOM) was fabricated via solution-induced intercalation. QCOM with different mass ratios of QCMC to OMMT were studied by various characterizations as well as moisture-adsorption and retention activities and UV-protection capacity. QCOM was then added into a cosmetic formulation to prepare a cosmetic cream. And the evaluation of its efficacy about skin care was conducted. The results showed that the optimal QCOM had better moisture-adsorption and retention behaviors than hyaluronic acid, and QCOM solution displayed good UV-protection ability. Furthermore, the QCOM-containing cosmetic cream meets the hygienic standard for cosmetics; it has negligible dermal irritation and a prominent moisture-retention efficacy on human stratum corneum.

In vivo confocal Raman microscopic determination of depth profiles of the stratum corneum lipid organization influenced by application of various oils

BackgroundThe intercellular lipids (ICL) of stratum corneum (SC) play an important role in maintaining the skin barrier function. The lateral and lamellar packing order of ICL in SC is not homogenous, but rather depth-dependent. ObjectiveThis study aimed to analyze the influence of the topically applied mineral-derived (paraffin and petrolatum) and plant-derived (almond oil and jojoba oil) oils on the depth-dependent ICL profile ordering of the SC in vivo. MethodConfocal Raman microscopy (CRM), a unique tool to analyze the depth profile of the ICL structure non-invasively, is employed to investigate the interaction between oils and human SC in vivo. ResultsThe results show that the response of SC to oils’ permeation varies in the depths. All oils remain in the upper layers of the SC (0–20% of SC thickness) and show predominated differences of ICL ordering from intact skin. In these depths, skin treated with plant-derived oils shows more disordered lateral and lamellar packing order of ICL than intact skin (p<0.05). In the intermediate layers of SC (30–50% of SC thickness), the oils do not influence the lateral packing order of SC ICL (p>0.1), except plant-derived oils at the depth 30% of SC thickness. In the deeper layers of the SC (60–100% of SC thickness), no difference between ICL lateral packing order of the oil-treated and intact skin can be observed, except that at the depths of 70–90% of the SC thickness, where slight changes with more disorder states are measured for plant-derived oil treated skin (p<0.1), which could be explained by the penetration of free fatty acid fractions in the deep-located SC areas. ConclusionBoth oil types remain in the superficial layers of the SC (0–20% of the SC thickness). Skin treated with mineral- and plant-derived oils shows significantly higher disordered lateral and lamellar packing order of ICL in these layers of the SC compared to intact skin. Plant-derived oils significantly changed the ICL ordering in the depths of 30% and 70–90% of the SC thickness, which is likely due to the penetration of free fatty acids in the deeper layers of the SC.

Galectin-7 is extracellularly released from epidermal keratinocytes in atopic dermatitis and serves as immunomodulator of Langerhans cells

A number of galectins are localized in various organs, and galectin-7 is expressed in epidermal keratinocytes (KCs). Our proteome analysis of extracts from human stratum corneum revealed that galectin-7 is highly expressed in atopic dermatitis (AD) patients (Sakabe J et al. JACI 2014). Meanwhile, galectin-7 might bind to hyaluronan, and its intercellular accumulation can yield spongiosis. Thus, it should be elucidated how galectin-7 is induced in the epidermis and whether it can suppress or rather promote skin inflammation. To address these issues, we used 3D cultured KCs to investigate the ability of IL-4/IL-13 to induce galectin-7, and employed murine epidermal Langerhans cells (LCs) for its ability to maturate LCs. Immunohistochemical staining showed that, in healthy skin, galectin-7 was expressed in the cytoplasm of KCs in the prickle layer. In AD lesional skin, however, galectin-7 was positively stained in the intercellular space of KCs. In human 3D skin culture, incubation of KCs with IL-4/IL-13 promoted its expression in the intercellular space, suggesting that these Th2 cytokines stimulate KCs to produce galectin-7 and to extracellularly release it. These findings urged us to further investigate its effect on LCs. Epidermal suspensions were freshly prepared from C57BL/6 mice, and cells were cultured for 24 h with or without 100 ng of mouse recombinant galectin-7. The expression levels of langerin, EpCAM, CD54, CD80, CD86, and MHC class II were examined by flow cytometry. LCs, gated as langerin+ and EpCAM+ cells, expressed increased levels of CD54 and CD86, when epidermal cells were incubated with galectin-7. These results suggest that galectin-7 is extracellularly secreted by epidermal KCs in AD and may play an immunomodulatory effect on LCs.

Skin permeation and antioxidant efficacy of topically applied resveratrol.

The permeation of resveratrol was assessed by in vitro and in vivo experiments 24h after topical administration. The in vitro profile of resveratrol was assessed by Raman spectroscopy. Human skin permeation was analysed in vivo by the tape stripping method with the progressive removal of the stratum corneum layers using adhesive tape strips. Moreover, the free radical scavenging activity of resveratrol after its topical application was determined using the DPPH assay. The Raman spectra indicated that the topically applied resveratrol penetrates deep into the skin. The results showed high amounts of resveratrol in the different stratum corneum layers close to the surface and a constant lower amount in the upper layers of the viable epidermis. The concentration of resveratrol present in the outermost stratum corneum layers was obtained by tape stripping after in vivo application. The results demonstrated that resveratrol mainly remained in the human stratum corneum layers. After topical application, resveratrol maintained its antiradical activity. The antioxidant efficacy of the compound was higher in the inner layers of the stratum corneum. As these results have demonstrated, topically applied resveratrol reinforces the antioxidant system of the stratum corneum and provides an efficient means of increasing the tissue levels of antioxidants in the human epidermis.

Large-scale human skin lipidomics by quantitative, high-throughput shotgun mass spectrometry

The lipid composition of human skin is essential for its function; however the simultaneous quantification of a wide range of stratum corneum (SC) and sebaceous lipids is not trivial. We developed and validated a quantitative high-throughput shotgun mass spectrometry-based platform for lipid analysis of tape-stripped SC skin samples. It features coverage of 16 lipid classes; total quantification to the level of individual lipid molecules; high reproducibility and high-throughput capabilities. With this method we conducted a large lipidomic survey of 268 human SC samples, where we investigated the relationship between sampling depth and lipid composition, lipidome variability in samples from 14 different sampling sites on the human body and finally, we assessed the impact of age and sex on lipidome variability in 104 healthy subjects. We found sebaceous lipids to constitute an abundant component of the SC lipidome as they diffuse into the topmost SC layers forming a gradient. Lipidomic variability with respect to sampling depth, site and subject is considerable, and mainly accredited to sebaceous lipids, while stratum corneum lipids vary less. This stresses the importance of sampling design and the role of sebaceous lipids in skin studies.

Measuring and Modeling Contractile Drying in Human Stratum Corneum

Measuring and Modeling Contractile Drying in Human Stratum Corneum

Measuring and Modeling Contractile Drying in Human Stratum Corneum.

Stratum corneum (SC) is the most superficial skin layer. Its contact with the external environment means that this tissue layer is subjected to both cleansing agents and daily variations in ambient moisture; both of which can alter the water content of the tissue. Reductions in water content from severe barrier dysfunction or low humidity environments can alter SC stiffness and cause a build-up of drying stresses. In extreme conditions, these factors can cause mechanical rupture of the tissue. We have established a high throughput method of quantifying dynamic changes in the mechanical properties of SC upon drying. This technique can be employed to quantify changes in the drying behavior and mechanical properties of SC with cosmetic cleanser and moisturizer treatments. This is achieved by measuring dynamic variations in spatially resolved in-plane drying displacements of circular tissue samples adhered to an elastomer substrate. In-plane radial displacements acquired during drying are azimuthally averaged and fitted with a profile based on a linear elastic contractility model. Dynamic changes in drying stress and SC elastic modulus can then be extracted from the fitted model profiles.

The role of RNase 7 in innate cutaneous defense against Pseudomonas aeruginosa.

The ribonuclease RNase 7 is a major skin-derived human antimicrobial protein expressed in keratinocytes. Here we show that the gram-negative pathogen Pseudomonas aeruginosa secretes factor(s) that induced RNase 7 gene and protein expression in human primary keratinocytes. The metalloprotease inhibitor marimastat, the epidermal growth factor receptor (EGFR) inhibitor AG1478 and the EGFR blocking antibody cetuximab significantly attenuated this induction, indicating an important role of the EGFR for the P.aeruginosa-mediated RNase 7 induction. In line with this, siRNA-mediated downregulation of ADAM17, a metalloprotease known to proteolytically mediate the release of soluble EGFR ligands, decreased the P.aeruginosa-mediated RNase 7 induction in keratinocytes. The impact of the EGFR was also demonstrated in a human 3D skin equivalent where blockade of the EGFR diminished induction of RNase 7 by P.aeruginosa. Blockade of Toll-like receptor 5 (TLR5), a pattern recognition receptor (PRR) known to be activated by P.aeruginosa, only moderately reduced the P.aeruginosa-mediated RNase 7 induction in keratinocytes. The functional relevance of RNase 7 to participate in cutaneous defense against P.aeruginosa was demonstrated by antibodies that neutralized the antimicrobial activity of RNase 7. These antibodies significantly inhibited the capacity of human stratum corneum skin extracts to control growth of P.aeruginosa. Taken together, our results indicate that P.aeruginosa induces the expression of RNase 7 in keratinocytes in an EGFR-dependent manner. Enhanced release of RNase 7 contributes to control cutaneous growth of P.aeruginosa.

Modulation mechanism of the stratum corneum structure during permeation of surfactant-based rigid and elastic vesicles

We evaluated the interaction between human stratum corneum (SC) and surfactant-based rigid or elastic vesicles during permeation using synchrotron X-ray diffraction to obtain the mechanism action of surfactant-based vesicles for enhanced skin permeation. The effects of vesicle elasticity on the interaction with SC were also investigated. Changes in the small-angle X-ray diffraction peaks of the human SC after buffer control and vesicle application were monitored. In the small-angle region, the control as the citrate buffer induced no significant changes of diffraction peaks for the lamellar structure of short periodicity phase (SPP), which is observed as a main peak in the human SC. Application of rigid vesicles resulted in small changes in the diffraction peaks attributed to lamellar phase of SPP. After application of elastic vesicles, however, a large shift to smaller angles due to swelling of the lamellar phase of SPP was clearly observed from the intensity difference profiles. All peaks due to the vesicles were still observed after 2h of application for all formulations, indicating that both vesicles interacted with the SC while maintaining their structures. These results strongly suggest that vesicles affect the lamellar phase of SPP of the intercellular lipids in the SC during permeation.

The rational design of biomimetic skin barrier lipid formulations using biophysical methods.

The focus of this communication was to study phospholipid-structured emulsions whose phase behaviour is modified with monoalkyl fatty amphiphiles. Ideally, these systems would mimic key physical and structural attributes observed in human stratum corneum (SC) so that they better alleviate xerotic skin conditions. Phosphatidylcholine-structured emulsions were prepared, and their phase behaviour modified with monoalkyl fatty amphiphiles. The effect of molecular volume, acyl chain length and head-group interactions was studied using a combination of physical methods. Water vapour transmission rate (WVTR) was used as a primary test to assess occlusive character. Changes in the vibrational modes observed in Fourier transform infrared (FTIR) spectroscopy and bilayer spacing measured by X-ray diffraction (XRD) were then applied to elucidate the lateral and lamellar microstructural characteristics in the systems. Water vapour transmission rate demonstrated that as the phosphatidylcholine acyl chain length increased from C14, to C18, to C22, there was a corresponding increase in occlusive character. The addition of monoalkyl fatty amphiphiles such as behenic acid, behenyl alcohol or cetostearyl alcohol to a base formulation incorporating dipalmitoyl and distearoylphosphatidylcholine (C18) was seen to further increase barrier characteristics of the emulsions. FTIR methods used to probe lipid-chain conformational ordering demonstrated that as phosphatidylcholine acyl chain lengths increased, there was a corresponding improvement in acyl chain ordering, with an increase in thermal transition temperatures. The addition of a monoalkyl fatty amphiphile resulted in conformational order and thermal transition temperature improvements trending towards those observed in stratum corneum. FTIR also demonstrated that systems containing behenic acid or behenyl alcohol exhibited features associated with orthorhombic character. X-ray diffraction data showed that addition of monoalkyl fatty amphiphile also resulted in thicker lamellar structures than when those agents are not present. The generalized approach described herein is shown to mechanistically describe the occlusive character of phospholipid-structured formulations in the presence of long-chain fatty acids or alcohols and that they exhibit characteristics mimicking those found in human SC lipids.

Cell surface glycans in the human stratum corneum: distributionand depth-related changes.

During the formation of the stratum corneum (SC) barrier, the extracellular spaces of viable epidermis, rich in glycans, are filled with a highly organized lipid matrix and the plasma membranes of keratinocytes are replaced by cornified lipid envelopes. These structures comprise cross-linked proteins, including transmembrane glycoproteins and proteoglycans, covalently bound to a monolayer of cell surface ceramides. Little is known about the presence and distribution of glycans on the SC corneocytes despite their possible involvement in SC hydration, cohesion and desquamation. In this work, we visualized ultrastructurally and quantified the distribution of glycans on the surface of native and delipidated corneocytes. The cells were harvested at different depths of the SC, allowing us to define the relationship between the distribution of various glycans, proteoglycans and glycoproteins, and other changes occurring in SC. At the cell periphery, we found a correlation between the depth-related alterations of corneodesmosome glycoproteins and α-d-mannosyl and N-acetyl-d-glucosamine-labelling patterns. Elimination of the terminal sugars, α-linked fucose and α-(2,3) linked sialic acid, was less abrupt, but also the initial extent of their peripheral distribution was overall lower than that of concanavalin A and wheat germ agglutinin lectin-detected glycans. Diffuse labelling of heparan sulphate glycosaminoglycans disappeared completely from the outermost corneocytes, whereas that of several simple carbohydrates could be detected at all SC levels. Our results suggest that specific glycan distribution may participate in the progressive changes of SC, as it evolves from the SC compactum to the SC disjunctum, towards desquamation.

In vivo intra- and inter-individual variability study of human stratum corneum by confocal Raman spectroscopy

The present research work deals with the in vivo determination of intra- and inter-individual human stratum corneum biochemical variability using confocal Raman spectroscopy. This study was carried out at three different spots on the volar forearm region of healthy human participants in the extended fingerprint and high wavenumber spectral regions. All the preprocessed spectra were compared by correlation matrices, which were calculated using Pearson's correlation coefficient (r). The results indicate that the participated volunteers in this study are homogenous with respect to the stratum corneum in the forearm region in both extended fingerprint and high wavenumber regions as there is high correlation between their Raman spectra at both intra- and inter-individual levels. This study confirms the application of confocal Raman spectroscopy as an accurate and precise technique for in vivo studies involving human skin.

A seasonal change of bleomycin hydrolase activity in the extract of human stratum corneum

A seasonal change of bleomycin hydrolase activity in the extract of human stratum corneum

Dynamics of glycerine and water transport across human skin from binary mixtures.

Skin transport properties of glycerine and water from binary mixtures contacting human skin were determined to better understand the mechanism of skin moisturization by aqueous glycerine formulations. Steady-state permeation for 3 H2 O and 14 C-glycerine across split-thickness human skin in vitro and desorption dynamics of the same permeants in isolated human stratum corneum (HSC) were experimentally determined under near equilibrium conditions. These data were compared to a priori values developed in the context of a thermodynamic model for binary mixtures of glycerine and water and a previously determined water sorption isotherm for HSC. This allowed the estimation of diffusion and partition coefficients for each permeant in the HSC, as well as HSC thickness, as a function of composition of the contacting solution. These data may be used to estimate water retention and associated HSC swelling related to the absorption and slow release of glycerine from the skin. It took 6+ days for glycerine to completely desorb from HSC immersed in glycerine/water binary solutions. Desorption of both 3 H2 O and 14 C-glycerine from HSC was slower in pure water than from binary mixtures, a result that is largely explained by the greater swelling of HSC in water. Parametric relationships were developed for water and glycerine intradiffusivities in HSC as functions of HSC water content, and a mutual diffusion coefficient was estimated by analogy with glycerine/water binary solutions. The intradiffusivity of 14 C-glycerine in HSC as inferred from sorption/desorption experiments was shown to be approximately 10-fold less than that inferred from permeation experiments, whereas the corresponding values for 3 H2 O were comparable. These studies confirm that glycerine enters HSC in substantial quantities and has a long residence time therein. The coupling between bulk water and glycerine transport projected from binary solution data suggests the net effect of glycerine is to slow water loss from the skin. The data support the concept of glycerine as a humectant with an excellent balance of skin penetration and retention characteristics; however, they do not rule out the possibility of an additional biological effect on skin barrier homoeostasis.

Characterization of Water and a Model Lipophilic Compound in Human Stratum Corneum by NMR Spectroscopy and Equilibrium Sorption

The stratum corneum (SC) is the outermost skin layer in humans and other mammals and the primary barrier to water loss and environmental exposure to chemicals and microorganisms. It consists of flattened, keratin-filled corneocytes surrounded by well-organized lipid layers. Human SC at varying degrees of hydration with and without addition of a model lipophilic compound, 2-(trifluoromethyl) benzonitrile (TFMB), was studied using proton (1H) and fluorine (19F) nuclear magnetic resonance techniques. Proton spectral analyses revealed that water mainly occupies the corneocytes in agreement with prior studies. Observations from 19F spectral and spin-lattice relaxation time (T1) analyses showed that TFMB is primarily present in the lipids with small amounts in water, which is located within the corneocytes. This is consistent with TFMB sorption, which was measured in SC with and without lipid extraction. The presence of TFMB within the corneocytes supports the hypothesis that transcellular diffusion of a lipophilic compound like TFMB may contribute to SC permeation.

Mechanistic Evaluation of Hydration Effects on the Human Epidermal Permeation of Salicylate Esters.

We sought to understand when and how hydration enhances the percutaneous absorption of salicylate esters. Human epidermal membrane fluxes and stratum corneum solubilities of neat and diluted solutions of three esters were determined under hydrated and dehydrated conditions. Hydration doubled the human epidermal flux seen for methyl and ethyl salicylate under dehydrated conditions and increased the flux of neat glycol salicylate 10-fold. Mechanistic analyses showed that this hydration-induced enhancement arises mainly from an increase in the stratum corneum diffusivity of the three esters. Further, we showed that unlike methyl and ethyl salicylate, glycol salicylate is hygroscopic and the ∼10-fold hydration-induced flux enhancement seen with neat glycol salicylate may be due to its ability to hydrate the stratum corneum to a greater extent. The hydration-induced enhancements in in vitro epidermal flux seen here for glycol and ethyl salicylate were similar to those reported for their percutaneous absorption rates in a comparable in vivo study, whilst somewhat higher enhancement was seen for methyl salicylate in vivo. This may be explained by a physiologically induced self enhancement of neat methyl salicylate absorption in vivo which is not applicable in vitro.

A Coarse-Grained Model of Stratum Corneum Lipids: Free Fatty Acids and Ceramide NS.

Ceramide (CER)-based biological membranes are used both experimentally and in simulations as simplified model systems of the skin barrier. Molecular dynamics studies have generally focused on simulating preassembled structures using atomistically detailed models of CERs, which limit the system sizes and time scales that can practically be probed, rendering them ineffective for studying particular phenomena, including self-assembly into bilayer and lamellar superstructures. Here, we report on the development of a coarse-grained (CG) model for CER NS, the most abundant CER in human stratum corneum. Multistate iterative Boltzmann inversion is used to derive the intermolecular pair potentials, resulting in a force field that is applicable over a range of state points and suitable for studying ceramide self-assembly. The chosen CG mapping, which includes explicit interaction sites for hydroxyl groups, captures the directional nature of hydrogen bonding and allows for accurate predictions of several key structural properties of CER NS bilayers. Simulated wetting experiments allow the hydrophobicity of CG beads to be accurately tuned to match atomistic wetting behavior, which affects the whole system, since inaccurate hydrophobic character is found to unphysically alter the lipid packing in hydrated lamellar states. We find that CER NS can self-assemble into multilamellar structures, enabling the study of lipid systems more representative of the multilamellar lipid structures present in the skin barrier. The coarse-grained force field derived herein represents an important step in using molecular dynamics to study the human skin barrier, which gives a resolution not available through experiment alone.

Proteomic profiling reveals candidate markers for arsenic-induced skin keratosis

Proteomics technology is an attractive biomarker candidate discovery tool that can be applied to study large sets of biological molecules. To identify novel biomarkers and molecular targets in arsenic-induced skin lesions, we have determined the protein profile of arsenic-affected human epidermal stratum corneum by shotgun proteomics. Samples of palm and foot sole from healthy subjects were analyzed, demonstrating similar protein patterns in palm and sole. Samples were collected from the palms of subjects with arsenic keratosis (lesional and adjacent non-lesional samples) and arsenic-exposed subjects without lesions (normal). Samples from non-exposed healthy individuals served as controls. We found that three proteins in arsenic-exposed lesional epidermis were consistently distinguishably expressed from the unaffected epidermis. One of these proteins, the cadherin-like transmembrane glycoprotein, desmoglein 1 (DSG1) was suppressed. Down-regulation of DSG1 may lead to reduced cell-cell adhesion, resulting in abnormal epidermal differentiation. The expression of keratin 6c (KRT6C) and fatty acid binding protein 5 (FABP5) were significantly increased. FABP5 is an intracellular lipid chaperone that plays an essential role in fatty acid metabolism in human skin. This raises a possibility that overexpression of FABP5 may affect the proliferation or differentiation of keratinocytes by altering lipid metabolism. KRT6C is a constituent of the cytoskeleton that maintains epidermal integrity and cohesion. Abnormal expression of KRT6C may affect its structural role in the epidermis. Our findings suggest an important approach for future studies of arsenic-mediated toxicity and skin cancer, where certain proteins may represent useful biomarkers of early diagnoses in high-risk populations and hopefully new treatment targets. Further studies are required to understand the biological role of these markers in skin pathogenesis from arsenic exposure.