Human Stratum Corneum Research Articles

070 Depth -related distribution of cell surface glycans in normal and pathological human stratum corneum

070 Depth -related distribution of cell surface glycans in normal and pathological human stratum corneum

Profile and quantification of human stratum corneum ceramides by normal-phase liquid chromatography coupled with dynamic multiple reaction monitoring of mass spectrometry: development of targeted lipidomic method and application to human stratum corneum of different age groups.

Skin, the largest organ of the human body, serves as the primary barrier to the external environment. Ceramides are one of the main constituents of stratum corneum (SC), playing an important role in skin barrier function. Therefore, comprehensive profiling and quantification of SC ceramide is important. Herein, a new targeted lipidomic method for human SC ceramide profiling and quantification is presented and tested. Normal-phase high-performance liquid chromatography coupled with dynamic multiple reaction monitoring mass spectrometry (NP-HPLC-dMRM-MS) was used to separate ceramides into subclasses and then characterize different ceramides within each subclass on the basis of their characteristics. In total, 483 ceramides were quantified in a single run within 20min, covering 12 subclasses as well as some glycosylated ceramides not previously reported. Each subclass had typical standard substances (if available) that served to establish representative standard curves and were used for related substances with no standards. Linearity range, limit of quantification (LOQ), limit of detection (LOD), precision, accuracy, stability, and matrix effects were validated. dMRM increased sensitivity and accuracy greatly compared with common MRM (cMRM). This method was successfully applied to the study of human SC from different age groups. A total of 193 potential biomarkers were found to indicate age differences between children and adults. This method is an innovative approach for high-throughput quantification of SC ceramide. Graphical Abstract Method establishment (MRM spectra by the established method) and method application (score scatter plots of authentic samples).

LB787 The effect of ethanol on phospholipase A2, lipase, β-glucocerebrosidase and sphingomyelinase activities in the human stratum corneum

LB787 The effect of ethanol on phospholipase A2, lipase, β-glucocerebrosidase and sphingomyelinase activities in the human stratum corneum

Vehicle effects on human stratum corneum absorption and skin penetration.

This study evaluated the effects of three vehicles-ethanol (EtOH), isopropyl alcohol (IPA), and isopropyl myristate (IPM)-on stratum corneum (SC) absorption and diffusion of the [14C]-model compounds benzoic acid and butenafine hydrochloride to better understand the transport pathways of chemicals passing through and resident in SC. Following application of topical formulations to human dermatomed skin for 30 min, penetration flux was observed for 24 h post dosing, using an in vitro flow-through skin diffusion system. Skin absorption and penetration was compared to the chemical-SC (intact, delipidized, or SC lipid film) binding levels. A significant vehicle effect was observed for chemical skin penetration and SC absorption. IPA resulted in the greatest levels of intact SC/SC lipid absorption, skin penetration, and total skin absorption/penetration of benzoic acid, followed by IPM and EtOH, respectively. For intact SC absorption and total skin absorption/penetration of butenafine, the vehicle that demonstrated the highest level of sorption/penetration was EtOH, followed by IPA and IPM, respectively. The percent doses of butenafine that were absorbed in SC lipid film and penetrated through skin in 24 h were greatest for IPA, followed by EtOH and IPM, respectively. The vehicle effect was consistent between intact SC absorption and total chemical skin absorption and penetration, as well as SC lipid absorption and chemical penetration through skin, suggesting intercellular transport as a main pathway of skin penetration for model chemicals. These results suggest the potential to predict vehicle effects on skin permeability with simple SC absorption assays. As decontamination was applied 30 min after chemical exposure, significant vehicle effects on chemical SC partitioning and percutaneous penetration also suggest that skin decontamination efficiency is vehicle dependent, and an effective decontamination method should act on chemical solutes in the lipid domain.

The global mechanical properties and multi-scale failure mechanics of heterogeneous human stratum corneum.

The outermost layer of skin, or stratum corneum, regulates water loss and protects underlying living tissue from environmental pathogens and insults. With cracking, chapping or the formation of exudative lesions, this functionality is lost. While stratum corneum exhibits well defined global mechanical properties, macroscopic mechanical testing techniques used to measure them ignore the structural heterogeneity of the tissue and cannot provide any mechanistic insight into tissue fracture. As such, a mechanistic understanding of failure in this soft tissue is lacking. This insight is critical to predicting fracture risk associated with age or disease. In this study, we first quantify previously unreported global mechanical properties of isolated stratum corneum including the Poisson's ratio and mechanical toughness. African American breast stratum corneum is used for all assessments. We show these parameters are highly dependent on the ambient humidity to which samples are equilibrated. A multi-scale investigation assessing the influence of structural heterogeneities on the microscale nucleation and propagation of cracks is then performed. At the mesoscale, spatially resolved equivalent strain fields within uniaxially stretched stratum corneum samples exhibit a striking heterogeneity, with localized peaks correlating closely with crack nucleation sites. Subsequent crack propagation pathways follow inherent topographical features in the tissue and lengthen with increased tissue hydration. At the microscale, intact corneocytes and polygonal shaped voids at crack interfaces highlight that cracks propagate in superficial cell layers primarily along intercellular junctions. Cellular fracture does occur however, but is uncommon. Human stratum corneum protects the body against harmful environmental pathogens and insults. Upon mechanical failure, this barrier function is lost. Previous studies characterizing the mechanics of stratum corneum have used macroscopic testing equipment designed for homogenous materials. Such measurements ignore the tissue's rich topography and heterogeneous structure, and cannot describe the underlying mechanistic process of tissue failure. For the first time, we establish a mechanistic insight into the failure mechanics of soft heterogeneous tissues by investigating how cracks nucleate and propagate in stratum corneum. We further quantify previously unreported values of the tissue's Poisson's ratio and toughness, and their dramatic variation with ambient humidity. To date, skin models examining drug delivery, wound healing, and ageing continue to estimate these parameters.

Fractional derivatives in the transport of drugs across biological materials and human skin

Abstract The diffusion of drugs across a composite structure such as a biological membrane is a rather complex phenomenon, because of its inhomogeneous nature, yielding a diffusion rate and a drug solubility strongly dependent on the local position across the membrane itself. These problems are particularly strengthened in composite structures of a considerable thickness like, for example, the human skin, where the high heterogeneity provokes the transport through different simultaneous pathways. In this note, we propose a generalization of the diffusion model based on Fick’s 2nd equation by substituting a diffusion constant by means of the memory formalism approach (diffusion with memory). In particular, we employ two different definitions of the fractional derivative, i.e., the usual Caputo fractional derivative and a new definition recently proposed by Caputo and Fabrizio. The model predictions have been compared to experimental results concerning the permeation of two different compounds through human skin in vivo , such as piroxicam, an anti-inflammatory drug, and 4-cyanophenol, a test chemical model compound. Moreover, we have also considered water penetration across human stratum corneum and the diffusion of an antiviral agent employed as model drugs across the skin of male hairless rats. In all cases, a satisfactory good agreement based on the diffusion with memory has been found. However, the model based on the new definition of fractional derivative gives a better description of the experimental data, on the basis of the residuals analysis. The use of the new definition widens the applicability of the fractional derivative to diffusion processes in highly heterogeneous systems.

A prodrug approach to enhance azelaic acid percutaneous availability

Azelaic acid is a dicarboxylic acid compound used in treatment of acne vulgaris. However, high concentration (ca 20%) is needed to guarantee the drug availability in the skin. The latter increases the incidence of side effects such as local irritation. The prodrug strategy to enhance azelaic acid diffusion through skin was not reported before. Thus, a lipophilic prodrug of azelaic acid (diethyl azelate [DEA]) was synthesized and investigated to improve percutaneous availability of azelaic acid, with a subsequent full physical, chemical, and biological characterization. Expectedly, DEA exhibited a significant increase in diffusion compared to azelaic acid through silicone membrane. In contrast, the diffusion results through human stratum corneum (SC) displayed weaker permeation for DEA with expected retention in the SC. Therefore, a desorption study of DEA from SC was conducted to examine the reservoir behavior in SC. Results showed an evidence of sustained release behavior of DEA from SC. Consequently, enhancement of keratolytic effect is expected due to azelaic acid produced from enzymatic conversion of DEA released from SC.

Permeation of topically applied Magnesium ions through human skin is facilitated by hair follicles.

Magnesium is an important micronutrient essential for various biological processes and its deficiency has been linked to several inflammatory disorders in humans. Topical magnesium delivery is one of the oldest forms of therapy for skin diseases, for example Dead Sea therapy and Epsom salt baths. Some anecdotal evidence and a few published reports have attributed amelioration of inflammatory skin conditions to the topical application of magnesium. On the other hand, transport of magnesium ions across the protective barrier of skin, the stratum corneum, is contentious. Our primary aim in this study was to estimate the extent of magnesium ion permeation through human skin and the role of hair follicles in facilitating the permeation. Upon topical application of magnesium solution, we found that magnesium penetrates through human stratum corneum and it depends on concentration and time of exposure. We also found that hair follicles make a significant contribution to magnesium penetration.

Age-Related Changes to Human Stratum Corneum Lipids Detected Using Time-of-Flight Secondary Ion Mass Spectrometry Following in Vivo Sampling.

This work demonstrates the ability to detect changes in both quantity and spatial distribution of human stratum corneum (SC) lipids from samples collected in vivo. The SC functions as the predominant barrier to the body, protecting against the penetration of xenobiotic substances. Changes to the SC lipid composition have been associated with barrier impairment and consequent skin disorders, and it is therefore important to monitor and quantify changes to this structure. This work demonstrates the first reported use of time-of-flight secondary ion mass spectrometry (ToF-SIMS) to assess physiological changes to human SC as a function of depth. This technique provides exceptional sensitivity and chemical specificity, allowing analysis of single tape stripped samples taken from volunteers. Using this methodology we were able to successfully identify chemical differences in human SC resulting from both intrinsic and extrinsic (photo) aging. Samples were collected from women of two age groups (under 27 and postmenopausal) and from two body sites with varying UV exposure (inner forearm and dorsal hand), and differences were identified using multivariate data analysis. The key finding was the significant aged-related increase and change in spatial distribution of the sterol cholesterol sulfate, a membrane stabilizing lipid. Significant changes in the prevalence of both lignoceric acid (C24:0) and hexacosanoic acid (C26:0) were also observed. This work describes previously unreported age-related chemical changes to human SC, providing an insight into aging mechanisms which may improve the design of both pharmaceutical and cosmetic topical products.

On the Variation of Water Diffusion Coefficient in Stratum Corneum With Water Content

Water permeability and transient water sorption data in human and porcine stratum corneum (SC) are analyzed in conjunction with equilibrium water sorption data and a dynamic skin swelling model to develop a quantitative model for water diffusivity in the SC as a function of its water content. The recommended function (model 1) is phenomenological and treats the SC as a uniform, swellable slab. This approach yields satisfactory agreement with experimental data over a wide range of RH and associated equilibrium SC water content, Cw. It is supported by two alternative approaches. Model 2 considers the SC to be a multilaminate membrane consisting of alternating lipid and protein layers. Diffusivity in the protein phase is estimated from water diffusivity in other keratinized tissues, whereas diffusivity in the lipid phase is assumed to be linearly related to the swelling strain on intercellular lipids. Model 3 uses an analysis previously suggested by Stockdale to rationalize transepidermal water loss data in humans over a wide range of relative humidity. All models yield similar results for 0.20 ≤ Cw ≤ 0.78 g/cm3, the usual range of SC water content in vivo.

Quantification of Multi-Scale Mechanics and Failure of Human Stratum Corneum

Human stratum corneum (SC) acts as an essential mechanical barrier to the inhospitable external environment. Maintaining skin barrier function by preventing rupture is of fundamental importance to healthcare. Cracking, chapping and the formation of exudative lesions all result from a mechanical failure of SC tissue. At macroscopic length scales, SC exhibits well defined mechanical properties. These properties, such as the elastic modulus, are strongly influenced by SC water content. Using a combination of uniaxial tensometry and macrophotography, we quantify variations in the Poisson's ratio and toughness of SC as a function of ambient humidity. These parameters are essential for accurate modeling of skin mechanics and have not previously been reported. Macroscopic measurements of SC mechanical properties however assume the tissue is homogeneous in composition and cannot describe the mechanisms of failure at the microscale. SC exhibits a rich topography across many length scales and large sample to sample variations in toughness suggest the microstructure of SC is likely to influence the onset of failure rather than the physical limits of the material. We demonstrate that structural heterogeneities cause the formation of heterogeneous micro-strains within uniaxially stressed tissue, with peak strains coinciding with sites of crack nucleation. We further study the influence of structural heterogeneities and SC water content on the failure mechanics of the SC. Crack propagation timescales, strain energy release rates and crack morphologies vary dramatically with tissue hydration. Examinations of crack interfaces at different length scales further illustrate that while topographical canyons and follicular holes influence cracking to a small degree, failure of the SC is governed by rupture of intercellular corneodesmosome plaques, leaving the structural integrity of corneocyte cells intact. These results provide key biophysical insights into the mechanics of fracture in soft heterogeneous tissues.

Lateral Phase Behavior of Human Skin Lipids

The human stratum corneum (SC) is the outermost layer of the epidermis, the first barrier against the environment. This largely impenetrable layer consists of anucleated, flattened, protein-rich cells called corneocytes suspended in an extracellular lipid matrix. This lipid matrix is composed of ceramides (Cers), cholesterol (Chol), and free fatty acids (FFAs). The SC lipids are unique from other biologically relevant lipids commonly found in cell membranes in that the Cers and FFAs have very long (>20 carbons) fatty acid chains, nearly all of which are fully saturated. Using surface pressure vs. molecular area isotherms and fluorescence microscopy, our research investigates the lateral packing and domain formation within Langmuir monolayers of the individual Cer, Chol, and FFA components and their binary and ternary mixtures to determine the role of each component in the material properties of the SC layer. Particular attention is paid to the effect of FFA length on membrane fluidity – FFA chains ranging from 16 to 30 carbons (24 carbons being the most common in the SC) were studied. We show that longer FFAs have a condensing effect in Cer monolayers, while Chol tends to disrupt this Cer-FFA organization. We also demonstrate the differences between the canonical 1:1:1 Cer:Chol:FFA mixture and more physiologically accurate mixtures in which Cers and Chol are in higher concentrations and several FFAs of different chain length are present.

Two-Photon Excitation STED-FCS with Far-Red Dyes in Tissue - Measuring Diffusion in Stratum Corneum

Understanding the diffusion and transport of molecules and particles through the human skin is important, both in terms of transdermal drug delivery but also to understand the possible risk of new chemicals and nano particles. Several models have been proposed for diffusion of various substances through the stratum corneum, which contains the barrier properties of skin, but they are based on macroscopic measurements and gene knock-outs. However there is a need for a thorough understanding of the micro- and nanoscopic properties of stratum corneum that governs the diffusion of chemicals through it. STED-FCS have shown great promises with respect to quantifying diffusion and distinguishes different diffusion models.In this study we have used FCS and STED-FCS to measure the diffusion of hydrophobic fluorescence dyes in human stratum corneum and in supported lipid bilayers made from extracted skin lipids. The results have been compared in terms of understanding the diffusion pathways through the lipids layers in stratum corneum.

The solution structure of the kallikrein-related peptidases inhibitor SPINK6

Kallikrein-related peptidases (KLKs) are crucial for epidermal barrier function and are involved in the proteolytic regulation of the desquamation process. Elevated KLK levels were reported in atopic dermatitis. In skin, the proteolytic activity of KLKs is regulated by specific inhibitors of the serine protease inhibitor of Kazal-type (SPINK) family. SPINK6 was shown to be expressed in human stratum corneum and is able to inhibit several KLKs such as KLK4, -5, -12, -13 and -14. In order to understand the structural traits of the specific inhibition we solved the structure of SPINK6 in solution by NMR-spectroscopy and studied its interaction with KLKs. Thereby, beside the conserved binding mode, we identified an alternate binding mode which has so far not been observed for SPINK inhibitors.

Skin penetration and antioxidant effect of cosmeto-textiles with gallic acid

In this work, the antioxidant gallic acid (GA) has been encapsulated in microspheres prepared with poly-ε-caprolactone (PCL) and incorporated into polyamide (PA) obtaining the cosmeto-textile. The topical application of the cosmeto-textile provides a reservoir effect in the skin delivery of GA. The close contact of the cosmeto-textile, containing microsphere-encapsulated GA (ME-GA), with the skin and their corresponding occlusion, may be the main reasons that explain the crossing of active principle (GA) through the skin barrier, located in the stratum corneum, and its penetration into the different compartments of the skin, epidermis and dermis.An ex vivo assessment was performed to evaluate the antioxidant effect of the ME-GA on the stratum corneum (SC) using the thiobarbituric acid-reactive species (TBARS) test. The test is based on a non-invasive ex vivo methodology that evaluates lipid peroxides formed in the outermost layers of the SC from human volunteers after UV radiation to determine the effectiveness of an antioxidant. In this case, a ME-GA cosmeto-textile or ME-GA formulation were applied to the skin in vivo and lipid peroxidation (LPO) in the horny layer were determined after UV irradiation. This methodology may be used as a quality control tool to determine ex vivo the percentage of LPO inhibition on human SC for a variety of antioxidants that are topically applied, in this case GA.Results show that LPO formation was inhibited in human SC when GA was applied directly or embedded in the cosmeto-textile, demonstrating the effectiveness of both applications. The percentage of LPO inhibition obtained after both topical applications was approximately 10% for the cosmeto-textile and 41% for the direct application of microspheres containing GA. This methodology could be used to determine the effectiveness of topically applied antioxidants encapsulated in cosmeto-textiles on human SC.

界面活性剤処理によるヒト皮膚角層の構造変化の小角・広角X線散乱法を用いた解析（第5報）

界面活性剤処理によるヒト皮膚角層の構造変化の小角・広角X線散乱法を用いた解析（第5報）

Depth profiles of hydrogen bound water molecule types and their relation to lipid and protein interaction in the human stratum corneum in vivo.

Confocal Raman microscopy has been used to measure depth-dependent profiles of human SC in vivo in the high wavenumber (HWN) region. In order to keep the linearity of HWN region boundaries and to not remove an informative signal from Raman spectra, a new baseline subtraction procedure has been introduced. After baseline subtraction, the HWN spectrum was deconvoluted using 10 Gaussian functions with individual chemical meanings. The results show that the hydrogen bound water molecule types contributed differently to the water diffusion process in the SC. The most concentrated double donor-double acceptor (DDAA) and single donor-single acceptor (DA) water molecule types in the SC represent more than 90% of the SC's water and mostly contribute to the water flux in the skin. Single donor-double acceptor (DAA) and weakly-bound water molecule types represent less than 10% of the SC's water content. The most tightly hydrogen bound water molecule type, DAA, reaches its maximum concentration near the skin surface and does not take part in the water diffusion process via the SC. The results show that the hydrogen bonding state of water (DA/DDAA water molecule type ratio) reaches its maximum at the depth of approx. 30% of the SC thickness, which correlates well with the maximum lateral packing order of intercellular lipids (ICL) and the natural moisturizing factor (NMF), and does not coincide with the folding/unfolding state of keratin. The NMF's contribution to the bonding of water in the SC is supposed to dominate over that of ICL.

A depth-dependent profile of the lipid conformation and lateral packing order of the stratum corneum in vivo measured using Raman microscopy.

The intercellular lipid structure of the stratum corneum (SC) plays a key role in skin barrier function. A depth profile of the intercellular lipid conformation and the lipid lateral packing order were measured in vivo in the human SC using confocal Raman microscopy. The depth profiles of the 2880 cm(-1)/2850 cm(-1) peak ratio intensity, which represent the C-H stretching and lateral packing order of lipids, and the 1080 cm(-1)/(1130 cm(-1) + 1060 cm(-1)) peak ratio, which represents the C-C skeleton vibration and trans-gauche conformation order of lipids, were investigated. The influence of keratin on the lipid peaks at 2850 cm(-1) and 2880 cm(-1) was excluded by the developed mathematical algorithm. The results show that the trans-conformation and lateral packing order of the intercellular lipids reach their maximum value in the SC at 20-40% of its depth and then decrease towards the stratum granulosum. These results show that at a depth of 20-40% (normally corresponding to a depth of 4-8 μm) the SC exhibits the most ordered lipids and therefore the highest skin barrier function. The lateral packing of lipids is more disordered on the surface and in the deeper parts of the SC, which may be associated with a reduced skin barrier function.

Plasma Induced Changes in Human Lipid Composition as Revealed through XPS-Analysis

In this study two plasma sources were used for an in vivo treatment of human stratum corneum. The sample preparation was realised with the Cyanoacrylat stripping method, whereby a few layers of corneocytes embedded in the lipid matrix were removed from the skin of healthy volunteers. For the plasma treatment, dielectric barrier discharges with pulse durations in the microsecond as well as in the nanosecond range were applied. A comparison of these sources with respect to their biologically active components including dissipated power, gas and electron temperature, irradiance in the ultraviolet range, ozone and nitric oxide concentration is presented. Furthermore, species generated during plasma treatment on the sample surface like hydrogen peroxide, nitride or nitrate were measured using reflectometry. In addition, safety aspects for both sources were evaluated. Resulting plasma induced changes in the sample composition were investigated through X-ray photoelectron spectroscopy. The main ingredients carbon, oxygen, and nitrogen in addition to minor concentrations of sulphur were considered. A significant influence of the pulse duration on plasma characteristics was shown. A more effective formation of reactive species as well as more intense UV emission for ns-plasma was observed. Based on the determined parameters, both plasma sources are suitable for therapeutic purpose. Furthermore, significant plasma induced changes in the stratum corneum composition were reported, including an increase in nitrogen and oxygen content.

Study on the Structural Change of Human Stratum Corneum Induced by the Treatment of Surfactant Solutions Using Small- and Wide-Angle X-ray Scattering, Part 4

Study on the Structural Change of Human Stratum Corneum Induced by the Treatment of Surfactant Solutions Using Small- and Wide-Angle X-ray Scattering, Part 4