Pores in the epidermis: aquaporins and tight junctions

Water homeostasis of the epidermis is important for the appearance and physical properties of the skin, as well as for water balance in the body. It depends on several factors, e.g. barrier quality, uptake of water into the epidermis, concentration of water-retaining humectants, and external humidity. Aquaporins (AQPs) are pores in the plasmamembranes of cells. Monomeric AQPs form barrel-like structures that are primarily water selective, some AQPs also transport glycerol and possibly other small solutes. In the epidermis, AQP3 is the predominant AQP. It is localized mainly in basal but also in suprabasal layers of the epidermis and is permeable for water as well as for glycerol, a humectant. Mice deficient in AQP3 exhibit reduced stratum corneum (SC) hydration and impaired SC barrier recovery after SC removal. In skin diseases associated with elevated transepidermal water loss (TEWL) and reduced SC hydration, altered expression of AQP3 was shown. Tight junctions (TJ) are cell-cell junctions, which play a central role in sealing the intercellular space of cell sheets and thereby establishing a paracellular barrier. Within the TJ, pores are postulated to exist, which allow the controlled diffusion of water and solutes via the paracellular pathway. In the epidermis, TJ structures were demonstrated in the stratum granulosum whereas TJ proteins were found in all viable layers. Mice which overexpress or are deficient of key-proteins of TJ die soon after birth because of a tremendous TEWL. In various skin diseases that are accompanied by elevated TEWL and reduced skin hydration, staining patterns of TJ proteins are altered. This review will summarize our current knowledge of the involvement of AQPs and TJ in the water homeostasis of the epidermis.     

Original semiologic standardized evaluation of stratum corneum hydration by Diagnoskin stripping sample

In a normal and healthy skin, the regular elimination of the superficial corneocytes, called desquamation, is a fundamental physiologic process intended to protect the barrier function of the skin. This invisible loss of corneocytes, individually or in small groups, is incessantly compensated by the divisions of the proliferative layer and the upward cellular maturation in order to maintain the harmonious renewal of the epidermis and the integrity of the stratum corneum. The harmony of this desquamation process is intimately conditioned by a sufficient hydration of the stratum corneum: (i) an abnormal desquamation leads to a disruption of the water barrier function and consequently to a dehydration tendency of the stratum corneum, and (ii) a cutaneous dryness (whatever the cause) is able to disturb the desquamation process. Protecting the water content of the stratum corneum has always been a major preoccupation of the cosmetic industry scientists. Consequently, the moisturizing properties of a cosmetic product are objectively measured by various explorations directly targeted on the hydration (corneometry) and on the level of the water barrier function (transepidermal water loss (TEWL) measurements), which depends directly on the skin hydration state. This intimate linkage of the desquamation process and the water content of the stratum corneum enable us to suggest an indirect assessment of the hydration from a direct study of the desquamation by examining a skin-stripping sample (D-Squames) by an optical microscope (linked to a computer). We will describe this already known technique and mainly its new and unpublished semiologic exploitation, named Diagnoskin, whose advantages are its simplicity and its reproducibility particularly interesting in the case of sequential appraisal of dermatologic or cosmetic treatments.     

Bioengineering and subjective approaches to the clinical evaluation of dry skin

Dry skin (also known as xerosis) is a cutaneous reaction pattern indicative of abnormal desquamation, which has not only cosmetic considerations, but can also lead to the penetration of irritants and allergens through the stratum corneum (SC). Over the last few decades, our understanding of the structure, composition, formation and function of the SC has advanced tremendously; however, despite these advancements, the occurrence of dry skin remains prevalent in the adult population. The clinical evaluation of dry skin is therefore of significant importance to the cosmetic industry not only for understanding the condition but also for measuring the effects of treatment. Traditionally, dry skin has been evaluated by visual inspection, however, recently a variety of bioengineering techniques have emerged enabling the investigator to objectively assess the extent of xerotic conditions. The most frequently employed methods for the evaluation of dry skin are discussed in this review, including regression testing, squametry, measurement of transepidermal water loss, epidermal hydration, profilometry, confocal Raman spectroscopy, optical coherence tomography, in vivo confocal microscopy and magnetic resonance imaging.     

A new technique for the evaluation of cosmetics effect on mechanical properties of stratum corneum and epidermis in vitro

The mechanical properties of stratum corneum and epidermis have been measured in vitro and a dispersity of approximately 30% to 50% between samples from the same donor has been found. To overcome such a limitation, a technique was developed in order to compare the mechanical properties of the same skin sample before and after treatment. The chosen parameter (initial slope of the stress-strain curve) appeared to be influenced by the topical application of products. A different time response was found with stratum corneum and epidermis reflecting their structural differences.
Une nouvelle technique d'evaluation de l'effet des produits cosmétique sur les propriétés mécaniques du stratum corneum et de l'épiderme humains (etude in vitro )     

Abstracts: Natural moisturizing factors in the stratum corneum I. Effects of lipid extraction and soaking

pp. 13–22 Natural moisturizing factor (NMF) is essential for appropriate stratum corneum hydration, barrier homeostasis, desquamation, and plasticity. It is formed from filaggrin proteolysis to small, hygroscopic molecules including amino acids. We hypothesized that common lipid extraction and soaking in water would alter the level of NMF in the upper stratum corneum (SC) and its biophysical properties. A novel method of measuring and quantifying the amino acid components of NMF is presented. Adhesive tapes were used to collect samples of the SC and were extracted with 6 mM perchloric acid for analysis by reverse‐phase high‐performance liquid chromatography (HPLC).The HPLC results were standardized to the amount of protein removed by the tapes. An increase in NMF was found with increased SC depth. Also, the combination of extraction and soaking was found to increase NMF loss relative to control or to extraction or soaking alone. Our results indicate that common skin care practices significantly influence the water binding materials in the upper SC. The findings have implications for the evaluation and formulation of skin care products.     

Confocal Raman microspectroscopy of stratum corneum: a pre-clinical validation study

Skin moisturization is not only important for maintaining skin functional properties but also has great impact on the skin's aesthetic properties. The top layer of the skin, the stratum corneum (SC), plays a key role in protecting and preventing against external aggressions as well as in regulating water flux in and out. Confocal Raman microspectroscopy is the first commercially available technique that provides a non-invasive, in vivo method to determine depth profiles of water concentration in the skin, however, in this case it was applied in an in vitro setting. As the first phase of validating the usefulness of confocal Raman microspectroscopy, we used porcine skin as a surrogate for human skin. Water concentration profiles were obtained using confocal Raman microspectroscopy from isolated pigskin SC and compared with that using the Karl Fischer titration method. The two methods correlated very well with a regression coefficient of 1.07 as well as a correlation coefficient, R(2) = 0.989, which demonstrated the consistency and accuracy of confocal Raman microspectroscopy for water concentration determination. To evaluate the instrument's response to different skin care/cleansing products, a wide range of products were tested to compare their skin moisturization ability. Among those tested were a lotion, commercial soap bar, syndet bar, traditional non-emollient shower gel (water, Sodium Laureth Ether Sulfate (SLES), cocamidopropyl betaine system) and emollient containing shower gel (water, sunflower oil, SLES, cocamidopropyl betaine, glycerin, petrolatum). The results were consistent with what was expected. The water content on skin treated with (A) lotion was significantly higher than the non-treated control; (B) syndet bar-treated skin had a significantly higher water content than soap-based bar-treated sites; (C) non-emollient shower gel washed sites were more moisturized than soap-based bar-treated samples; and (D) emollient shower gel-treated skin was significantly more hydrated than non-emollient shower gel washed skin. The unique and direct quantitative water content information provided by confocal Raman microspectroscopy offers a whole new perspective for fundamental skin moisturization studies and will play an important role in evaluating moisturizing profiles and the hydration potential of products designed for personal care in the cosmetic industry.     

A review on the extensive skin benefits of mineral oil

This review was initially prepared in 2011 before Professor Johann Wiechers tragically passed away. It has been updated and is being published in his memory. It discusses the importance of mineral oil and its benefits to skin. Its source, structure, properties and efficacy are discussed. Mineral oil has been shown to improve skin softness and barrier function better than some other emollients using the gas‐bearing dynamometer and standard water vapour transmission testing as well as in vivo studies showing its effects on suppressing transepidermal water loss (TEWL). It has also been subjected to the rigour of the newer in vivo confocal microscopic measurements now used for testing the performance of moisturizers by following the swelling characteristics of the stratum corneum and been found favourable compared with many vegetable oils. Its introduction as a cosmetic oil was in the late 1800s, and still today, it is used as one of the main components of moisturizers, a true testament to its cost to efficacy window. Naturally, it has physical effects on the stratum corneum, but it is expected that these will translate into biological effects simply through its mechanism of hydrating and occluding the stratum corneum from which many benefits are derived.     

Peau sèche-rêche et "Hydratation". Concept de la capture de l'eau organisée comme de la glace

About sixty years ago Frank and Evans showed, by entropy measurements, that when a "non-polar molecule dissolves in water it modifies the water structure in the direction of greater 'cristallinity', the water builds a microscopic iceberg around it" Now, we propose the "concept of ice-like-water capture": a lowering of organized ice-like water promotes aggregation (loss of solubility) of the filaggrin/keratin1/keratin10 associations through their hydrophobic patches. The capture of ice-like water may be performed by the glucoceramides-rich bilayers in stratum granulosum. Probably, the same process aggregates the proteins of corneocytes envelope as well as corneodesmosomes proteins. According to the "concept of ice-like-water capture", to regulate the keratinization, it is not total water that must be added to the stratum corneum, but ice-like water that must be removed from stratum granulosum. Both petrolatum (lipophilic ingredient) and glycerol (hydrophilic ingredient) would capture the ice-like water, most probably after combination with the lipid bilayers of stratum corneum. Moisturizing cream, when organized in secondary droplets is likely to perform the same action. Measurements by near-infrared reflectance spectroscopy of the skin show that petrolatum; glycerol and/or moisturizing cream enhance the quantity of bulk water (1890-1897 nm band). As the ice-like water is the complement of bulk water, the enhanced bulk water let presume an ice-like water lessening. Some desynchronization (late or forward) of the keratinization/differentiation which confer the somatosensory problems associated with "dry and flaky skin" may be linked to an excess or lack of ice-like. For instance, the winter xerosis, very common by chilling weather, could be explained by an increase of ice-like water driven by the fall of the temperature.     

Age‐related changes in skin barrier function – Quantitative evaluation of 150 female subjects

The protection against water loss and the prevention of substances and bacteria penetrating into the body rank as the most important functions of the skin. This so‐called ‘skin barrier function’ is the natural frontier between the inner organism and the environment, and is primarily formed by the epidermis. An impairment of the skin barrier function is often found in diseased and damaged skin. An influence of ageing on skin barrier function is widely accepted, but has not been conclusively evaluated yet. Therefore, the aim of this clinical study was to assess the potential influence of ageing on skin barrier function, including transepidermal water loss (TEWL), stratum corneum hydration, sebum content and pH value. One hundred and fifty healthy women aged 18–80, divided into five age groups with 30 subjects each, were evaluated in this study. TEWL, hydration level, sebum secretion and pH value of hydro‐lipid acid film were measured with worldwide acknowledged biophysical measuring methods at cheek, neck, décolleté, volar forearm and dorsum of hand. Whereas TEWL and stratum corneum hydration showed only very low correlation with subject's age, the sebum production decreased significantly with age, resulting in the lowest skin surface lipids levels measured in subjects older than 70 years. The highest skin surface pH was measured in subjects between 50 and 60 years, whereas the eldest age group had the lowest mean pH. The dorsum of the hand was the location with the highest TEWL and lowest stratum corneum hydration in all age groups. The results show that only some parameters related to skin barrier function are influenced by ageing. Whereas sebum production decreases significantly over lifetime and skin surface pH is significantly increased in menopausal woman, TEWL and stratum corneum hydration show only minor variations with ageing.     

Stratum corneum keratin structure, function and formation - a comprehensive review

A comprehensive review on stratum corneum keratin organization, largely based on the recently published cubic rod-packing and membrane templating model [J. Invest. Dermatol., 123, 2004, 715], is presented. Keratin is the major non-aqueous component (wt/wt) of stratum corneum. As 90-100% of the stratum corneum water is thought to be located intracellularly one may presume that keratin also is a major factor (together with filaggrin-derived free amino acids) determining stratum corneum hydration level and water holding capacity. This water holding capacity depends in turn on the structural organization of the corneocyte keratin intermediate filament network. The cubic rod-packing model for the structure and function of the stratum corneum cell matrix postulates that corneocyte keratin filaments are arranged according to a cubic-like rod-packing symmetry. It is in accordance with the cryo-electron density pattern of the native corneocyte keratin matrix and could account for the swelling behaviour and the mechanical properties of mammalian stratum corneum. The membrane templating model for keratin dynamics and for the formation of the stratum corneum cell matrix postulates the presence in viable epidermal cellular space of a highly dynamic small lattice parameter (<30 nm) membrane structure with cubic-like symmetry, to which keratin is associated. It further proposes that membrane templating, rather than spontaneous self-assembly, is responsible for keratin intermediate filament formation and dynamics. It is in accordance with the cryo-electron density patterns of the native keratinocyte cytoplasmic space and could account for the characteristic features of the keratin network formation process, the dynamic properties of keratin intermediate filaments, the close lipid association of keratin, the insolubility in non-denaturating buffers and pronounced polymorphism of keratin assembled in vitro, and the measured reduction in cell-volume and hydration level between stratum granulosum and stratum corneum.     

Epidermal tight junction: The master skin barrier regulator

IFSCC Magazine , 12 (2009) (2) 87–91
Tight junctions between adjacent epithelial cells control paracellular permeability of solutes and maintain cell polarity by fencing lipid bilayer components into demarcated apical and basolateral domains. In the present study we investigated the role of epidermal tight junctions in two skin physiological processes, 'formation of the epidermal calcium ion (Ca²⁺) gradient' and 'polarized lamellar body secretion'. The former is based on the fact that the epidermal Ca2+ gradient is closely related to epidermal differentiation and the latter on the knowledge that polarized lamellar body secretion is essential to supply intercellular lipids to the stratum corneum. We hypothesized that tight junctions might form the Ca²⁺ gradient in the epidermis by sealing cell-cell contact at the stratum granulosum and allow the lamellar bodies to be normally secreted toward the stratum corneum by giving granular cells polarity. In our experiments we discovered that tight junctions control not only both intercellular Ca²⁺ permeability and fluorescent ceramide analog (Cer-FL) secretion in cultured normal human keratinocyte cells but also both intercellular calcium distribution and polarized lamellar body secretion in the skin equivalent. Thus, tight junctions should be responsible for linking a series of processes from epidermal differentiation to stratum corneum barrier formation. Our findings suggest that tight junctions should have a crucial role not only in epidermal barrier function but also in both epidermal differentiation and stratum corneum barrier function.
Keywords: Calcium gradient, cell polarity, lamellar body, skin barrier, tight junction
IFSCC Basic Research Award winning paper presented at the 25th IFSCC Congress 2008, Barcelona, Spain     

Chitin–glucan,a natural cell scaffold for skin moisturization and rejuvenation

Two clinical studies were conducted to evaluate the tolerance and effects of a copolymer of chitin and beta‐glucan, forming the exoskeleton of fungal cell walls, now supplied for cosmetic applications. A 6‐week randomized, double‐blind, placebo‐controlled study was conducted on 13 volunteers with sensitive skin to compare with 0.5–2% formulations chitin–glucan applied twice daily. Biometrological evaluations showed that erythema did not develop, the water retention capacity of the stratum corneum increased and the transepidermal water loss moderately decreased. Another 16‐week randomized, double‐blind, placebo‐controlled study was conducted on 20 men showing signs of ageing skin. A 1.5% chitin–glucan formulation was applied twice daily. Objective biometrological assessments showed a progressive increase in skin firmness and stratum corneum hydration when desquamation and skin roughness decreased. In conclusion, the chitin–glucan formulations appear safe. They significantly mitigate some signs of skin ageing and improve both stratum corneum hydration and skin barrier function.     

Occlusive power evaluation of O/W/O multiple emulsions on gelatin support cells

The water content of the stratum corneum plays an important role in providing skin suppleness and smoothness. The diffusion of water through the skin is limited primarily by the stratum corneum and the noncornified part of the epidermis has negligible water barrier properties. Multiple emulsions are vesicular systems utilized for the prolonged liberation of active ingredients. The O/W/O multiple emulsion type is employed in cosmetics because its high oil content is able to maintain an occlusive film (barrier) on the skin surface. The objective of this study was to determine the occlusive power of O/W/O multiple emulsions on gelatin support cells. The results showed that occlusive products form a uniform layer on the surface of gelatin after the test, whereas nonocclusive products form two layers: an aqueous phase on the gelatin, and an oil phase above the aqueous phase. Thus, the different occlusive powers are due to the homogeneity of this layer and to its ability to prevent water evaporation.     

A liquid crystal application in skin care cosmetics

Intercellular lipids of the stratum corneum contribute threefold to the maintenance of a healthy skin: by hydration, cell adhesion, and reduction of transepidermal water loss. All of these functions can be attributed to the self-organizing property of the amphiphilic molecules of the stratum corneum lipids. A new type of skin care product called Lamellar Gel was developed, which contains a (synthesized) pseudo-ceramide. Its structure is similar to that of ceramide found among the stratum corneum lipids, which allows it to control intramolecular interactions. Compared to regular emulsions the Lamellar Gel demonstrated better skin care characteristics regarding permeability, skin hydration, and skin occlusion. This was attributed to the fact that it formed the same self-organizing structure as natural stratum corneum lipids, hence showing a high affinity to the skin. A high moisturizing effect was observed as the Lamellar Gel combines the benefits of both O/W and W/O emulsions: it provides the same initial hydration as an O/W emulsion and at the same time the same occlusivity as a W/O emulsion. Transepidermal water loss increases under dry environmental conditions. This especially affects the skin around the eyes, where the skin is very thin, and wrinkles are very easily formed. Treatment with the Lamellar Gel recovered these wrinkles promptly and hydrated the stratum corneum for a long time.     

Moisturizing effect of vesicles formed from monoglycerides on human skin

The moisturizing effect of vesicles formed from monoglycerides on human skin was studied by measurement of conductance on and transepidermal waterloss from the skin surface. Although sonication of the monoglycerides in water with Ca2+ gave multilamellar vesicles, the lamellar structure of the vesicles disappeared during their storage without any other additive. With the addition of poly(vinylpyrrolidone) after the sonication, the stability of the vesicles increased and their lamellar structure was maintained for 3 months at 40 degrees C. These vesicles led to a significantly higher water content of the stratum corneum of human skin compared with non-lamellar monoglyceride, and consequently they improved rough human skin.     

Surfactants and the skin

The skin surface is the interface between us, the organism, and the outside world. When we clean the skin we remove not only the bacteria, dirt and grease which have accumulated, but also part of our natural barrier - the stratum corneum. Corneocytes, both singly and in clumps, are released from the skin surface by the action of detergents and mechanical stimulation. So too are the lipids and proteins which make up the intercorneocyte region of the stratum corneum. The analysis of the types and amounts of materials released by a standard scrub procedure may prove useful in the selection of surfactants with particular properties. Changes in the physical properties of the skin occur after washing. For example, changes in skin surface pH and transepidermal water loss (a sensitive index of barrier function) are easily demonstrable. Excessive exposure to surfactants results in repeated damage to the stratum corneum which can in turn lead to an irritant dermatitis. Individual susceptibility to irritant dermatitis varies and this may be demonstrated using a simple patch test technique. This test is a way of potentially increasing the sensitivity of human based assays such as the soap chamber test by preselection of subjects. Alternatively it may be possible to use measurements of function such as transepidermal water loss or laser Doppler blood flow as an index of damage rather than conventional cutaneous irritancy. These approaches may help in the search for the ideal of a non-irritant cleanser.     

Infrared spectroscopy of the skin: influencing the stratum corneum with cosmetic products

The stratum corneum is the outermost layer of the skin and, as such, represents the decisive barrier between the body and the environment. The combination of horny cells and lamellar lipid layers prevents water loss from the body and protects it against penetration by exogenous substances. For selective investigation of the thin outermost stratum corneum layer ATR-FTIR (attenuated total reflection Fourier transform infrared) spectroscopy has proved itself in practice. It provides information about the sebum content, type of fatty substances, water content and degree of order of the lamellar lipid film in the stratum corneum. Different types of skin (dry, normal and greasy) differ mainly in their sebum content but also in the composition of their fats and the degree of order of their lipids. A comparison with model lipid mixtures shows that the stratum corneum lipids are mainly present in a fixed gel form under physiological conditions. In cleansing processes a large part of the sebum is removed from the skin. The skin reacts to the defatting process by the rapid secretion of sebum. After cleansing with a mild face cleansing emulsion the re-achievement of the same fat status takes longer than with a surfactant shower gel. Skin creams cause alterations to the skin moisture and in the degree of order of the stratum corneum lipids. A lamellar cream with a structure similar to that of the stratum corneum lipids increases the degree of order of the alkyl chains of the skin lipid film (biomimetic principle), whereas a conventional w/o cream reduces this degree of order. Skin moisture increases after the use of the creams.     

High spatial resolution study of human skin using synchrotron infrared microscopy: application to the penetration of external agents

Human skin sections were studied using synchrotron-powered infrared microscopy. The superior lateral resolution achieved with this technique (diffraction-limited spot sizes) allows the detailed analysis of the biocomposition of the skin layers. It is shown that highly organized lipids are present in the stratum corneum, as evidenced by a slight frequency difference in the symmetric stretch mode of the methylene groups. Biochemical images were generated showing the precise location of the lipids, proteins and collagen across the skin section. More enhanced images were generated using a statistical approach (fuzzy C-means clustering). Penetration of two external agents (cyanophenol and a cosmetic compound, ethylhexyl methoxycinnamate) was studied. The penetration pathway across the stratum corneum is favored at locations of lower lipid concentration. Both compounds penetrated into the epidermis, but neither of them induced a change in the protein secondary structure. It was shown that hair follicle is a favored penetration pathway for these agents.     

Measurement of total water and bound water contents in human stratum corneum by in vitro proton nuclear magnetic resonance spectroscopy

The reliability of in vitro proton nuclear magnetic resonance (NMR) spectroscopy to specifically measure the water content of rehydrated human stratum corneum samples was assessed by comparison with the previously validated thermal desorption-mass spectrometry technique. The interest of proton NMR is that it can determine, in the same sample, both the amount of total water by recording the spectra at ambient temperature, and that of bound water (non-freezing water) by recording the spectra below 0 degrees C. Provided that enough samples of stratum corneum are analysed to average out the observed and well-known sample-to-sample variation, proton NMR may be of value in the evaluation of hygroscopic properties of raw materials for moisturizer formulations.     

Non-invasive assessment of stratum corneum structure and function

Methods for the study of the stratum corneum in situ are described and their application to clinical problems and cosmetic science are discussed. In vivo measurements of the thickness of skin and the components of skin can be made using high frequency pulsed ultrasound. Improved resolution of the device should allow measurement of epidermal thickness, and even that of the stratum corneum, which may vary with hydration. The protective ability of the stratum corneum can be estimated in a number of ways, the most familiar technique being transepidermal water loss measurements. Mechanical properties of the stratum corneum have been widely investigated in vivo by numerous researchers. A recent technique devised by us to deform the stratum corneum using an extendable metal frame has been used on patients with ichthyotic disorders. The deformation or compliance of the skin surface has been measured using profilometric methods, and compared to normal individuals. The results appear to be in agreement with the loss of flexural ability experienced by ichthyotic patients. Other mechanical methods more usually involve the measurement of the forces generated by skin while undergoing extension. Three techniques are described which have been used to investigate hydrational change with emollient application. Finally, the phenomenon of desquamation is discussed and methods presented for its evaluation. Passive collection of corneocytes using chambers is useful for estimation of the rates of desquamation, but are laborious and inconvenient. Forced desquamation using the desquamator enables rapid controlled harvesting of corneocytes. In addition to this, the dansyl chloride fluorescence technique has been adopted for estimation of stratum corneum kinetics using visual comparison and fluorimetric techniques. These techniques require further refinement and validation, but the tests available now are nonetheless important in understanding the biology and pharmacology of the stratum corneum.