Approche osmotique de l'hydratation du stratum corneum

Afin de reprendre l'hypothèse selon laquelle l'absorption de l'eau par le stratum corneum est un phénomène osmotique et afin de d'effectuer des études, in vitro , de la biophysique de la diffusion de l'eau par osmose, nous avons mis au point un Modèle Osmotique de Cornéocyte (MOC), qui est constitué par de la kératine isolée par une membrane semi-perméable.
Immeré dans de l'eau, le MOC voit sa masse augmenter. En effet, bien qu'insoluble, la kératine est hydrophile et en accord avec le Deuxième principe de la thermodynamique, elle fait diffuser par osmose, de l'eau vers l'intérieur du MOC.
A saturation, l'eau absorbée représente 13 fois la masse de kératine.
La diffusion s'effectue selon une cinétique classique: la masse de l'eau qui pénètre est proportionelle à la racine carrée du temps.
Les éléments constitutifs du Facteur Naturel d'Hydratation comme l'urée, l'acide lactique, l'acide pyrrolidone carboxylique, accroissent la vitesse de diffusion et la quantité totale d'eau absorbée. Un humectant comme la glycérine produit un effet semblable. L'urée est le composé qui donne les meilleures performances.
L'accroissement de la vitesse de diffusion par osmose dans le MOC en présence de composés hydratants peut s'interpréter comme un accroissement de l'hydrophilie de la kératine induite par ces composés.
Le MOC semble pouvoir permettre, outre une étude théorique sur le phénomène osmotique dans le stratum corneum , des études préliminaires in vitro des produits hydratants hydrosolubles utilisables en cosmétologie.     

Increased basal transepidermal water loss leads to elevation of some but not all stratum corneum serine proteases

There are indications of elevation of some inflammatory serine proteases in barrier damaged skin (e.g. plasmin and urokinase). Moreover, many other serine protease activities are present such as desquamatory enzymes as well as a newly detected tryptase‐like serine protease. However, the activities of these proteases have never been correlated with stratum corneum (SC) barrier function. The activity of extractable key serine proteases (SC trypsin‐like kallikreins, SC chymotrypsin‐like kallikreins, SC tryptase‐like serine protease, urokinase and plasmin) was measured from the outermost layers of SC obtained from facial tape strippings in clinically normal subjects. The protein content of the tape strippings was quantified by absorption measurements with the novel infrared densitometer SquameScan^TM 850A and the protease activities by the use of fluorogenic peptide substrates. SC barrier function, SC hydration and skin surface pH were measured using AquaFlux^TM, NOVA dermal phase meter and Skin‐pH‐Meter^®, respectively. As expected, SC hydration was reduced with increased transepidermal water loss (TEWL) values indicative of barrier impairment. Surprisingly, SC chymotrypsin‐like activity showed no correlation with hydration or TEWL, whereas all other enzymes positively correlated with impaired barrier function and some were statistically significant: SC trypsin‐like kallikreins (R² = 0.66, P < 0.01), SC tryptase‐like enzyme (R² = 0.95, P < 0.001), plasmin (R² = 0.86, P < 0.001) and urokinase (R² = 0.50, P < 0.05). All enzymes except urokinase also negatively correlated with SC hydration. Elevated levels of SC serine proteases have been associated with some dermatological disorders, such as atopic dermatitis, psoriasis and rosacea but these results indicate that these enzymes are also elevated with milder forms of barrier disruption, which is not clinically evident as irritated skin. As these proteases are elevated in the SC, they will also be elevated in the epidermis where they can be involved in neurogenic inflammation and epidermal barrier impairment via activation of the protease‐activated receptors. These results highlight the need for using serine protease inhibitors especially for urokinase and plasmin, SC tryptase‐like serine protease and possibly SC trypsin‐like kallikreins even in milder forms of barrier damage.     

Effect of Vichy water on catalase activity in the stratum corneum

Vichy spa water is essentially known for its therapeutic action on liver and bile duct functions. Its mechanism of action may be partially explained by the activation of certain digestive enzymes. A literature survey showed that Vichy water has also been used for local application in the treatment of certain dermatoses. Based on these data, the effects of Vichy spa water on the skin were studied using cutaneous enzymatic systems. The first studies were carried out on catalase, an oxidoreductase. The results showed a statistically significant increase ( p <0.05) in the activity of the enzyme in the presence of Vichy spa water both in vitro and in vivo. Considering the involvement of catalase in skin defence against oxygen-derived free radicals generated, its increased activity may explain the beneficial role of Vichy water observed in various dermatoses.     

Investigation of interactions between silicones and stratum corneum lipids

Ingredients of topically applied skin care formulations have not only positive effects on the appearance of human skin but can also disturb the Stratum corneum (SC) lipid barrier. In the present study, the influence of silicones (PDMS), as often used cosmetic ingredients, on the microstructure of SC lipids was investigated. For this purpose the interactions of four different PDMS with excised human SC were examined first using differential scanning calorimetry (DSC) and wide angle X-ray diffraction for physical characterization. Because the physical properties of human stratum corneum strongly depend on the lipid composition, showing inter-and intra-individual differences, the interactions with an in vitro model lipid system containing SC fatty acids were also studied, using polarized light microscopy, transmission electron microscopy, small angle X-ray diffraction and DSC. The results revealed that the investigated PDMS do not change either the microstructure of excised human SC or the biphasic lamellar/inverse hexagonal structure of the in vitro model. We concluded that PDMS will not cause any side-effects when topically applied and that our simplified in vitro model could be helpful for estimating interactions between cosmetic ingredients and other topically applied substances and the skin barrier at an early moment of formulation development.     

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 )     

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.     

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.     

Role of ceramides 2 and 5 in the structure of the stratum corneum lipid barrier

We utilized Fourier transform infrared (FTIR) spectroscopy to investigate headgroup and chain interactions in model SC lipid barriers containing equimolar amounts of deuterated hexadecanoic acid, cholesterol, and ceramide 2 (non-hydroxy sphingosine) or ceramide 5 (alpha-hydroxy sphingosine). In the ceramide 2 model the thermotropic response of the CD _ 2 and CH _ 2 stretching modes indicates that hexadecanoic acid begins to disorder at 42 degrees C while ceramide 2 remains ordered until 52 degrees C. Additionally, splitting of the CD _ 2 bending and CH _ 2 rocking modes provides evidence for separate orthorhombic hexadecanoic acid and ceramide domains. The ceramide amide I mode (1650 cm ; -1) is split into two components indicating strong intermolecular hydrogen bonding between headgroups. In the ceramide 5 model, the CH _ 2 and CD _ 2 stretching frequencies again reveal highly conformationally ordered ceramide 5 and hexadecanoic acid chains. Splitting of both the CD _ 2 bending and CH _ 2 rocking modes is observed. However, the CH _ 2 rocking frequencies indicate distorted packing of the ceramide. The collapse of these highly ordered phases, and the onset of conformational disorder, occurs at 50 degrees C for both ceramide 5 and hexadecanoic acid. The amide I and II frequencies of ceramide 5 indicate strong H-bonding, although neither mode is split. Our results demonstrate that model SC lipid systems have quite different physical properties depending on whether they contain ceramide 2 or 5. From this we infer that ceramide 2 and 5 make distinct contributions to the structural biophysics of the SC lipid barrier. Our observation of ordered lipid domains is also consistent with the recently proposed domain mosaic model of the skin barrier.     

The ultraviolet microscopic transmission characteristics of human stratum corneum

This study has evaluated the ultraviolet light transmission characteristics of human stratum corneum at the single cell level using a low light level video microscope to measure the mean percentage transmission of light at different wavelengths and the variation in transmission across the stratum corneum. Stratum corneum was isolated by an enzymic technique and examined on a low light level UV video microscope. Quantitative evaluation of the transmitted monochromatic light for the underside of the layer was measured directly using a Kontron UNIPS image processor or indirectly with a Quantimet 920 image processor after video recording. Transmission distribution histograms were obtained from samples of stratum corneum taken from human breast, scalp, abdomen and leg. Mean transmission values were also derived and compared with diffuse transmission values obtained using the same tissue mounted on an integrating sphere. The UV microscopic transmission characteristics of enzyme separated stratum corneum clearly demonstrated that this structure was not an ideal diffuser. Uniform light intensity on the surface of the stratum corneum led to areas of transmitted intensity in close proximity that differed by factors ofthree to six fold, e.g. between regions of high (>70%) and low (<20%) brightness. However, the average transmission was found to be compatible with published data obtained by diffuse transmission spectrophotometry, taking into account the enhanced transmission arising from stratum corneum immersion in phosphate buffered saline. This was confirmed by the elevated values obtained by diffuse transmission spectrophotometry in this study for samples of stratum corneum prepared for UV microscopy being higher than these found in published data. It is obvious from these findings that viable cells in the epidermis are not exposed to a uniform incident light intensity even when this is true for the surface of skin. Studies of skin response to ultraviolet light at the single cell level must take account of the possibility of preferential exposure of specific sites in any subsequent explanation of cell sensitivity. This is in addition to the already well established cell cycle dependent ultraviolet sensitivity.     

Drying stress and damage processes in human stratum corneum

The drying stresses that develop in stratum corneum (SC) are crucial for its mechanical and biophysical function, its cosmetic feel and appearance, and play a central role in processes of dry skin damage. However, quantitative methods to characterize these stresses are lacking and little understanding exists regarding the effects of drying environment, chemical exposures and moisturizing treatments. We describe the application of a substrate curvature technique adapted for biological tissue to accurately characterize SC drying stresses as a function of time following environmental pre‐conditioning and chemical treatment in a range of drying environments. SC stresses were observed to increase to stress levels of up to ～ 3 MPa over periods of 8 h depending on pretreatment and drying environment. A unique relationship between the SC stress and water in the drying environment was established. The effect of glycerol on lowering SC stresses and damaging surfactants on elevating SC stresses were quantified. Extensions of the method to continuous monitoring of SC stresses in response to changes in environmental moisture content and temperature are reported. Finally, a biomechanics framework to account for the SC drying stress as a mechanical driving force for dry skin damage is presented.     

In vivo stratum corneum over-hydration and water diffusion coefficient measurements using opto-thermal radiometry and TEWL Instruments

Skin over-hydration is a common problem that affects many people who wear incontinence pads or diapers. The aim of this study is to develop a new method for stratum corneum (SC) over-hydration and SC water diffusion coefficient measurements using opto-thermal transient emission radiometry (OTTER) and evaporimetry. With OTTER, we can measure the SC surface hydration and hydration gradient. With evaporimetry, we can measure the time-dependent evaporative drying curves of water vapour flux density (WVFD). The combination of hydration results and WVFD results can yield information on the SC water diffusion coefficient and how it depends on the SC surface hydration level. The results show that SC water diffusion coefficient is non-linearly proportional to the SC surface hydration level. The results also show strong correlations between evaporative drying flux measured using the Evaporimeter and surface hydration estimated from OTTER measurements.     

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.     

Profiling of serine protease activities in human stratum corneum and detection of a stratum corneum tryptase-like enzyme

Seasonal variation in stratum corneum (SC) biophysical and biological characteristics has been described previously. In particular, the winter season has been shown to affect more severely the properties of facial skin compared with forearm skin. Moreover, when compromised, such as in dry skin conditions, facial SC has been shown to contain increased inflammatory cytokines and proteases. Nevertheless, there have been no comparative studies of the activities and depth activity of several proteases in the SC on different body sites and at different times of the year. In this study, we examined the distribution of key serine protease activities (kallikrein 5, kallikrein 7, urokinase, plasmin and a tryptase-like enzyme) in different layers of the SC on the cheek and the forearm by analysis of consecutive tape strippings of healthy Caucasian subjects during winter and summer. The protein content of the tape strippings was quantified by absorption measurements with a recently developed and novel infrared densitometer SquameScan 850A while the SC enzyme activities were determined using fluorogenic peptide substrates. Transepidermal water loss (TEWL), skin pH and skin hydration were higher on the cheek than on the forearm. In the same way, the activity of the inflammatory-related proteases plasmin, urokinase and tryptase was approximately five to eight times and the activity of the desquamatory-related proteases kallikrein 5 and kallikrein 7 approximately two to four times higher on the cheek than on the forearm. There were no gender-related differences in these enzyme activities except for the increased kallikrein 7 in the forearm skin of the female subjects in winter. Reduced kallikrein 5 was associated with increased SC cohesion, as judged by increased protein removal, in forearm skin in the winter months of the year although the skin was clinically normal. It can be concluded that (i) protected skin areas show lower TEWL, skin pH and skin hydration and less protease activities than skin areas that are exposed to the environment, possibly indicating subclinical inflammation on these body sites, (ii) in normal healthy forearm skin, the outer SC exhibits greater serine protease activity than its deeper layers, (iii) compared with the forearm, urokinase- and plasmin-like activities are elevated on SC strippings from the cheek, confirming activation of the plasminogen cascade, and (iv) tryptase-like activity in the SC is also elevated in samples from the cheek, possibly indicating involvement of mast cells in these barrier-compromised body sites or the synthesis of a novel tryptase-like enzyme by keratinocytes. Although elevation of the activities of urokinase, plasmin, kallikrein 5, kallikrein 7 and now a tryptase-like enzyme was observed on SC derived from skin of clinically normal cheeks, we anticipate even higher activities in skin conditions where the epidermal barrier is further impaired.