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.     

Eucalyptus increases ceramide levels in keratinocytes and improves stratum corneum function

The objectives of this study were to identify a plant extract that would improve stratum corneum functions and to elucidate the mechanism(s) involved. Based on the information that stratum corneum functions depend on the level of ceramide in the stratum corneum, we identified a Eucalyptus extract that was able to increase the level of ceramide in human keratinocytes in culture and in human stratum corneum and that improves the stratum corneum water holding and barrier functions. Addition of the Eucalyptus extract to human keratinocytes in culture increased the level of ceramide in a dose-dependent manner and also increased the biosynthesis of ceramide, glucosylceramide and sphingomyelin. Topical application of the Eucalyptus extract on the dry skin of human subjects induced by acetone and diethylether treatment resulted in a significant increase in ceramide level in the stratum corneum, a significant improvement in its water-holding function and an improvement in its barrier function. The addition of macrocarpal A, one of the main components of the Eucalyptus extract, to human keratinocytes in culture increased the level of ceramide and the mRNA expression of serine palmitoyltransferase, acid sphingomyelinase, neutral sphingomyelinase, glucosylceramide synthase and glucocerebrosidase in a dose-dependent manner. Our results indicate that the increased content of ceramides in the stratum corneum may underlie the therapeutic effect of the Eucalyptus extract. Our results also indicate the possibility that macrocarpal A is the key component that stimulates the synthesis of ceramide in the stratum corneum.     

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.     

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.     

The chemistry,function and (patho)physiology of stratum corneum barrier ceramides

Research on understanding of the chemistry, function and (patho)physiology of stratum corneum (SC) lipids and especially ceramides has evolved over the last two decades. This has been made successful through the application of separation techniques that have become increasingly more sophisticated, and it has become increasingly evident that our understanding of these molecules remains in its infancy. Thirteen classes of ceramides with over 300 and possibly up to 1000 distinct ceramide species have been identified suggesting an exquisitely subtle relationship between the types of ceramides and physical and chemical behaviour. Nevertheless, research has demonstrated the importance of the correct SC lipid lamellar architecture with conformationally‐ordered lipid bilayers, the presence of long‐chain ceramides, as either free or covalently bound lipids, greater quantities of phytosphingosine‐containing ceramides and a high SC lipid/protein ratio is essential for optimal barrier function. These features are known to change in a variety of physiological and pathophysiological conditions. Clearly, there is more to be learned but as we further decipher the complexity of SC lipids and understand their individual roles in the SC, we will learn how to better treat the disorders of cornification.     

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.     

Effects of environmentally realistic levels of ozone on stratum corneum function

The impact of troposphere ozone (O(3)), the major oxidant in photochemical smog, on the overall wellbeing of skin is of considerable interest. To date, limited information is available on the impact of O(3) on human skin. Using a specially designed O(3) exposure chamber, we provide the first evidence that exposure of human skin to O(3) (0.8 ppm, 2-h time-weighted average) significantly reduced vitamin E by 70% and concomitantly increased lipid hydroperoxides by 2.3 fold in the superficial stratum corneum (SC). Although the dose of O(3) used here reduced the resident microflora population by 50% and created a state of oxidative stress within the SC, it did not affect several key enzymes involved in SC homeostasis including the redox-sensitive transglutaminase and the SC tryptic (KLK5) and chymotryptic (KLK7) proteases. Importantly, no signs of skin dryness or erythema were observed. We hypothesize that the limited effects of low doses of O(3) on SC function is attributable to several factors including: (i) protection provided by the anti-oxidant defence system; (ii) inability of O(3) to penetrate the SC; and (iii) limited water available to catalyse the Criegee reaction. Although chronic exposure to O(3) may produce a different outcome than that reported here, our data suggest that exposure to environmentally relevant doses of O(3), at best, induces a moderate state of oxidative stress, without producing a visible clinical response. In our opinion, exposure of skin to UV radiation is a much more significant threat than exposure to ground-level O(3).     

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.     

A new covalently bound ceramide from human stratum corneum -omega-hydroxyacylphytosphingosine

The outermost layer of the skin, the stratum corneum, consists of non-viable keratin-filled cells, or corneocytes, embedded in a matrix of lipids. The boundary of the cells consists of cross-linked proteins with covalently bound lipids on the outer surface. The spaces between cells are filled with a mixture of ceramides, cholesterol and fatty acids. The stratum corneum provides a protective barrier against water loss through the skin and limits the penetration of potentially harmful substances from the environment. Among the covalently bound lipids on the corneocyte surface are omega-hydroxyacylsphingosine and omega-hydroxyacyl-6-hydroxysphingosine. The previously suspected presence of omega-hydroxyacylphytosphingosine is confirmed in this report through its specific isolation and characterization based on chromatographic behaviour and proton magnetic resonance spectral data.     

Trends in stratum corneum research and the management of dry skin conditions

The structure, composition, formation and function of the stratum corneum have been the subject of intense research over the last few decades. As has become apparent, stratum corneum barrier function is not only dependent on one single component but also on its total architecture. Recent developments in understanding lipid composition have led to a new ceramide nomenclature system, a new proposal for a molecular model of the interactions between ceramides, cholesterol and fatty acids, and the demonstration of the presence of crystalline orthorhombic and gel hexagonal lipid phases in the stratum corneum. Linoleate-containing ceramide one, now known as CER EOS, have been shown to be essential for the formation of the 13 nm long periodicity phase (LPP) observed by electron microscopy and X-ray diffraction studies, whereas long-chain fatty acids are important for the formation of the crystalline lipid phases essential for barrier function. The role of the corneocyte envelope, its constituent proteins and its transglutaminase-mediated maturation processes have been shown to be essential for good skin condition. Several proteases may have a role in corneodesmolysis, particularly serine and cathepsin-like enzymes. Novel filaggrin polymorphisms have been identified that may be involved in the expression of a dry skin phenotype. Disturbances in lipid packing states, reduction in ceramide levels (particularly the phytosphingosine-containing ceramides), reductions in the levels of long-chain fatty acids and loss of the LPP largely account for the perturbations in lipid structure that occur in dry skin. The reduced corneodesmolysis that occurs in this xerotic skin disorder is now well accepted and is caused by reductions in the levels and activities of stratum corneum proteases together with elevated levels of corneodesmosomal glycoproteins in the superficial layers of the stratum corneum. Additionally, increased levels of fragile corneocytes are associated with reduced transglutaminase activity and corneocyte envelope cross-linking events. However, in comparison with the advances in our understanding of the textural changes that occur in dry skin, the somatosensory changes are poorly understood and the itching associated with dry skin is still an under-researched area. The unique biosensor role of the stratum corneum essential for a competent natural moisturizing barrier may also have a role to play in the action of anti-ageing technologies by controlling the expression and secretion of epidermal cytokines and growth factors. Technologies to treat the surface textural skin problems, enhance the differentiation process, particularly lipid biosynthesis, and to control the somatosensory problems in dry skin have received much attention in the last decade. This paper will review the state of the art of stratum corneum biology and the trends in the management of dry skin.     

Involvement of transglutaminase in ex vivo maturation of cornified envelopes in the stratum corneum

The cornified envelope (CE) is a thin insoluble structure enveloping corneocytes, and is essential for the barrier function of the stratum corneum (SC). Our previous studies revealed that immature CEs are detected in the outermost layer of SC of barrier-impaired epidermis including the face and in various inflammatory disorders, using a non-invasive method to evaluate CE maturity. However, factors attributable for immaturity of CEs are still unclear. The aim of the present study is to clarify whether immature CEs in the SC have the potential to mature. SC samples, in which immature CEs abundantly exist, were collected from the cheek of healthy volunteers by tape-stripping, and were incubated ex vivo under the humidified air at 37 degrees C. Then, CE maturity was evaluated by staining with a combination of anti-involucrin and Nile red to detect involucrin antigenicity in the immature CEs and hydrophobicity in the mature CEs, respectively. Ex vivo incubation of the SC resulted in the conversion of immature CEs into mature CEs in terms of loss of involucrin antigenicity and acquisition of hydrophobicity. Application of buffer solutions of various pH onto the SC prior to incubation revealed that maturation of CEs was proceeded at range of pH 5-7, corresponding to intrinsic pH range within the SC. Chelating agents, ethylenediamine-N, N, N', N',-tetraacetic acid (EDTA) and ethyleneglycol bis(beta-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA), and thiol alkylating agents, N-ethylmaleimide and iodoacetamide, inhibited the maturation. Labelled cadaverine as an exogenous substrate for transglutaminase (TGase) could be incorporated into CEs during maturation. Extractable involucrin-like protein detected in the SC samples before incubation concomitantly disappeared with CE maturation, suggesting incorporation of endogenous substrates into the CEs. These results obviously demonstrate that maturation of CEs was mediated by TGase activity in the SC, and that immature CEs found in the outermost face SC have potential to mature by cross-linking of endogenous CE precursors present in the SC. Reduction of environmental humidity during ex vivo incubation of the SC resulted in marked suppression of maturation of CEs, and application of a moisturizer, glycerine, onto the SC replenished the suppression of maturation, suggesting that water content in the SC may affect the TGase reaction in the SC. Therefore, various factors, including a decrease in the water content in the SC, may account for impaired maturation of CEs in the face SC.     

Hydration of the stratum corneum

Topically applied water, occlusion and topically applied glycerol were used to investigate and characterize some of the changes which occur in the hydrated stratum corneum. The effects of these treatments were monitored using non-invasive techniques under controlled conditions. The Servomed Evaporimeter was used to determine natural water flux from the skin surface before and after treatment. The performance of the Evaporimeter in this type of study had previously been improved by attaching a paper baffle to the detector. This eliminated the variance in output caused by atmospheric movement. Experiments were carried out at temperatures below the threshold of thermal sweating and emotional sweating was minimized. Skin surface topography was characterized by means of a new type of profilometer. The instrument's design allowed a diamond stylus to traverse the living skin surface without significantly altering its structure. Changes in skin surface roughness were further elucidated using scanning electron microscopy and macrophotography. In vivo penetration of glycerol was assessed by chemical analysis of stratum corneum layers of treated skin. Samples were obtained by sequential stripping of the stratum corneum using adhesive tape. Topically applied water produced only a transient benefit because of rapid evaporation. More prolonged hydration was achieved by suppressing transepidermal water loss with polyethylene film. This occlusive hyperhydration was characterized by a significant reduction in profile roughness and by a smoother macroscopic appearance. Glycerol achieved the same effects by reducing the magnitude of the natural water flux from the skin surface and by reducing the rate of evaporation of water from applied aqueous glycerol solution or cosmetic product. Both effects were seen as the result of lowered water activity in the proximity of glycerol. Smoothing effects of glycerol on the skin surface, and improved appearance, persisted for at least 24 h. This persistence was explained by evidence for diffusion of glycerol into the stratum corneum where it formed a reservoir. Hydration of the skin is known to affect its barrier function and thereby exert a profound effect on penetration of both lipophilic and hydrophilic molecules. Clinically, this effect may be achieved using liberal applications of occlusive petroleum jelly and ointments. The results presented in this paper suggest that the use of humectants could achieve useful hydration using cosmetically acceptable materials.     

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.     

The spectral stability of several sunscreening agents on stratum corneum sheets

Film layers of seventeen commercially available sunscreen products and sixteen active ingredients on stratum corneum sheets were spectrophotometrically monitored before and after simulated solar irradiation. Fixed irradiation doses were given within the daily terrestrial limits. From the changes in the absorption spectra after irradiation the spectral stability was determined. The spectral stability reflects the photochemical stability and the assay quickly provides an insight in the sunscreen's stability towards (solar) radiation in situ . Good to excellent spectral stability was observed for the benzophenones, paraaminobenzoic acid and its esters, homomenthylsalicylate, guanine, 2-phenylbenzimidazol-5-sulphonic acid (potassium salt) and guiazulene. Moderate spectral stability was observed in case of the cinnamates and 3-[4-methylbenzylidene] camphor.
Poor spectral stability was exhibited by phenylsalicylate and 4-isopropyldibenzoylmethane. Among the commercial products good to poor spectral stability was established.
We recommend the performance of this assay on every sunscreen agent or cosmetic formulation which may be exposed to the sun or any other ultra-violet/visible radiation source.