Outermost Layers Of Stratum Corneum Research Articles

Depth profiling of epidermal hydration inducing improvement of skin roughness and elasticity: in vivo study by confocal Raman spectroscopy

Skin hydration in the stratum corneum plays an important role in skin condition, and skin efficacy properties are influenced by its hydration level. However, few studies have identified the correlation between changes in skin hydration content and skin characteristics by skin depth level. This study aims to determine how changes in skin hydration at specific depth levels affect skin condition by long-term tracking changes in hydration of stratum corneum and viable epidermis after usage of moisturizer. Ten volunteers were recruited and subjected to in vivo confocal Raman spectroscopy to perform water content profiling at skin depths of up to 52μm. Mechanical properties of skin were measured using Cutometer and Antera 3D. Skin-elasticity and roughness values observed before and after 15days of moisturizing emulsion use were compared to demonstrate the correlation between observed changes in skin efficacy parameters and skin water content at specific depths. Significant increase in relative water content at specific depths was observed in this study. Among mechanical properties of skin, only R4, R6, and R8 parameters demonstrated significant changes. Additionally, rates of change in values of the R6 and R8 parameters revealed a high correlation with water content changes at viable epidermis depths below the stratum corneum. On the other hand, skin roughness parameter showed a correlation with water content changes at the outermost layer of stratum corneum. Results of this study indicate that skin elasticity is influenced by its hydration level at viable epidermis depths and skin roughness at stratum corneum each. This suggests that monitoring depth profiles of water content using in vivo confocal Raman spectroscopy provides a breakthrough in tracking the skin efficacy effect of topically applied substances.

Accuracy and feasibility of piezoelectric inkjet coating technology for applications in microneedle-based transdermal delivery

Coated microneedles have shown immense promise for use in transdermal delivery and diagnostics, due to their ability to painlessly breach the skin's outermost stratum corneum layer and interact with the epidermal layers immediately beneath. In this work, we use an off-the-shelf piezoelectric dispensing system to demonstrate the feasibility of depositing material directly on to steeply-sloping microneedle sidewalls, without the need for specific needle array positioning or material pretreatment. In the first instance, an analysis of deposition accuracy shows that over 95% of dispensed droplets land within 20μm of the target. Through the use of sequential dispense and drying steps, 3.2nL of a model drug formulation has been deposited onto both silicon and polymeric microneedles with highly sloped (71°) sidewalls; these are the steepest surfaces that have been coated to date. Finally, preliminary ex-vivo skin studies have been performed to show that the material may be successfully transferred from the needle to skin. Despite the smooth surfaces, ultrasharp tips and steep sidewalls of these structures, piezoelectric dispense techniques are clearly feasible for microneedle coating and may offer a promising alternative to conventional coating processes.

Safety Assessment by Multiphoton Fluorescence/Second Harmonic Generation/Hyper-Rayleigh Scattering Tomography of ZnO Nanoparticles Used in Cosmetic Products

Zinc oxide nanoparticles (ZnO NPs) are commonly used as UV filters in commercial sunscreen products. Their penetration into the skin is intensively discussed in the literature. In the present in vivo study, penetration of ZnO NPs (30 nm in size) into human skin was investigated by multiphoton tomography. Based on the non-linear effects of a second harmonic generation and hyper-Rayleigh scattering, the distribution of ZnO NPs in the horny layers of the epidermis, as well as the furrows, wrinkles and orifice of the hair follicles was analyzed. This method permitted distinguishing between the particulate and dissolved forms of Zn. A detection limit of 0.08 fg/µm³ was estimated. Taking advantage of this sensitivity, it was clearly shown that ZnO NPs penetrate only into the outermost layers of stratum corneum, furrows and into the orifices of the hair follicles and do not reach the viable epidermis.

Status of surfactants as penetration enhancers in transdermal drug delivery.

Surfactants are found in many existing therapeutic, cosmetic, and agro-chemical preparations. In recent years, surfactants have been employed to enhance the permeation rates of several drugs via transdermal route. The application of transdermal route to a wider range of drugs is limited due to significant barrier to penetration across the skin which is associated with the outermost stratum corneum layer. Surfactants have effects on the permeability characteristics of several biological membranes including skin. They have the potential to solubilize lipids within the stratum corneum. The penetration of the surfactant molecule into the lipid lamellae of the stratum corneum is strongly dependent on the partitioning behavior and solubility of surfactant. Surfactants ranging from hydrophobic agents such as oleic acid to hydrophilic sodium lauryl sulfate have been tested as permeation enhancer to improve drug delivery. This article reviews the status of surfactants as permeation enhancer in transdermal drug delivery of various drugs.

Infant Skin Microstructure Assessed In Vivo Differs from Adult Skin in Organization and at the Cellular Level

Functional differences between infant and adult skin may be attributed to putative differences in skin microstructure. The purpose of this study was to examine infant skin microstructure in vivo and to compare it with that of adult skin. The lower thigh area of 20 healthy mothers (ages 25-43) and their biological children (ages 3-24 months) was examined using in vivo noninvasive methods including fluorescence spectroscopy, video microscopy, and confocal laser scanning microscopy. Stratum corneum and supra-papillary epidermal thickness as well as cell size in the granular layer were assessed from the confocal images. Adhesive tapes were used to remove corneocytes from the outer-most layer of stratum corneum and their size was computed using image analysis. Surface features showed differences in glyph density and surface area. Infant stratum corneum was found to be 30% and infant epidermis 20% thinner than in adults. Infant corneocytes were found to be 20% and granular cells 10% smaller than adult corneocytes indicating a more rapid cell turnover in infants. This observation was confirmed by fluorescence spectroscopy. Dermal papillae density and size distribution also differed. Surprisingly, a distinct direct structural relationship between the stratum corneum morphology and the dermal papillae was observed exclusively in infant skin. A change in reflected signal intensity at approximately 100 mum indicating the transition between papillary and reticular dermis was evident only in adult skin. We demonstrate in vivo qualitative and quantitative differences in morphology between infant and adult skin. These differences in skin microstructure may help explain some of the reported functional differences.

Analysis of human face skin surface moleculesin situby Fourier-transform infrared spectroscopy

For medical and dermatological researchers, it is important to realize the molecular dynamics and its control in the stratum corneum (SC) of human skin, which may be related to some skin abnormalities such as atopic dermatitis and skin pruritus. We have tried to analyze the periodic molecular dynamics of the outermost layers of SC in vivo. We measured the skin surface molecules of human face in situ non-invasively using a Fourier-transform infrared (FTIR) spectroscopy system attached with a newly designed attenuated total reflection (ATR) probe. The water-extracted components from the SC were also analyzed using mass spectrometry, an enzymatic assay and high-performance liquid chromatography characterization. The infrared spectral changes of some components on the face skin at around 1000-1200 cm(-1) with circa-monthly rhythms were observed when monitored for 10 months, and the components also showed a seasonal change. The analysis of different FTIR spectrum of the changeable components with circa-monthly rhythm suggested the presence of a lactate compound. The presence of magnesium lactate in a conjugated form was detected in the water extract of SC. We demonstrate that the periodically changed components of the human face skin contained magnesium lactate conjugate as a major component.

An ex vivo methodology to assess the lipid peroxidation in stratum corneum

Environmental risks, particularly UV radiation, provide a challenge to the function of the skin barrier. Protective measures such as the use of antioxidant products represent a possible method of providing protection to the skin. This paper reports the development of a non-invasive ex vivo method using tape strips of the outermost layers of stratum corneum (SC) from human volunteers in order to determine the effectiveness of an antioxidant emulsion topically applied to prevent lipid peroxidation (LPO) in the horny layer after an UV irradiation exposure. Two different formulations were used: formulation (A), containing Vitamin A, E and C, and formulation (B) containing fish extract. Both formulations were topically applied in vivo on volunteer forearms; then, a tape stripping of the SC of each volunteer was carried out. The lipid peroxidation was measured ex vivo after an UV irradiation of the SC samples. The amount of SC stripped to evaluate differences in lipid peroxidation, the UV irradiation intensity to form lipid peroxides and the accuracy of lipid peroxide analysis were optimized in this methodology using formulation (A). After an exposure application of seven days, a group of three strips of the outermost layers of SC of volunteers was irradiated with an intensity of 182.7 J/cm 2 to quantify the LPO inhibition. The percentage of LPO inhibition obtained after topical application of both formulations was in the range of 40–58% demonstrating the effectiveness of the formulations topically applied against lipid peroxidation on human SC. This methodology may be used as a quality control tool to determine ex vivo the percentage of the LPO inhibition on human SC for a variety of antioxidants topically applied.

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 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, 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 (R(2 )=0.66, P < 0.01), SC tryptase-like enzyme (R(2 )=0.95, P < 0.001), plasmin (R(2 )=0.86, P < 0.001) and urokinase (R(2 )=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.

Imaging of zinc oxide nanoparticle penetration in human skin in vitro and in vivo

Zinc oxide (ZnO-nano) and titanium dioxide nanoparticles (20 to 30 nm) are widely used in several topical skin care products, such as sunscreens. However, relatively few studies have addressed the subdermal absorption of these nanoparticles in vivo. We report on investigation of the distribution of topically applied ZnO in excised and in vivo human skin, using multiphoton microscopy (MPM) imaging with a combination of scanning electron microscopy (SEM) and an energy-dispersive x-ray (EDX) technique to determine the level of penetration of nanoparticles into the sub-dermal layers of the skin. The good visualization of ZnO in skin achieved appeared to result from two factors. First, the ZnO principal photoluminescence at 385 nm is in the "quiet" spectral band of skin autofluorescence dominated by the endogenous skin fluorophores, i.e., NAD[P]H and FAD. Second, the two-photon action cross section of ZnO-nano [sigma(ZnO) ((TPEF)) approximately 0.26 GM; diameter, 18 nm] is high: approximately 500-fold of that inferred from its bulk third-order nonlinear susceptibility [Im chi(ZnO) ((3))], and is favorably compared to that of NAD[P]H and FAD. The overall outcome from MPM, SEM, and EDX studies was that, in humans in vivo, ZnO nanoparticles stayed in the stratum corneum (SC) and accumulated into skin folds and/or hair follicle roots of human skin. Given the lack of penetration of these nanoparticles past the SC and that the outermost layers of SC have a good turnover rate, these data suggest that the form of ZnO-nano studied here is unlikely to result in safety concerns.

Depth Dependence of Stratum Corneum Lipid Ordering: A Slow-Tumbling Simulation for Electron Paramagnetic Resonance

We investigated the structural ordering of stratum corneum (SC) lipid by means of electron paramagnetic resonance (EPR) slow-tumbling simulation in conjunction with spin probe studies. The SC of human mid-volar forearm was stripped consecutively from three to six times. The EPR probe method detected a characteristic peak of sebaceous matter in the first SC stripping. The order parameter values obtained by the slow-tumbling simulation (S(0)) showed significant differences between each layer compared with those indicated by the conventional order parameter (S) using hyperfine couplings. Although the conventional S values were in the range of 0.56 (outermost layer) to 0.61 (bottom layer), the S(0) values by the simulation changed from 0.22 to 0.96. The present results suggest that the structural ordering of the outermost SC layer is less tight, whereas the structure of inner layers becomes more rigid. Therefore, we concluded that the EPR probe method recognizes sebaceous matters, whereas EPR in conjunction with the simulation allows quantitative evaluation of SC lipid ordering in relation to skin depth.

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.

Induction of stress proteins and MMP-9 by 0.8 ppm of ozone in murine skin

Ozone (O 3) is among the most reactive environmental oxidant pollutants to which cutaneous tissues are exposed. O 3 exposure has been shown to induce antioxidant depletion as well as the oxidation of lipids and proteins within the outermost skin layer, the stratum corneum. However, relatively little is known regarding the potential effects of O 3 on the cellular constituents of the underlying skin epidermis and dermis. In the present study, hairless mice exposed for 6 h to 0.8 ppm O 3 showed increases in lipid peroxidation, as quantitated by increases in 4-hydroxynonenal-protein adducts. O 3 exposure caused an induction of the stress proteins HSP27 and heme oxygenase-1 (HO-1), starting at 6 h and increasing up to 18 h after O 3 exposure. This was accompanied by an increase in matrix metalloproteinase-9 (MMP-9) mRNA and activity levels, indicative of possible injurious-reparative processes. Collectively, our data demonstrate that skin exposure to O 3 not only affects antioxidant levels and oxidation markers in the outermost stratum corneum layer, but also induces cellular stress responses in the deeper cellular layers of the skin.

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.

The Outermost Stratum Corneum Layer is an Effective Barrier Against Dermal Uptake of Topically Applied Micronized Titanium Dioxide

In order to help clarify the controversially discussed dermal uptake properties of micronized titanium dioxide (TiO _ 2), we conducted extensive in vitro dermal absorption studies with 'Franz-type' diffusion cells on excised porcine skin. After biopsies and chemical fixation, the overall localization of TiO _ 2 in the skin was analyzed by means of transmission electron microscopy (TEM). The lateral and vertical distribution of TiO _ 2 within the stratum corneum (SC) was investigated by tape stripping and subsequent scanning electron microscopy (SEM) in combination with energy dispersive X-ray analysis (EDXA). TiO _ 2 was found exclusively on the outermost SC layer. The surface deposit, as displayed by TEM, featured clearly distinguishable agglomerates as well as single particles with a characteristic cubic shape and a primary particle size of about 20-50 nm. Concurrently, SEM/EDXA micrographs first showed an even distribution of TiO _ 2 on the skin surface. After 10-fold stripping, however, TiO _ 2 was found to be localized only in the furrows and not on the partially removed ridges of the skin surface. SEM/EDXA micrographs of the adhesive tape strips revealed a characteristic pattern of stripped material and free regions. This pattern was an imprint of the skin's topography. Hence, tape stripping initially removed TiO _ 2 and SC layers only from the ridges and not from the deeper furrows. Continued stripping increasingly yielded material from the deeper contours of the SC surface. TiO _ 2 was found only in traces in the upper part of the follicle without any evidence of uptake into the follicular epithelium. This indicates that there is not any relevant penetration via the follicular route. We conclude that due to the microtopography of the skin, the strip number normally does not reflect the SC layer number. Accordingly, tape stripping results should always be interpreted with care, especially in the case of topically applied particles, as even higher numbers of subsequent strips may still sample material from the outermost SC layer of the deeper furrows, which could be interpreted falsely as penetrated material. Our results clearly demonstrate that TiO _ 2 homogeneously and completely covers the outermost SC layer. It is neither delivered to the SC nor to the underlying skin layers when applied topically to porcine skin in vitro in the cosmetic vehicle used here. These findings underscore the safety of this micronized inorganic UV filter.

Homogeneous transport in a heterogeneous membrane: water diffusion across human stratum corneum in vivo

The objective of this study was to determine whether a structurally heterogeneous biomembrane, human stratum corneum (SC), behaved as a homogeneous barrier to water transport. The question is relevant because the principal function of the SC in vivo is to provide a barrier to the insensible loss of tissue water across the skin. Impedance spectra (IS) of the skin and measurements of the rate of transepidermal water loss (TEWL) were recorded sequentially in vivo in human subjects as layers of the SC were progressively removed by the serial application of adhesive tape strips. The low-frequency (< or = 100 rad s-1) impedance of skin was much more significantly affected by tape stripping than the higher frequency values; removal of the outermost SC layer had the largest effect. In contrast, TEWL changed little as the outer SC layers were stripped off, but increased dramatically when 6-8 microns of the tissue had been removed. It follows that the two noninvasive techniques probe SC barrier integrity in somewhat different ways. After SC removal, recovery of barrier function, as assessed by increasing values of the low-frequency impedance, apparently proceeded faster than TEWL decreased to the prestripping control. The variation of TEWL as a function of SC removal behaved in a manner entirely consistent with a homogeneous barrier, thereby permitting the apparent SC diffusivity of water to be found. Skin impedance (low frequency) was correlated with the relative concentration of water within the SC, thus providing an in vivo probe for skin hydration. Finally, the SC permeability coefficient to water, as a function of SC thickness, was calculated and correlated with the corresponding values of skin admittance derived from IS.

Keratinization of the outer surface of the avian scutate scale: Interrelationship of alpha and beta keratin filaments in a cornifying tissue

The outer surface of adult Gallus domesticus scutate scale was studied as a model for epidermal cornification involving accumulation of both alpha and beta keratins. Electron-microscopic analysis demonstrated that the basal cells of the adult epidermis contained abundant lipid droplets and that filament bundles and desmosomes were distributed throughout the cell layers. Indirect immunofluorescence microscopy and double-labeling immunogold-electron microscopy confirmed that the stratum germinativum contained alpha keratin but not beta keratin. Beta keratins were first detected in the stratum intermedium and were always found intermingled with filament bundles of alpha keratin. As the differentiating cells moved into the outer regions of the stratum intermedium and the stratum corneum, the large mixed keratin filament bundles labeled increasingly more with beta keratin antiserum and relatively less so with alpha keratin antiserum. Sodium dodecyl sulfate-polyacrylamide gel analysis of vertical layers of the outer surface of the scutate scale confirmed that cells having reached the outermost layers of stratum corneum had preferentially lost alpha keratin. The mixed bundles of alpha and beta keratin filaments were closely associated with desmosomes in the lower stratum intermedium and with electron-dense aggregates in the cytoplasm of cells in the outer stratum intermedium. Using anti-desmosomal serum it was shown that these cytoplasmic plaques were desmosomes.

Epidermal Kinetics and Skin Condition: I. Stripping Technique for Quantitating Stratum Corneum Turnover in Hairless Mouse Skin

AbstractThe turnover kinetics of hairless mouse epidermis were explored. 3H-labeled lysine was incorporated into epidermal proteins by injecting it systemically through the tail vein. Radioactivity in the outermost layer of stratum corneum was initially documented by stripping the skin daily with cellophane tape and assaying the tissue adhering to the tape. A broad band of radioactivity was detected between 4 and 7 days, indicating that lysine labels proteins throughout the forming epidermis. To ensure that stripping did not accentuate turnover, stripping was also performed every third day per mouse on rotation through three groups of radiotagged mice. Although there was more variability in the data gathered this way, the curves for daily stripping and every third day stripping actually superimposed. [3H]Glycine was introduced as the marker. This amino acid evidenced a single radioactive peak coinciding in time with the first lysine peak. The mass of stripped cells on each tape was quantified using a sens...