The mechanical properties of stratum corneum: I. The effect of water and ambient temperature on the tensile properties of newborn rat stratum corneum

The tensile properties of the outermost layer of skin of neonatal rats, the stratum corneum, were investigated at a constant strain rate as a function of moisture content and ambient test temperature. The results show that the mechanical behavior of this membrane, whose primary constituent is the fibrous protein keratin, can be significantly altered by variations in both the sorbed water content and ambient temperature. In particular, a brittle to ductile transition was observed at 25°C once the hydration level exceeded 70% relative humidity. Similarly, an identical phenomenon moisture concentrations were maintained at 10 g H₂O/100 g dry protein. Differential scanning calorimetry measurements showed the presence of a molecular relaxation process which migrated from 42°C at 40% relative humidity to −18°C at 95% relative humidity. It is postulated that this relaxation process, possibly corresponding to the glass transition of the fibrous protein component of stratum corneum, is primarily respnsible for the observed behavior.     

Experimental penetration of Trichophyton mentagrophytes into human stratum corneum

We present confirmation of the experimental penetration of Trichophyton mentagrophytes into human stratum corneum under designated conditions of temperature and humidity. When stratum corneum, obtained from healthy human heel region, was incubated at 100% humidity, mycelium was observed in the corneum layer on day 2 at 35 °C and 27 °C, and on day 4 at 15 °C. At 90% humidity, the hyphae penetrated into the stratum corneum on day 4 at 35 °C, and on day 6 at 27 °C. Whereas, at 80% humidity, no fungal elements were observed in the stratum corneum at both 27 °C and 35 °C for up to 7 day. These data suggested that humidity was a more important environmental factor for penetration than temperature, and that at least 90% humidity is necessary for dermatophytes to penetrate into the stratum corneum within a few days. Mean humidity in the interdigital space between the fourth and fifth toes was found to be approximately 98%. This revised version was published online in June 2006 with corrections to the Cover Date.     

The stratum corneum lipid thermotropic phase behavior.

The stratum corneum, the outermost layer of mammalian skin, is considered the least permeable skin layer to the diffusion of water and other solutes. It is generally accepted that the intercellular lipid multilayer domain is the diffusional pathway for most lipophilic solutes. Fluidization of the lipid multilayers is believed to result in the loss of barrier properties of the stratum corneum. Current investigations address the lipid thermotropic phase behavior in terms of lipid alkyl chain packing, mobility and conformational order as measured by Fourier transform infrared (FTIR) spectroscopy. A solid-solid phase transition is observed with increased alkyl chain mobility followed by a gel to liquid-crystalline phase transition near 65 degrees C. These results further elucidate the role of lipid fluidity that may contribute to the transport properties of the stratum corneum.     

Molecular properties of a stratum corneum model lipid system: large unilamellar vesicles.

Stratum corneum lipids are relatively complex, and there is little detailed understanding of their chemical and physical properties at the molecular level. Large unilamellar vesicles (LUVs) with lipid compositions similar to those of stratum corneum were prepared at pH 9 with commercially available lipids. This system was used as a model system for molecular studies of stratum corneum lipids. LUVs were chosen as the model system as they are comparatively more stable and can be characterized more quantitatively in terms of lipid concentration, surface area, and volume than model systems such as lipid mixture suspensions, lipid films, and small unilamellar vesicles. Results from freeze-fracture and cryo electron microscopy studies of our LUVs showed spherical vesicles. Quasi-elastic light scattering measurements revealed a narrow size distribution, centering around 119 nm. At room temperature, the LUVs were stable for several weeks at pH 9 and for more than 15 h but less than 24 h at pH 6. Differential scanning calorimetry measurements indicated broad endothermic transitions centered near 60-65 degrees C, closely matching the transition temperature reported for stratum corneum lipid extracts. Spin probes, 5-doxylstearic acid and 12-doxylstearic acid, were used for electron paramagnetic resonance (EPR) studies of the molecular dynamics of the lipids. EPR results indicated more restricted motion near the polar headgroup region than near the center of the alkyl chain region. Motional profiles of the spin labels near the polar headgroup and within the alkyl chain region in the LUVs were obtained as a function of temperature, ranging from 25 to 90 degrees C. We also found that the partitioning between the lipid and aqueous phases for each spin probe was temperature dependent and was generally correlated with phase transitions observed by differential scanning calorimetry and with alkyl chain mobility observed by EPR. Thus, this LUV system is well suited for additional molecular studies under different experimental conditions.     

Models of stratum corneum intercellular membranes: 2H NMR of macroscopically oriented multilayers.

Deuterium NMR was used to characterize model membrane systems approximating the composition of the intercellular lipid lamellae of mammalian stratum corneum (SC). The SC models, equimolar mixtures of ceramide:cholesterol:palmitic acid (CER:CHOL:PA) at pH 5.2, were contrasted with the sphingomyelin:CHOL:PA (SPM:CHOL:PA) system, where the SPM differs from the CER only in the presence of a phosphocholine headgroup. The lipids were prepared both as oriented samples and as multilamellar dispersions, and contained either perdeuterated palmitic acid (PA-d31) or [2,2,3,4,6-2H5]CHOL (CHOL-d5). SPM:CHOL:PA-d31 formed liquid-ordered membranes over a wide range of temperatures, with a maximum order parameter of approximately 0.4 at 50 degrees C for positions C3-C10 (the plateau region). The quadrupolar splitting at C2 was significantly smaller, suggesting an orientational change at this position, possibly because of hydrogen bonding with water and/or other surface components. A comparison of the longitudinal relaxation times obtained at theta = 0 degrees and 90 degrees (where theta is the angle between the normal to the glass plates and the magnetic field) revealed a significant T1Z anisotropy for all positions. In contrast to the behavior observed with the SPM system, lipid mixtures containing CER exhibited a complex polymorphism. Between 20 and 50 degrees C, a significant portion of the entire membrane (as monitored by both PA-d31 and CHOL-d5) was found to exist as a solid phase, with the remainder either a gel or liquid-ordered phase. The proportion of solid decreased as the temperature was increased and disappeared entirely above 50 degrees C. Between 50 and 70 degrees C, the membrane underwent a liquid-ordered to isotropic phase transition. These transitions were reversible but displayed considerable hysteresis, especially the conversion from a fluid phase to solid. The order profiles, relaxation behavior, and angular dependence of these parameters suggest strongly that both the liquid-ordered CER- and SPM-membranes are bilayers. The unusual phase behavior observed for the CER-system, particularly the observation of solid-phase lipid at physiological temperatures, may provide insight into the functioning of the permeability barrier of stratum corneum.     

An analysis on the rates and regulation of insensible water loss through the eccrine sweat glands

An analysis is presented on insensible water loss from the human body at rest through exposed skin surfaces into still air. Possible sites of moisture release are identified as the stratum corneum of the skin, free surfaces of dilute sweat liquids perpetually present in the microscopic ducts of a large population of eccrine sweat glands, and moist microvillous processes which line part of the periductal surfaces in the glands, particularly in the helical coils within the stratum spinosum of the epidermis. Water supply to the sites involves transepidermal migration across skin tissue layers, secretion and partial reabsorption of solutes and water within eccrine glands, and transport across periductal lining of eccrine glands from the surrounding connective tissues respectively. Evaporation and gas phase diffusion within eccrine ducts were modelled. Basal loss rates of water (as regulated by the ambient temperature and relative humidity and by aspects of the anatomy of and physiological factors for eccrine glands, the epidermis and the dermis) were calculated at between 1 and 20 g hr-1 at an ambient temperature of 25 degrees C and a relative humidity of 60% as an example. Such rates are significant fractions of experimental values for insensible water loss rates reported at between 4 and 35 g hr-1 in air at 22-30 degrees C and a relative humidity of 30-60%.     

Filaggrin breakdown to water binding compounds during development of the rat stratum corneum is controlled by the water activity of the environment

Filaggrin is a specific epidermal protein which is the precursor of the free amino acids, urocanic acid and pyrrolidone carboxylic acid which are largely responsible for the ability of the stratum corneum of the skin to remain hydrated at low environmental humidity. The distribution of filaggrin shown by immunofluorescence in the stratum corneum of the rat changed dramatically during the first hours of postnatal life. During late foetal development, filaggrin accumulated through the entire thickness of the stratum corneum, indicating that there was a block on the subsequent processing of the protein which normally would convert it to free amino acids. Immediately after birth this block was lifted and normal proteolysis of the filaggrin took place in the outer part of the stratum corneum, leaving the normal adult pattern of a thin zone of cells containing filaggrin at the bottom of the stratum corneum. This activation of filaggrin proteolysis was dependent on the drop in external water activity caused by the transition from an aqueous environment in utero to a dryer environment after birth and it could be blocked by maintaining a 100% humidity atmosphere around the newborn rat after birth. In isolated stratum corneum in vitro, filaggrin proteolysis took place only between 80 and 95% relative humidity, both higher and lower relative humidity blocked the proteolysis. Application of occlusive patches to adult rats prevented the normal proteolysis of filaggrin, indicating that this mechanism controls not only the massive filaggrin proteolysis occurring after birth but also the proteolysis occurring during normal stratum corneum maturation. The stratum corneum therefore has the ability to respond to changes in external humidity by altering the level of the stratum corneum where it converts its reserves of filaggrin into water binding amino acids, such that under humid conditions water binding components will be produced in only the most superficial stratum corneum, or even not produced at all.     

Comparison of rat epidermal keratinocyte organotypic culture (ROC) with intact human skin: lipid composition and thermal phase behavior of the stratum corneum.

The present report is a part of our continuing efforts to explore the utility of the rat epidermal keratinocyte organotypic culture (ROC) as an alternative model to human skin in transdermal drug delivery and skin irritation studies of new chemical entities and formulations. The aim of the present study was to compare the stratum corneum lipid content of ROC with the corresponding material from human skin. The lipid composition was determined by thin-layer chromatography (TLC) and mass-spectrometry, and the thermal phase transitions of stratum corneum were studied by differential scanning calorimetry (DSC). All major lipid classes of the stratum corneum were present in ROC in a similar ratio as found in human stratum corneum. Compared to human skin, the level of non-hydroxyacid-sphingosine ceramide (NS) was increased in ROC, while alpha-hydroxyacid-phytosphingosine ceramide (AP) and non-hydroxyacid-phytosphingosine ceramides (NP) were absent. Also some alterations in fatty acid profiles of ROC ceramides were noted, e.g., esterified omega-hydroxyacid-sphingosine contained increased levels of oleic acid instead of linoleic acid. The fraction of lipids covalently bound to corneocyte proteins was distinctly lower in ROC compared to human skin, in agreement with the results from DSC. ROC underwent a lipid lamellar order to disorder transition (T2) at a slightly lower temperature (68 degrees C) than human skin (74 degrees C). These differences in stratum corneum lipid composition and the thermal phase transitions may explain the minor differences previously observed in drug permeation between ROC and human skin.     

Study of human stratum corneum and extracted lipids by thermomicroscopy and DSC

A study on the thermal behavior of human stratum corneum and lipids is described. The use of high scanning rate DSC for both SC and extracted lipids allows the consistent determination of transition temperatures, including those of lower energy. Changes are found both at physiological and higher temperatures. There is a clear correspondence between the thermotropic behavior of these two systems. However, one of the transitions found in human SC (approximately 55 degrees C) is absent in extracted lipids and may be ascribed to those covalently-linked to corneocytes. Lipidic thermotropic behavior is clearly found above 100 degrees C, in which proteins do not play an exclusive role. Changes related to most transitions are observed directly by polarized light thermal microscopy in extracted lipids. This technique also allowed for the observation of large segregated domains in the extracted lipids. A drastic change is observed at approximately 60 degrees C, corresponding to the disruption of the lamellar structure.     

Deuterium NMR investigation of polymorphism in stratum corneum lipids

The intercellular lipid lamellae of stratum corneum constitute the major barrier to percutaneous penetration. Deuterium magnetic resonance and freeze-fracture electron microscopic investigation of hydrated lipid mixtures consisting of ceramides, cholesterol, palmitic acid and cholesteryl sulfate and approximating the stratum corneum intercellular lipid composition, revealed thermally induced polymorphism. The transition temperature of bilayer to hexagonal transition decreased as the ratio of cholesterol to ceramides in these mixtures was lowered. Lipid mixtures in which the stratum corneum ceramides were replaced by synthetic dipalmitoylphosphatidylcholine did not show any polymorphism throughout the temperature range used in the present study. The ability of the ceramide-containing samples to form hexagonal structures establishes a plausible mechanism for the assembly of the stratum corneum intercellular lamellae during the final stages of epidermal differentiation. Also, the bilayer to hexagonal phase transition of these nonpolar lipid mixtures could be used to enhance the penetration of drugs through skin.     

Infrared spectroscopic study of stratum corneum model membranes prepared from human ceramides, cholesterol, and fatty acids

The outermost layer of the skin, the stratum corneum, consists of corneocytes surrounded by lipid domains. The main lipid classes in stratum corneum are cholesterol, ceramides (CER), and free fatty acids forming two crystalline lamellar phases. However, only limited information is available on whether the various lipid classes participate in the same crystalline lattices or if separate domains are formed within the lipid lamellae. In this article infrared spectroscopic studies are reported of hydrated mixtures prepared from cholesterol, human CER, and free fatty acids. Evaluation of the methylene stretching vibrations revealed a conformational disordering starting at approximately 60 degrees C for all mixtures. Examination of the rotational ordering (scissoring and rocking vibrations) of mixtures prepared from equimolar cholesterol and CER with a variation in the level of free fatty acids showed that at lower free fatty acid content orthorhombic and hexagonal domains coexist in the lipid lamellae. Increasing the fatty acid level to an equimolar cholesterol/CER/fatty acid mixture reveals the dominant presence of an orthorhombic lattice, confirming x-ray diffraction studies. Replacing the protonated free fatty acid chains by their perdeuterated counterparts demonstrates that free fatty acids and CER participate in the same orthorhombic lattice up to a level of slightly less than 1:1:0.75 cholesterol/CER/free fatty acids molar ratio but that free fatty acids also form separate domains within the lipid lamellae at equimolar ratios at room temperature. However, no evidence for this has been observed at 32 degrees C. Extrapolating these findings to the situation in stratum corneum led us conclude that in stratum corneum, fatty acids and CER participate in the orthorhombic lattice at 32 degrees C, the skin temperature.     

Stratum corneum hydration: phase transformations and mobility in stratum corneum, extracted lipids and isolated corneocytes

The outermost layer of skin, stratum corneum (SC), functions as the major barrier to diffusion. SC has the architecture of dead keratin filled cells embedded in a lipid matrix. This work presents a detailed study of the hydration process in extracted SC lipids, isolated corneocytes and intact SC. Using isothermal sorption microcalorimetry and relaxation and wideline (1)H NMR, we study these systems at varying degrees of hydration/relative humidities (RH) at 25 degrees C. The basic findings are (i) there is a substantial swelling both of SC lipids, the corneocytes and the intact SC at high RH. At low RHs corneocytes take up more water than SC lipids do, while at high RHs swelling of SC lipids is more pronounced than that of corneocytes. (ii) Lipids in a fluid state are present in both extracted SC lipids and in the intact SC. (iii) The fraction of fluid lipids is lower at 1.4% water content than at 15% but remains virtually constant as the water content is further increased. (iv) Three exothermic phase transitions are detected in the SC lipids at RH=91-94%, and we speculate that the lipid re-organization is responsible for the hydration-induced variations in SC permeability. (v) The hydration causes swelling in the corneocytes, while it does not affect the mobility of solid components (keratin filaments).     

Phenotypic flexibility in cutaneous water loss and lipids of the stratum corneum in house sparrows (Passer domesticus) following acclimation to high and low humidity

Resistance to water-vapor diffusion through the skin is thought to be conferred by lipids in the stratum corneum (SC), the outer layer of the epidermis. We tested the effect of ambient humidity on cutaneous water loss (CWL) and lipid composition of the SC by acclimating house sparrows (Passer domesticus) to either a dry (6.5 g/m(3) absolute humidity) or a humid (31 g/m(3)) environment for 3 wk at a thermoneutral temperature (30 degrees C). Sparrows in the dry-acclimated group reduced CWL by 36% compared with those in the humid environment. Relative to initial values, both groups of sparrows decreased CWL, 45% in the dry-acclimated group and 23% in the humid group, suggesting that temperature is also an important stimulus for CWL apart from humidity. Both groups of acclimated sparrows decreased quantities of cholesterol, free fatty acids, and cerebrosides and increased the proportion of ceramides in their SC. Lipid amounts or proportions in the SC did not differ between dry- and humid-acclimated sparrows, but the free fatty acid : ceramide ratio was significantly lower in dry-acclimated birds. Also, lipid composition was only correlated with CWL in dry-acclimated sparrows, suggesting that structural changes to SC lipids are more tightly linked to CWL regulation in response to low humidity. Our results demonstrate phenotypic flexibility in CWL and lipid composition of the SC and provide support for a functional relationship between these traits.     

Ethanol effects on the stratum corneum lipid phase behavior.

The stratum corneum is considered to be the diffusional barrier of mammalian skin for water and most solutes. The intercellular lipid multilayer domains of the stratum corneum are believed to be the diffusional pathway for most lipophilic solutes. Fluidization of the lipid multilayers in the presence of ethanol is frequently conceived to result in enhanced permeation. Current investigations address the effect of ethanol on the phase behavior in terms of stratum corneum lipid alkyl chain packing, mobility and conformational order as measured by Fourier transform infrared (FTIR) spectroscopy. Phospholipid multilamellar vesicles were also studied as model systems. There appeared to be no effect of ethanol on either the solid-solid phase transition or the gel phase interchain coupling of the stratum corneum lipids. However, there was a reduction in the mobility of the alkyl chains in the presence of ethanol. Possible mechanistic relationships between the current FTIR spectroscopic results with available literature data of ethanol induced lipophilic solute penetration enhancement through the skin are discussed.     

Heterogeneous drying stresses in stratum corneum

We study the drying of stratum corneum, the skin's outermost layer and an essential barrier to mechanical and chemical stresses from the environment. Even though stratum corneum exhibits structural features across multiple length-scales, contemporary understanding of the mechanical properties of stratum corneum is based on the assumption that its thickness and composition are homogeneous. We quantify spatially resolved in-plane traction stress and deformation at the interface between a macroscopic sample of porcine stratum corneum and an adherent deformable elastomer substrate. At length-scales greater than a millimeter, the skin behaves as a homogeneous elastic material. At this scale, a linear elastic model captures the spatial distribution of traction stresses and the dependence of drying behavior on the elastic modulus of the substrate. At smaller scales, the traction stresses are strikingly heterogeneous and dominated by the heterogeneous structure of the stratum corneum.     

beta-Glucocerebrosidase activity in the stratum corneum of house sparrows following acclimation to high and low humidity

Skin is an important avenue of water loss in terrestrial birds, so environmental conditions that necessitate water conservation should favor physiological mechanisms that reduce cutaneous water loss (CWL). Skin resistance to CWL is conferred by a barrier of lipid molecules located in the stratum corneum (SC), the outer layer of the epidermis. In mammals, SC barrier function depends on the conversion of cerebrosides to ceramides by the enzyme beta -glucocerebrosidase ( beta -GlcCer'ase). Avian SC contains both cerebrosides and ceramides, suggesting that observed plasticity in CWL may be mediated by changes in beta -GlcCer'ase activity and resultant SC lipid composition. We tested the hypothesis that changes in ambient humidity would alter beta -GlcCer'ase activity by acclimating house sparrows (Passer domesticus) to either dry (6.5 g H(2)O m(-3) absolute humidity) or humid (31 g H(2)O m(-3)) conditions for 5 and 21 d at 30 degrees C and then measuring beta -GlcCer'ase activity from SC homogenates. Our results provide the first characterization of beta -GlcCer'ase activity in any nonmammalian vertebrate. Relative to nonacclimated controls, both dry- and humid-acclimated sparrows had significantly elevated beta -GlcCer'ase activity at 21 d postacclimation. Across individuals, we observed negative correlations between beta -GlcCer'ase activity and both CWL and SC ceramide content. Although dry- and humid-acclimated sparrows did not differ in beta -GlcCer'ase activity, these results are consistent with our findings that both humidity treatments caused a reduction in CWL and similar changes in SC lipid composition. Our results demonstrate physiological plasticity in CWL and provide tentative support for a role of beta -GlcCer'ase in mediating this response.     

Hydration of stratum corneum

A model for the hydration behavior of human stratum corneum has been developed from measurements on in vitro samples isolated in a manner which minimized tissue treatment and trauma. Water sorption/desorption rate measurements as a function of water activity, temperature, and tissue integrity are reported. These data, together with thermodynamic data (infrared and nmr results reported earlier) are consistent with a model in which rapidly sorbed/desorbed water (ca. 0.5 mg water/mg stratum corneum) is associated with (“bound” by) the tissue, while slowly sorbed/desorbed “free” water (up to 12 mg water/mg stratum corneum) is kinetically restricted and probably intracellular in location. Both equilibrium water binding and desorption kinetic data suggest structural changes of this cellular water barrier upon hydration. Evidence for hysteresis in water sorption isotherms (reported by others) could not be reproduced.     

Mechanical and failure behaviour of the stratum corneum

The load-deformation-time behaviour of heat-separated human stratum corneum was investigated using a pure shear specimen geometry. The tissue displayed non-linear load-deformation behaviour and stress relaxation, although the extensibility and amount of stress relaxation was considerably less than that shown by other soft connective tissues. Controlled failure tests were carried out after an edge cut had been made in the pure shear specimen. Sources of secondary failure, either at the free edge of the specimen, or due to the presence of inhomogeneities in the tissues, were common. Analysis of the test results suggested that the fracture surface energy of stratum corneum has a mean value of 3.6 kJ m-2 which is comparable with the tougher synthetic polymers.     

Stratum corneum hydration: Phase transformations and mobility in stratum corneum, extracted lipids and isolated corneocytes

The outermost layer of skin, stratum corneum (SC), functions as the major barrier to diffusion. SC has the architecture of dead keratin filled cells embedded in a lipid matrix. This work presents a detailed study of the hydration process in extracted SC lipids, isolated corneocytes and intact SC. Using isothermal sorption microcalorimetry and relaxation and wideline ¹H NMR, we study these systems at varying degrees of hydration/relative humidities (RH) at 25 °C. The basic findings are (i) there is a substantial swelling both of SC lipids, the corneocytes and the intact SC at high RH. At low RHs corneocytes take up more water than SC lipids do, while at high RHs swelling of SC lipids is more pronounced than that of corneocytes. (ii) Lipids in a fluid state are present in both extracted SC lipids and in the intact SC. (iii) The fraction of fluid lipids is lower at 1.4% water content than at 15% but remains virtually constant as the water content is further increased. (iv) Three exothermic phase transitions are detected in the SC lipids at RH = 91-94%, and we speculate that the lipid re-organization is responsible for the hydration-induced variations in SC permeability. (v) The hydration causes swelling in the corneocytes, while it does not affect the mobility of solid components (keratin filaments).     

Stratum corneum lipid phase transitions and water barrier properties

In mammals, the outer skin layer, the stratum corneum, is the ultimate barrier to water loss. In order to relate barrier function to stratum corneum structure, samples from porcine skin were investigated by using differential scanning calorimetry (DSC), infrared (IR) spectroscopy, and water permeability techniques. Results of DSC and IR studies show that stratum corneum lipids undergo thermal transitions between 60 and 80 degrees C similar to lipid thermotropic transitions seen in a variety of synthetic and biological membranes. Results of water flux experiments performed under conditions similar to those of the DSC and IR studies show an abrupt change in permeability at about 70 degrees C. At low temperatures, water flux values are similar to those obtained for human skin in vivo, yielding an activation energy of 17 kcal/mol, in excellent agreement with values obtained for water flux through a variety of lipid biomembranes. In contrast, at temperatures above about 70 degrees C, water flux is characterized by an activation energy only slightly higher than that of free diffusion, suggesting that the stratum corneum offers little diffusional resistance under these conditions. These combined results suggest that increased disorder in stratum corneum lipid structure, brought about by thermotropic transitions, results in dramatically altered diffusional resistance of this tissue to water flux. Thus, as found for numerous biological membranes, water flux and lipid order in porcine stratum corneum are inversely related.