Linear shear response of the upper skin layers

This study presents an in vitro experimental method to determine shear properties of the epidermis. Shear tests were performed with a parallel plate rheometer on samples of stratum corneum and the viable epidermis. The method was validated on very thin silicon sheets. Preliminary test were performed to determine the linear viscoelastic range, the effect of normal loading on the sample and the time to reach equilibrium after changes of temperature and relative humidity. The study shows that reproducible results can be obtained for the shear properties of epidermis in an in vitro set up. The dynamic shear modulus for stratum corneum ranges from about 4-12 kPa, decreasing with increasing relative humidity. The values are considerably lower than the shear modulus value based on tensile Young's moduli in the literature, indicating a considerable anisotropic material behavior. Results for the epidermis were of the same order of magnitude, but were less consistent possibly due to a less well-defined tissue composition.     

Free sphingosines in porcine epidermis

Sphingosines and phytosphingosines serve as intermediates in the synthesis of ceramides and glucosylceramides, which are prominent components of mammalian epidermis. In the present study, we have investigated the possibility that free sphingoid bases also may be present in epidermal tissue. Samples of pig epidermis were trypsinized to separate the stratum corneum from the unkeratinized portion of the epidermis. After drying, the lipids were extracted and analyzed by thin-layer chromatography using ninhydrin to detect free amino groups. Both the stratum corneum and the unkeratinized epidermal material contained a ninhydrin-positive material with the same mobility as the sphingosine standard. Quantitation of the chromatograms by photodensitometry indicated that free sphingosine bases account for 0.44% by weight of the total stratum corneum lipid and 0.09% of the lipid in the viable portion of the epidermis. To further identify this material, it was treated with 1-fluoro-2,4-dinitrobenzene, which resulted in the production of an intensely yellow N-2,4-dinitrophenyl derivative with the same mobility as N-2,4-dinitrophenylsphingosine on thin-layer chromatography. Oxidation of the isolated dinitrophenyl derivative with lead tetraacetate produced a mixture of aldehydes which were analyzed by gas-liquid chromatography. This analysis indicates that the free sphingoid bases from the stratum corneum consist of a mixture of mainly 16- through 20-carbon sphingenines and sphinganines, the most abundant components being d17:0, d17:1, d18:1 and d20:1. The production of these free sphingosine bases may be significant in regulating epidermal differentiation.     

The effects of layer properties on shear disturbance propagation in skin

The effects of the stratum corneum and dermis on shear wave propagation along the skin surface was investigated using a mathematical model. The skin was modeled as two distinct viscoelastic layers, one representing the stratum corneum and the other representing the dermis. The layers were supported by a semi-infinite visco-elastic half-space representing the subcutaneous fat. Physical and mechanical properties of the materials in the model were determined from the literature and from our own experimental measurements. Although the stratum corneum is very thin (12-15 microns), results showed that it could have a strong effect on the wave propagation due to its high stiffness relative to the dermis. Results of the analysis are discussed with respect to an experimental procedure used to determine age-related changes in mechanical properties of skin.     

Modified distribution of epidermal glycoproteins in the nude mouse

The nude mouse is an athymic mutant whose immunological deficiency has been exploited for transplantation of normal and diseased xenogeneic tissue. Histologically, its skin has no unusual features apart from the absence of hair. We report here a biochemical study of its epidermis, with comparison to the hairless mouse (which is devoid of hair but otherwise functionally normal). The epidermal glycoproteins were probed with the lectin, concanavalin A (Con A). Fluorescein isothiocyanate (FITC)-Con A overlays of cryostat skin sections gave a similar fluorescent pattern for both mouse strains: all the viable epidermal cell layers were labeled but not the stratum corneum. In contrast, when different populations of keratinocytes that were separated on Percoll gradients were analyzed by gel electrophoresis, and the gels then overlaid with iodinated Con A, all the epidermal layers, including the stratum corneum, were labeled. For all the epidermal cell layers there are substantial differences between the two mouse strains. We observe changes in the glycoprotein distribution with the stage of differentiation. Comparison with our earlier data for human epidermis indicates that the discrepancies between the nude mouse and the hairless mouse are much greater than those between the latter and man. The most striking difference is the absence in the stratum corneum of the nude mouse of a 40 K glycoprotein which is the dominant feature for the hairless mouse and for man. The gel patterns point to functional discrepancies in the epidermis of the nude mouse, particularly in the stratum corneum, not evident histologically or with FITC-Con A.     

In vitro measurement of the mechanical properties of skin by nano/microindentation methods

The elastic behaviors of stratum corneum, viable epidermis, dermis, and whole skin were investigated by nano/microindentation techniques. Insignificant differences in reduced elastic modulus of skin samples obtained from three different porcine breeds revealed breed type independent measurements. The reduced elastic modulus of stratum corneum is shown to be about three orders of magnitude higher than that of dermis. As a result, for relatively shallow and deep indentations, skin elasticity is controlled by that of stratum corneum and dermis, respectively. Skin deformation is interpreted in the context of a layered structure model consisting of a stiff and hard surface layer on a compliant and soft substrate, supported by microscopy observations and indentation measurements.     

Skin viscoelasticity studied in vitro by microprobe-based techniques

Time-dependent deformation of porcine skin was studied in vitro using specialized microprobe instruments. The deformation behavior of stratum corneum, dermis, and whole skin is examined in the context of results of creep strain, elastic stiffness, and viscoelastic constants obtained in terms of the hold time, loading/unloading rate, and maximum indentation depth (load). Skin time-dependent deformation is significantly influenced by dermis viscoelasticity up to a critical indentation depth (load) beyond which it is controlled by the outermost hard epidermis, particularly stratum corneum. Skin viscoelastic behavior under constant load (creep) and constant displacement (stress relaxation) is interpreted in the light of phenomenological observations and experimental trends.     

Human epidermal lipids: characterization and modulations during differentiation

Using thin-layer chromatography and glass capillary gas-liquid chromatography, we have quantitated the lipids in the germinative, differentiating, and fully cornified layers in human epidermis. As previously noted in nonhuman species, we found progressive depletion of phospholipids coupled with repletion of sterols and sphingolipids during differentiation. The sphingolipids, present only in small quantities in the lower epidermis, accounted for about 20% of the lipid in the stratum corneum, and were the major repository for the long-chain fatty acids that predominate in the outer epidermis. Although the absolute quantities of sphingolipids increased in the outer epidermis, the glycolipid:ceramide ratio diminished in the stratum corneum, and glycolipids virtually disappeared in the outer stratum corneum. Squalene and n-alkanes were distributed evenly in all epidermal layers, suggesting that these hydrocarbons are not simply of environmental or pilosebaceous origin. Cholesterol sulfate, previously considered only a trace metabolite in epidermis, was found in significant quantities, with peak levels immediately beneath the stratum corneum in the stratum granulosum. These studies: 1) provide new quantitative data about human epidermal lipids; 2) implicate certain classes of lipids for specific functions of the stratum corneum; and, 3) shed light on possible product-precursor relationships of these lipids.     

Avian permeability barrier function reflects mode of sequestration and organization of stratum corneum lipids: reevaluation utilizing ruthenium tetroxide staining and lipase cytochemistry.

The epidermis of avians and terrestrial mammals has evolved distinct, but related mechanisms to survive in a terrestrial environment. In both phyla, stratum corneum lipids form the basis of the cutaneous permeability barrier, but barrier function is less efficient in avians. Whereas in mammals the epidermal lamellar body (LB) secretes its contents into the intercellular spaces, in the feathered epidermis of avians, its distinctive secretory organelle, the multigranular body (MGB), does not secrete its contents into the stratum corneum intercellular spaces. Yet, neutral lipid-enriched droplets, derived from the cytosolic breakdown of MGB, ultimately are squeezed through membrane pores into the stratum corneum interstices. In this study we determined: a) using ruthenium tetroxide (RuO4) fixation, whether these droplets form membrane structures after deposition in the stratum corneum interstices; and b) the similarities and differences between avian MGB and mammalian LB, using enzyme cytochemistry as a marker for secretion, and optical diffraction computer-aided image analysis and reconstruction to compare the internal structure of MGB vs. LB. MGB were shown to possess a similar lamellar substructure to LB in RuO4-fixed specimens, exhibiting comparable dimensions on optical diffraction and computer transform analysis. Moreover, the intercellular lipids of avian stratum corneum lacked membrane-substructure, as was present in parallel samples of mammalian stratum corneum. Thus, both the absence of MGB secretion, and the failure of intercellular lipids to form membrane bilayers may explain the inherent differences in barrier function in these two taxa.     

Two-photon fluorescence lifetime imaging of the skin stratum corneum pH gradient

Two-photon fluorescence lifetime imaging is used to identify microdomains (1-25 microm) of two distinct pH values within the uppermost layer of the epidermis (stratum corneum). The fluorophore used is 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF), whose lifetime tau (pH 4.5, tau = 2.75 ns; pH 8.5, tau = 3.90 ns) is pH dependent over the pH range of the stratum corneum (pH 4.5 to pH 7.2). Hairless mice (SKH1-hrBR) are used as a model for human skin. Images (< or =50 microm x 50 microm) are acquired every 1.7 microm from the stratum corneum surface to the first viable layer (stratum granulosum). Acidic microdomains (average pH 6.0) of variable size (~1 microm in diameter with variable length) are detected within the extracellular matrix of the stratum corneum, whereas the intracellular space of the corneocytes in mid-stratum corneum (25 microm diameter) approaches neutrality (average pH 7.0). The surface is acidic. The average pH of the stratum corneum increases with depth because of a decrease in the ratio of acidic to neutral regions within the stratum corneum. The data definitively show that the stratum corneum acid mantle results from the presence of aqueous acidic pockets within the lipid-rich extracellular matrix.     

Avian sebokeratocytes and marine mammal lipokeratinocytes: structural, lipid biochemical, and functional considerations

In terrestrial mammals, stratum corneum lipids derive from two sources: deposition of lamellar body lipids in stratum corneum interstices and excretion of sebaceous lipids onto the skin surface, resulting in a two-compartment ("bricks and mortar") system of lipid-depleted cells surrounded by lipid-enriched intercellular spaces. In contrast, intracellular lipid droplets, normally not present in the epidermis of terrestrial mammals, are prominent in avian and marine mammal epidermis (cetaceans, manatees). We compared the transepidermal water loss, ultrastructure, and lipid biochemistry of the viable epidermis and stratum corneum of pigeon apterium, fledgling (featherless) zebra finches, painted storks, cetaceans, and manatees to those of humans and mice. Marine mammals possess an even more extensive lamellar-body secretory system than do terrestrial mammals; and lamellar-body contents, as in terrestrials, are secreted into the stratum corneum interstices. In cetaceans, however, glycolipids, but not ceramides, persist into the stratum corneum; whereas in manatees, glycolipids are replaced by ceramides, as in terrestrial mammals. Acylglucosylceramides, thought to be critical for lamellar-body deposition and barrier function in terrestrial mammals, are present in manatees but virtually absent in cetaceans, a finding that indicates that they are not obligate constituents of lamellar-body-derived membrane structures. Moreover, cetaceans do not elaborate the very long-chain, saturated N-acyl fatty acids that abound in terrestrial mammalian acylglucosylceramides. Furthermore, cold-water marine mammals generate large, intracellular neutral lipid droplets not found in terrestrial and warm-water marine mammals; these lipid droplets persist into the stratum corneum, suggesting thermogenesis, flotation, and/or cryoprotectant functions. Avians generate distinctive multigranular bodies that may be secreted into the intercellular spaces under xerotic conditions, as in zebra fledglings; ordinarily, however, the internal lamellae and limiting membranes deteriorate, generating intracellular neutral lipid droplets. The sphingolipid composition of avian stratum corneum is intermediate between terrestrials and cetaceans (approximately equal to 50% glycolipids), with triglycerides present in abundance. In the midstratum corneum of avians, neutral lipid droplets are released into the interstices, forming a large extracellular, lipid-enriched compartment, surrounding wafer-thin corneocytes, with a paucity of both lipid and keratin ("plates-and-mortar" rather than the "bricks-and-mortar" of mammals).(ABSTRACT TRUNCATED AT 400 WORDS)     

Biological and physical factors influencing the successful engraftment of a cultured human skin substitute

Skin tissue may be engineered in a variety of ways. Our cultured skin substitute (Graftskin, living skin equivalent or G-LSE), Apligraftrade mark, is an organotypic culture of skin, containing both a "dermis" and "epidermis." The epidermis is an important functional component of skin, responsible for biologic wound closure. The epidermis possesses a stratum corneum which develops with time in culture. The stratum corneum provides barrier function properties and gives the LSE improved strength and handling characteristics. Clinical experience indicated that the stratum corneum might play an important role in improving the clinical utility of the LSE. Handling and physical characteristics improved with time in culture. We examined the LSE at different stages of epidermal maturation for barrier function and ability to persist as a graft. LSE grafted onto athymic mice before significant development of barrier function did not withstand bandage removal at 7 days postgraft. LSE grafted after barrier function had been established in vitro were able to withstand bandage removal at day 7. Corneum lipid composition and structure are critical components for barrier function. Media modifications were used in an attempt to improve the fatty acid composition of the stratum corneum. The barrier developed more rapidly and was improved in a serum-free, lipid-supplemented condition. Lipid lamellar structure was improved with 10% of the stratum corneum exhibiting broad-narrow-broad lipid lamellar arrangements similar to human skin. Fatty acid metabolism was not appreciably altered. Barrier function in vitro was 4- to 10-fold more permeable than human skin. Epidermal differentiation does not compromise engraftment or the wound healing ability of the epidermis. The stratum corneum provides features beneficial for engraftment and clinical use. (c) 1996 John Wiley & Sons, Inc.     

Topical antioxidant vitamins C and E prevent UVB-radiation-induced peroxidation of eicosapentaenoic acid in pig skin

Eicosapentaenoic acid protects against UV-radiation-induced immunosuppression and photocarcinogenesis, but it is also prone to oxidative degradation, which may reduce or abolish its beneficial effects. The protective effect of topically applied vitamin E, vitamin C, or both against UVB-radiation-induced lipid peroxidation in the presence of eicosapentaenoic acid was investigated using an ex vivo pig skin model. Changes in the bioavailability of both antioxidants induced by UV radiation were studied in different skin compartments. The UVB-radiation dose used (25 kJ/m2) was similar to that required to induce immunosuppression in BALB/c mice. Exposure of pig skin with an epidermal eicosapentaenoic acid content of 1.0 +/- 0.3 mol% to UVB radiation resulted in an 85% increase of epidermal lipid peroxidation (P < 0.005). Topical application of vitamin E or vitamin C 60 min prior to UVB irradiation resulted in a major increase in both antioxidants in the stratum corneum and viable epidermis (P < 0.05). Vitamin E and vitamin C completely protected against UVB-radiation-induced lipid peroxidation (P < 0.005), but compared to vitamin E, a 500-fold higher vitamin C dose was needed. UVB irradiation induced a vitamin E consumption of up to 100% in the stratum corneum and viable epidermis, and a vitamin C consumption of only 21% in the stratum corneum. Simultaneously applied vitamin E and vitamin C also completely protected against UVB-radiation-induced lipid peroxidation (P < 0.05), and lower antioxidant doses were needed compared to vitamin E or vitamin C alone. In the presence of vitamin C, epidermal vitamin E was more stable upon UVB irradiation (P < 0.05), suggesting interaction between vitamin E and vitamin C. In conclusion, topically applied vitamin E and/or vitamin C efficiently protect against UVB-radiation-induced lipid peroxidation in the presence of eicosapentaenoic acid. The beneficial biological effects of eicosapentaenoic acid may therefore be improved if vitamin E and/or vitamin C are present in sufficient amounts. The ex vivo pig skin model provides a useful tool for assessing short-term biochemical effects related to UVB radiation, without the use of living experimental animals.     

AKT-dependent HspB1 (Hsp27) activity in epidermal differentiation

AKT activity has been reported in the epidermis associated with keratinocyte survival and differentiation. We show in developing skin that Akt activity associates first with post-proliferative, para-basal keratinocytes and later with terminally differentiated keratinocytes that are forming the fetal stratum corneum. In adult epidermis the dominant Akt activity is in these highly differentiated granular keratinocytes, involved in stratum corneum assembly. Stratum corneum is crucial for protective barrier activity, and its formation involves complex and poorly understood processes such as nuclear dissolution, keratin filament aggregation, and assembly of a multiprotein cell cornified envelope. A key protein in these processes is filaggrin. We show that one target of Akt in granular keratinocytes is HspB1 (heat shock protein 27). Loss of epidermal HspB1 caused hyperkeratinization and misprocessing of filaggrin. Akt-mediated HspB1 phosphorylation promotes a transient interaction with filaggrin and intracellular redistribution of HspB1. This is the first demonstration of a specific interaction between HspB1 and a stratum corneum protein and indicates that HspB1 has chaperone activity during stratum corneum formation. This work demonstrates a new role for Akt in epidermis.     

Structure of the epidermis of Australian Merino sheep over a 12-month period

Light-microscopic examination of frozen sections of skin taken from the dorsal thoraco-lumbar region of Australian Merino sheep in winter revealed that the thickness of the epidermis plus a sudanophilic layer was 24.9 micron in the interfollicular region. The uncornified epidermis (10.9 micron) was separated from the sudanophilic layer (14.0 micron) by a thin stratum corneum. It was concluded that the bulk of the sudanophilic layer was emulsified sebum in which was embedded a disorganized collection of desquamated cornified cells. Although large variances were observed in the thickness of the uncornified epidermis and of the sudanophilic layers between sheep and both within the between blocks of tissue obtained from individual sheep, there were no strong seasonal effects on either epidermal structure or layer thickness over a 12-month period. These results suggest that the Australian Merino differs from Finnish Landrace X Dorset Horn ewes, which are reported to possess, at least in winter, a thicker uncornified epidermis and a thicker stratum corneum that could be divided into two zones and was uniformly permeated by lipid.     

Degenerative and regenerative changes in epidemrmal organ culture: A morphological study wity reference to membrane-coating granules

Summary Membrane-coating granules (MCG) are poorly understood lamellate organelles unique to keratinized epithelia. This study provides data on a skin model for future in vitro investigations of MCG. Porcine ear epidermal, organ cultures were used under standard cell culture conditions. This system was selected because it is easily established and, following a degenerative period in which MCG are lost, regenerates to form a highly differentiated epidermis. The epidermis appeared healthy during the first 2 din vitro and contained MCG but lost keratohyalin granules (KHG). Overt degenerative changes were evident in the upper epidermis on Day 3, and MCG were now bloated. By Day 4 only one to three layers of viable undifferentiated cells remained. In the overlying necrotic epidermis MCG were rare, presumably due to the bursting of bloated MCG. Epidermal regeneration began around Day 5 and by Day 7 there, were 8 to 13 layers, including a, rudimentary parakeratotic stratum corneum (up to 4 layers). The stratum granulosum (two to three layers) now containe immature KHG and poorly lamellate MCG, but only amorphous material extracellularly. By Day 11 there were three to four layers of granular cells as in vivo, and an orthokeratotic stratum corneum (two to four layers). Improved cornification coincided with an increased number of mature KHG and cross-banded MCG, and lamellate MCG contents extracellularly. This model of epidermal regeneration will factiliate studies into the role played by MCG in keratinization because the epithelium initially lacked MCG but later expressed all the major morphologic features of epidermis. Furthermore the mechanisms by which MCG translaction and extrusion are effected may be probed, by the inclusion of such agents as antimicrotubular drugs and calcium ionophores. This work was supported by a grant from Unilever Research, Port Sunlight, England.     

Interactions Between non-ionic Surfactant Vesicles and human stratum corneum in vitro.

Abstract

The effects of non-ionic surfactant vesicles (NSVs) on human skin in vitro were studied in relation to the physico-chemical properties of the vesicles. The interactions between NSVs and skin were visualized using both freeze fracture electron microscopy and confocal laser scanning microscopy. The physico-chemical properties of the NSVs were varied in a systematic way, using a broad series of polyoxyethylene monoalkyl ether type surfactants (C_nEO_m). The number of oxyethylene units (m) was varied between 3, 7 and 10, and the number of carbon atoms (n) was either 12 or 18. Both the effects of liquid state vesicles composed of ^C12^Eo3,7 ^and C9=9^EO10 surfactants and gel state vesicles (C18EO37) were investigated. After the application of the NS V suspension on the stratum corneum surface two essentially different types of vesicle-skin interactions were visualized. Firstly, an interfacial interaction, involving the adsorption of vesicles, and the deposition of bilayer sheets on the outermost layers of the stratum corneum observed for all NSV formulations tested in this study. Secondly, effects on the ultrastructure of the stratum corneum were observed: the appearance of water pools observed for the liquid state vesicles only, and ultrastructural changes of the intercellular lipid domains are induced only observed after treatment with C₁₂EO₃ NSVs. Neither changes in the ultrastructure of the viable epidermis, nor changes in deeper skin layers were observed.     

Mechanical properties of the axial skeleton in gorgonians

Axial skeletons of thirteen species representing a wide range of genera of Gorgonians were investigated using Young's modulus as a measure of stiffness and Torsion modulus as a measure of resistance to shear or twist. Atomic absorption spectroscopic determination of magnesium and calcium content as measures of mineralization were done. Relative quantities of calcareous material in the axial skeletons showed a strong linear correlation with Young's modulus and suggests an important role for calcareous material in the modulation of the mechanical properties of axoskeleton. Torsion moduli also showed a mathematical but non-linear relationship to calcareous content. Axis stiffness correlated well with zonation-related water movement. Stiffest axes occur in deeper water with no wave surge, most flexible in shallower water with moderate surge and intermediate stiffness in shallow, high energy habitats. An extremely high MgCO₃ containing carbonate that may be a previously unreported biological structural material was found in the Plexauridae.     

Identification of a Rapidly Labelled 350K Histidine-Rich Protein in Neonatal Mouse Epidermis

Abstract. The major histidine-rich protein (HRP) found in the stratum corneum of neonatal mouse epidermis (band 2 protein, molecular weight 27,000) is a relatively late product of epidermal differentiation and incorporates labelled amino acids in vivo only after a 6–9 h lag period. A number of putative precursor HRPs in the 70–300 K molecular weight range were initially identified using short pulse labelling times and our previously described methods for isolation of epidermis and extraction of proteins. However, when steps were taken to minimise proteolysis during preparation, a single species of approximately 350 K molecular weight was the most strongly labelled protein following a 1 h in vivo pulse of [³H]-histidine. This protein was stable in sodium dodecyl sulphate dithiothreitol at 100°C and in 4 M urea, suggesting a single covalently linked polypeptide. The kinetics of labelling and the localisation of the 350 K HRP in the lower granular layers suggest that it is a precursor of the stratum corneum HRP. The processing of the 350 K HRP to the stratum corneum species appears to involve a complex series of specific cleavage steps which give rise to a number of HRPs of intermediate molecular weight.     

Similarities between stratum corneum basic protein and histidine-rich protein II from newborn rat epidermis

The stratum corneum basic protein and histidine-rich protein II were each isolated from newborn rat epidermis and compared by biochemical and immunologic methods. The proteins were indistinguishable by immunodiffusion using antiserum elicited to either protein. The migration of the proteins on SDS-polyacrylamide gel electrophoresis was identical giving a molecular weight of 49 000. These proteins, which have similar but unusual amino acid compositions, give very similar tryptic peptide maps. Both proteins aggregate with keratin filaments to form macrofibrils. These results suggest that histidine-rich protein II and stratum corneum basic protein are the same protein. We suggest that this protein be called histidine-rich basic protein.     

Identification of a rapidly labelled 350K histidine-rich protein in neonatal mouse epidermis

The major histidine-rich protein (HRP) found in the stratum corneum of neonatal mouse epidermis (band 2 protein, molecular weight 27,000) is a relatively late product of epidermal differentiation and incorporates labelled amino acids in vivo only after a 6-9 h lag period. A number of putative precursor HRPs in the 70-300 K molecular weight range were initially identified using short pulse labeling times and our previously described methods for isolation of epidermis and extraction of proteins. However, when steps were taken to minimise proteolysis during preparation, a single species of approximately 350 K molecular weight was the most strongly labelled protein following a 1 h in vivo pulse of [3H]-histidine. This protein was stable in sodium dodecyl sulphate dithiothreitol at 100 degrees C and in 4 M urea, suggesting a single covalently linked polypeptide. The kinetics of labelling and the localisation of the 350 K HRP in the lower granular layers suggest that it is a precursor of the stratum corneum HRP. The processing of the 350 K HRP to the stratum corneum species appears to involve a complex series of specific cleavage steps which give rise to a number of HRPs of intermediate molecular weight.