Human skin equivalents are an excellent tool to study the effect of moisturizers on the water distribution in the stratum corneum.

In this study, the mode of action of moisturizers on the level of water in the stratum corneum was studied using cryo-scanning electron microscopy. As model for dry skin, we used human skin equivalents (HSEs) generated at 93% or 60% relative humidity (RH). During the generation of the HSEs, the moisturizers were applied during a period of maximal 2 weeks. In HSEs generated under normal culture conditions (93% RH), application of 10% glycerol or 5% urea formulations resulted in increased water levels. Whereas the 5% urea formulations resulted mainly in the formation of intercellular water domains, after 10% glycerol both swelling of corneocytes and formation of intercellular water domains were noticed. A reduction in RH to 60% during treatment reduced the stratum corneum water levels drastically. Treatment with the non-occlusive lipophilic moisturizer isopropyl isostearate resulted in increased water level in the central part of the stratum corneum compared with the untreated control. Our results show that HSEs can be used as a model to study the water distribution.

Leiden reconstructed human epidermal model as a tool for the evaluation of the skin corrosion and irritation potential according to the ECVAM guidelines.

In the ECVAM validation studies two common skin protocols have been developed, the skin corrosion and skin irritation protocol. Both protocols include next to general and functional conditions that the skin model must meet, also the correct prediction of the activity of certain reference chemicals. For the skin corrosion protocol, the OECD TG 431 defined 12 reference chemicals that should be correctly predicted by the epidermal skin model. For skin irritation 20 test substances should meet the defined criteria. In this study we aimed to subject our Leiden human epidermal (LHE) model to both common protocols according to the ECVAM guidelines. The LHE model generated in this study has been fully characterized and shows very high similarities with the native skin. After minor technical changes in both protocols, corrosion classifications were obtained in concordance with those reported for the validated human skin models EpiSkin and EpiDerm. The results obtained with the common skin irritation protocol were very similar to that of earlier studies with the SkinEthic, EpiSkin and EpiDerm models. This means that the protocols and prediction models developed during the validation studies with a specific skin model can be used with other similar skin models. This study demonstrates that reconstructed human skin equivalents have been proven to be efficient and reliable alternatives to animal testing.

Human adipose tissue-derived cells delay re-epithelialization in comparison with skin fibroblasts in organotypic skin culture.

BACKGROUND: Wound healing of deep and extensive burns can induce hypertrophic scar formation. During the early steps of wound healing fibroblasts migrate into the wounded area. Fibroblastic cells present in tissues other than dermis may also migrate into the wounded area and participate in the wound healing process. OBJECTIVES: To examine the influence of human fibroblastic cells derived from subcutaneous fat or dermis on epidermal morphogenesis in vitro. METHODS: We prepared human skin equivalents (HSEs) made of a collagen type I matrix populated either with dermal fibroblasts or adipose tissue-derived cells (ADCs), on top of which keratinocytes were seeded and subsequently grown at the air-liquid interface. RESULTS: A fully differentiated epidermis was formed on matrices populated with ADCs. However, the HSE formed differed in a number of features from HSE generated with dermal fibroblasts. The major differences included: marked contraction of the dermal matrix, low lateral migration of keratinocytes, high keratin 17 expression indicating increased keratinocyte activation, delayed deposition of collagen IV at the epidermal/matrix junction, accumulation of alpha-smooth muscle actin-positive cells only underneath the epidermal compartment and positioning of these cells in a direction parallel to the epidermal compartment. The latter two phenomena have also been found in scar tissue. CONCLUSIONS: The possibility of generating HSEs with different cell types represents an attractive approach for in vitro studies focusing on the mechanism of wound healing.

Analysis of epidermal lipids of the healthy human skin: factors affecting the design of a control population.

The intervariability of studies on the lipids of human epidermis and stratum corneum is high because of the different origin of the skin samples and the variety of extraction methods used. In the present work, a high-performance thin-layer chromatographic technique has been used to study the parameters age, sex, and anatomical site for their effects on the lipid profiles recovered from healthy epidermal skin biopsy specimens. It was found that sex-related differences were seen at the level of the total ceramide concentration. Observed decreases in lipid concentration, due to ageing, depended on the anatomical site. Therefore, these variables should be controlled in a reproducible and standardized way in order to be able to study the direct relationship between skin condition and barrier lipid composition. Only when this relation is established, results of topical treatment can be scientifically evaluated.

The phase behaviour of skin lipid mixtures based on synthetic ceramides.

The lipid lamellae present in the outermost layer of the skin, the stratum corneum (SC), form the main barrier for diffusion of molecules across the skin. The main lipid classes in SC are cholesterol (CHOL), free fatty acids (FFA) and at least nine classes of ceramides (CER), referred to as CER1 to CER9. In the present study the phase behaviour of four synthetic CER, either single or mixed with CHOL or CHOL and FFA, has been studied using small and wide angle X-ray diffraction. The lipid mixtures showed complex phase behaviour with coexistence of several phases. The results further revealed that the presence of synthetic CER1 as well as a proper composition of the other CER in the mixture were crucial for the formation of a phase with a long periodicity, characteristic for SC lipid phase behaviour. Only a mixture containing synthetic CER1 and CER3, CHOL and FFA showed similar phase behaviour to that of SC.

Effect of fibroblasts on epidermal regeneration.

BACKGROUND: There is little information on specific interactions between dermal fibroblasts and epidermal keratinocytes. The use of engineered skin equivalents consisting of organotypic cocultures of keratinocytes and fibroblasts offers an attractive approach for such studies. OBJECTIVES: To examine the role fibroblasts play in generation and maintenance of reconstructed epidermis. METHODS: Human keratinocytes were seeded on collagen matrices populated with increasing numbers of fibroblasts and cultured for 2 weeks at the air-liquid interface. RESULTS: In the absence of fibroblasts, stratified epidermis with only three or four viable cell layers was formed. In the presence of fibroblasts, keratinocyte proliferation was stimulated and epidermal morphology was improved. Epidermal morphogenesis was also markedly improved in epidermis generated in organotypic keratinocyte monocultures grown in medium derived from dermal equivalents or from organotypic keratinocyte-fibroblast cocultures. These observations clearly indicate the proliferation-stimulating activity of soluble factors released from fibroblasts. Under all experimental conditions, onset of keratinocyte differentiation was shown by the expression of keratin 10 in all suprabasal cell layers. With increasing numbers of fibroblasts incorporated into the collagen matrix, the expression of markers associated with keratinocyte activation, e.g. keratins 6, 16 and 17 and the cornified envelope precursor SKALP decreased, and involucrin localization shifted toward the granulosum layer. This fibroblast-mediated effect was even more pronounced when the fibroblasts were precultured in the collagen matrices for 1 week instead of overnight. The basement membrane proteins collagen VII and laminin 5 were present at the epithelial-matrix border. The expression of integrin alpha 6 beta 4 and of E-cadherin was comparable with that seen in native skin and was not significantly modulated by fibroblasts. Under all experimental conditions the expression of integrin subunits alpha 2, alpha 3 and beta 1 was upregulated, indicating keratinocyte activation. CONCLUSIONS: Our results illustrate that numbers of fibroblasts in the collagen matrix and their functional state is a critical factor for establishment of normal epidermal morphogenesis.

Proteomic analysis of skin irritation reveals the induction of HSP27 by sodium lauryl sulphate in human skin.

BACKGROUND: There is an increasing need for screening of mild irritants in vitro to reduce animal testing. OBJECTIVES: Proteomics were used to search for new markers of which the expression changes after mild irritation. METHODS: Sodium lauryl sulphate (SLS) was applied topically on excised human skin. Epidermal proteins were isolated from SLS-treated skin specimens that showed hardly any morphological changes. The proteins were analysed by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and proteins that significantly increased or decreased after SLS treatment in a dose-dependent way were characterized by mass spectrometry. Subsequently, immunohistochemistry was performed on skin samples treated with SLS in vivo and nonanoic acid (NAA) or benzalkonium chloride (BC) in vitro to evaluate one of the identified proteins for its predictive value. RESULTS: We identified seven proteins as potentially new epidermal markers for skin irritation. Among these seven proteins, the 27 kDa heat shock protein (HSP27) was identified as the most prominently upregulated protein. A strong nuclear HSP27 staining was seen in the SLS-treated skin, whereas in the vehicle controls only cytoplasmic staining was observed. Moreover, nuclear staining was also observed after topical application of SLS in vivo and after exposure to NAA and BC in vitro. CONCLUSIONS: Our findings suggest that HSP27 may serve as a sensitive marker of skin irritation and eventually as a novel tool in clinics for testing the sensitivity of the patient for a panel of irritants.

Non-animal testing strategies for assessment of the skin corrosion and skin irritation potential of ingredients and finished products.

The dermatotoxicologist today is faced with a dilemma. Protection of workers and consumers from skin toxicities (irritation and allergy) associated with exposure to products, and the ingredients they contain, requires toxicological skin testing prior to manufacture, transport, or marketing. Testing for skin corrosion or irritation has traditionally been conducted in animals, particularly in rabbits via the long established Draize test method. However, this procedure, among others, has been subject to criticism, both for its limited predictive capacity for human toxicity, as well as for its use of animals. In fact, legislation is pending in the European Union which would ban the sale of cosmetic products, the ingredients of which have been tested in animals. These considerations, and advancements in both in vitro skin biology and clinical testing, have helped drive an intensive effort among skin scientists to develop alternative test methods based either on in vitro test systems (e.g. using rat, pig or human skin ex vivo, or reconstructed human skin models) or ethical clinical approaches (human volunteer studies). Tools are now in place today to enable a thorough skin corrosion and irritation assessment of new ingredients and products without the need to test in animals. Herein, we describe general testing strategies and new test methods for the assessment of skin corrosion and irritation. The methods described, and utilized within industry today, provide a framework for the practicing toxicologist to support new product development initiatives through the use of reliable skin safety testing and risk assessment tools and strategies.

Lack of desquamation - the Achilles heel of the reconstructed epidermis.

The use of human skin equivalents for screening tests aiming to assess repetitive application of various test agents is hampered by the lack of desquamation in vitro. The present study was undertaken to examine whether the desquamation can be induced by various treatments including mechanical stress, application of various agents that should decrease the surface pH and calcium level, activate the enzymes involved in desquamation process or UV irradiation. In addition, the effect of alpha-hydroxyacids, known to enhance desquamation and to improve the stratum corneum barrier function in vivo, was examined as well. Human epidermis reconstructed on de-epidermized dermis or on fibroblast-populated collagen matrices during a 2-week culture at the air-liquid interface underwent various treatments during an additional 3-week period. The effects of treatments were evaluated on the basis of tissue morphology and lipid composition. The results of the present study revealed that cell shedding could only be induced by a mild repetitive mechanical treatment. The lack of desquamation, under most in vitro conditions, has a practical consequence, since it may hamper the use of reconstructed epidermis for various screening studies aiming to examine the repetitive exposure to topical agents or UV irradiation. The gradual thickening of the stratum corneum will lead to its higher resistance to the environmental stimuli and in this way affect the outcome of the tests. Furthermore, from the results obtained in the present study, it became evident that one should be careful in selecting endpoints when, for example, the effects of agents known to modulate melanogenesis are examined.

The lipid organisation of the skin barrier: liquid and crystalline domains coexist in lamellar phases.

The superficial layer of the skin, the stratum corneum, is the main barrier for diffusion of substances across the skin. The stratum corneum is composed of corneocytes embedded in lipid lamellae. In previous studies two lamellar phases have been identified with periodicities of 6.4 and 13.4 nm of which the 13.4 nm phase (long periodicity phase = LPP) is considered to be very important for the skin banier function. The main lipid classes in stratumcorneum are ceramides, free fatty acids and cholesterol. Until now 8 subclassesof ceramides are identified in human stratum corneum referred to as ceramide 1 to 8. Studies with mixtures prepared with isolated human ceramides revealed that cholesterol and ceramides are very important for the formation of the lamellar phases. After addition of free fatty acids the lipids are organised in an orthorhombic packing with a small proportion of lipids in a liquid phase. Our most recent results show that the presence of ceramide 1 and the formation of a liquid phase are crucial elements for the formation of the LPP. These observations and the broad-narrowbroad sequence of lipid layers in the LPP led us to propose a molecular model for this phase. This consists of one narrow central lipid layer with fluid domains with on both sides a broad layer with a crystalline structure. This model is referred to as `the sandwich model'.

Phase behavior of lipid mixtures based on human ceramides: coexistence of crystalline and liquid phases.

The lipid regions in the outermost layer of the skin (stratum corneum) form the main barrier for diffusion of substances through the skin. In this layer the main lipid classes are ceramides, cholesterol (CHOL), and FFA. Previous studies revealed a coexistence of two crystalline lamellar phases with periodicities of approximately 13 nm (referred to as long periodicity phase) and 6 nm (short periodicity phase). Additional studies showed that lipid mixtures prepared with isolated pig ceramides (pigCER) mimic lipid phase behavior in stratum corneum closely. Because the molecular structure of pigCER differs in some important aspects from that of human ceramides (HCER), in the present study the phase behavior of mixtures prepared with HCER has been examined. Phase behavior studies of mixtures based on HCER revealed that in CHOL:HCER mixtures the long periodicity phase dominates. In the absence of HCER1 the short periodicity phase is dominant. Addition of FFA promotes the formation of the short periodicity phase and induces a transition from a hexagonal sublattice to an orthorhombic sublattice. Furthermore, the presence of FFA promotes the formation of a liquid phase. Finally, cholesterol sulfate, a minor but important lipid in the stratum corneum, reduces the amount of cholesterol that phase separates in crystalline domains. From these observations it can be concluded that the phase behavior of mixtures prepared from HCER differs in some important aspects from that prepared from pigCER. The most prevalent differences are the following: i) the addition of FFA promotes the formation of the short periodicity phase; and ii) liquid lateral packing is obviously present in CHOL:HCER:FFA mixtures. These changes in phase behavior might be due to a larger amount of linoleic acid moiety in HCER mixtures compared with that in pigCER mixtures.

New aspects of the skin barrier organization.

In the superficial layer of the skin, the stratum corneum (SC), the lipids form two crystalline lamellar phases with periodicities of 6.4 and 13.4 nm (long-periodicity phase). The main lipid classes in SC are ceramides, free fatty acids and cholesterol. Studies with mixtures prepared with isolated ceramides revealed that cholesterol and ceramides are very important for the formation of the lamellar phases, and the presence of ceramide 1 is crucial for the formation of the long-periodicity phase. This observation and the broad-narrow-broad sequence of lipid layers in the 13.4-nm phase led us to propose a molecular model for this phase. This consists of one narrow central lipid layer with fluid domains on both sides of a broad layer with a crystalline structure. This model is referred to as 'the sandwich model'. While the presence of free fatty acids does not substantially affect the lipid lamellar organization, it is crucial for the formation of the orthorhombic sublattice, since the addition of free fatty acids to cholesterol/ceramide mixtures results in transition from a hexagonal to a crystalline lipid phase. Studies examining lipid organization in SC derived from dry or lamellar X-linked ichthyosis skin revealed that in native tissue the role of ceramide 1 and free fatty acids is similar to that observed with mixtures prepared with isolated SC lipids. From this we conclude that the results obtained with lipid mixtures can be used to predict the SC lipid organization in native tissue.

Barrier function in reconstructed epidermis and its resemblance to native human skin.

One of the prerequisites for the use of human skin equivalents for scientific and screening purposes is that their barrier function is similar to that of native skin. Using human epidermis reconstructed on de-epidermized dermis we demonstrated that the formation of the stratum corneum (SC) barrier in vitro proceeds similarly as in vivo as judged from the extensive production of lamellar bodies, their complete extrusion at the stratum granulosum/SC interface, and the formation of multiple broad lamellar structures in the intercorneocyte space. The presence of well-ordered lipid lamellar phases was confirmed by small-angle X-ray diffraction. Although the long periodicity lamellar phase was present in both the native and the reconstructed epidermis, the short periodicity lamellar phase was present only in native tissue. In addition, the SC lipids predominantly formed the hexagonal sublattice. Analysis of lipid composition revealed that all SC lipids are synthesized in vitro. Differences in SC lipid organization in reconstructed epidermis may be ascribed to the differences in fatty acid content and profile indicating that further improvement in culture conditions is required for generation of in vitro reconstructed epidermis with stratum barrier properties of the native tissue.

Determination of stratum corneum lipid profile by tape stripping in combination with high-performance thin-layer chromatography.

Intercellular lipids in the stratum corneum (SC) are responsible for the barrier function of mammalian skin. The main components of the SC lipids are ceramides, cholesterol, and free fatty acids, as established by thin-layer chromatographic analysis of lipids extracted from the human and mammalian SC. Up to now, for lipid analysis the extracts of the entire SC has been used and information on whether the lipid composition changes with the depth in the SC is scarce. Tape stripping is a technique which removes corneocyte layers step by step with an adhesive film. The use of this technique for lipid analysis was hampered by the contamination of lipid extracts with compounds co-extracted from the tape with organic solvents used for the extraction of SC lipids. The aim of the present study was to establish a suitable analytical method for the determination of the local SC lipid composition. For this purpose, the SC samples were collected by sequential stripping with Leukoplex tape in five healthy volunteers. The lipids were extracted with ethyl acetate:methanol mixture (20:80) and separated by means of HPTLC. The results of this study revealed that the free fatty acid level is highest and the cholesterol and ceramide levels lowest in the uppermost SC layers (about 4 strippings). The levels remained unchanged in the underlying SC layers. In these layers, the ceramide level was about 60 wt% and the free fatty acid and cholesterol levels were about 20 wt% each. Ceramides could be separated into seven different fractions and the relative amounts of individual ceramide fractions did not significantly change with the SC depth. Cholesterol sulfate levels were about 5% of total cholesterol and did not change with the SC depth, except for the for the first strip where the level was about 1%. The method developed makes it possible to study the differences in the SC lipid profile in healthy and diseased human skin with relation to the SC lipid organization and to the skin barrier function in vivo.

Phase behaviour of skin barrier model membranes at pH 7.4.

The main function of the skin is to protect the body against exogenous substances. The skin barrier is located in the outermost layer of the skin, the stratum corneum (SC). This layer consists of keratin enriched cells embedded in lipid lamellae that form the main barrier for diffusion of substances through the skin. The main lipid classes in this barrier are ceramides, cholesterol and free fatty acids. Cholesterol sulfate and calcium are also present in SC. Furthermore it has been suggested that a pH gradient exists. In a previous paper the effect of cholesterol sulfate and calcium on the lipid phase behaviour of mixtures prepared from cholesterol, ceramides and free fatty acids at pH 5 was reported (approximate pH at the skin surface). In the present study the phase behaviour of mixtures prepared from cholesterol, ceramides and free fatty acids prepared at pH 7.4 (the pH of viable cells) has been examined between 25 and 95 degrees C. Our studies reveal that a reversed hexagonal phase has been formed at elevated temperatures. Addition of calcium inhibits the formation of the reversed hexagonal phase, while cholesterol sulfate promotes the presence of the reversed hexagonal phase at increased temperatures. From our results we can conclude that the lipid mixtures prepared at pH 5 resemble more closely the lipid phase behaviour in intact SC than the lipid mixtures prepared at pH 7.4.

Lipid and ultrastructural characterization of reconstructed skin models.

The study aimed at evaluating tissue architecture and quality of the permeability barrier in commercially available reconstructed human skin models; EpiDerm, SkinEthic and Episkin in comparison to native tissue. For this purpose, tissue architecture was examined by electron microscopy and epidermal lipid composition was analyzed by HPTLC. Stratum corneum lipid organization was investigated by electron microscopy in combination with RuO(4) post-fixation and by SAXD. Ultrastructurally, the overall tissue architecture showed high similarities with native epidermis. In the stratum corneum extracellular space, lipid lamellae consisting of multiple alternating electron-dense and electron-lucent bands were present. This regular pattern was not seen throughout the whole stratum corneum probably due to the observed irregular lamellar body extrusion in some areas. Lipid analyses revealed the presence of all major epidermal lipid classes. Compared with native epidermis the content of polar ceramides 5 and 6 was lower, ceramide 7 was absent, and the content of free fatty acids was very low. These differences in lipid composition may account for differences observed in SAXD pattern of Episkin and EpiDerm penetration models. In the latter only the long-distance periodicity unit of about 12 nm was observed and the short periodicity unit was missing. In conclusion, all three skin models provide a promising means for studying the effects of topically applied chemicals, although the observed deviations in tissue homeostasis and barrier properties need to be optimized.

The lipid organisation in the skin barrier.

The main function of the skin is to protect the body against exogenous substances. The skin barrier is located in the outermost layer of the skin, the stratum corneum. This layer consists of keratin enriched cells embedded in lipid lamellae. These lamellae form the main barrier for diffusion of substances through the skin. In diseased skin the barrier function is often impaired. For a full understanding of the properties of the human skin barrier, insight in the stratum corneum lipid organisation is of great importance. In this paper a short description of the lipid organisation in normal human stratum corneum will be given, after which the role the main lipid classes play in the stratum corneum lipid organisation will be described. In addition the effect of cholesterol sulfate and calcium on the lipid organisation will be discussed. Finally a new model, the "sandwich model", will be proposed that describe the localisation of the fluid phases in the stratum corneum.

Epidermal growth factor and keratinocyte growth factor differentially regulate epidermal migration, growth, and differentiation.

Various growth factors such as epidermal growth factor and keratinocyte growth factor have been reported to promote wound closure and epidermal regeneration. In the present study epidermis reconstructed on de-epidermized dermis was used to investigate the effects of epidermal growth factor and keratinocyte growth factor on keratinocyte proliferation, migration and differentiation. Our results show that epidermal growth factor supplemented cultures share many of the features which are observed during regeneration of wounded epidermis: a thickening of the entire epidermis, an enhanced rate of proliferation and migration, and an increase in keratin 6, keratin 16, skin-derived antileukoproteinase, involucrin and transglutaminase 1 expression. The increase in transglutaminase 1 protein is accompanied by an increase in the amount of active transglutaminase 1 enzyme. Surprisingly no increase in keratin 17 is observed. Prolonging the culture period for more than two weeks results in rapid senescence and aging of the cultures. In contrast, keratinocyte growth factor supplemented cultures have a tissue architecture that is similar to healthy native epidermis and remains unchanged for at least 4 weeks of air-exposure. The rate of proliferation and the expression of keratins 6, 16 and 17, skin-derived antileukoproteinase, involucrin and transglutaminase 1 is similar to that found in healthy epidermis and furthermore keratinocyte migration does not occur. When the culture medium is supplemented with a combination of keratinocyte growth factor and a low concentration of epidermal growth factor, skin-derived antileukoproteinase, involucrin and keratins 6, 16 and 17 expression is similar to that found in cultures supplemented with keratinocyte growth factor alone and in healthy epidermis. Only high transglutaminase 1 expression remains similar to that observed in cultures supplemented with epidermal growth factor alone. Our results show that the regulation of keratinocyte growth, migration and differentiation depends on the availability of these growth factors. Epidermal growth factor may play a dominant early role in wound healing by stimulating keratinocyte proliferation and migration while keratinocyte growth factor may play a role later in the repair process by stabilizing epidermal turnover and barrier function.

Covalently bound lipids in reconstructed human epithelia.

The composition of free and covalently bound lipids in reconstructed epithelia generated with normal human keratinocytes, HaCaT cells and squamous carcinoma cells was investigated and compared with native skin. Stratum corneum isolated from native human and reconstructed epidermis was subjected to extensive extraction with chloroform-methanol mixtures followed by alkaline hydrolysis to release covalently bound lipids. High-performance thin layer chromatography was used for analysis of solvent-extractable and non-extractable lipids and gas liquid chromatography was performed to assess the fatty acid profile in extractable lipids. In both native and reconstructed tissue covalently bound lipids consisted of omega-hydroxyceramides, omega-hydroxyacids and free fatty acids. Small amounts of omega-hydroxyacids could already be detected in solvent-extractable fractions. omega-Hydroxyceramides consisted of Ceramide A, Ceramide B and a small fraction of unknown ceramides with intermediate polarity. The relative proportions of individual omega-hydroxyceramides were similar in both native and reconstructed stratum corneum. In contrast, differences were found in profiles of both solvent-extractable and non-extractable lipids isolated from epithelia reconstructed with transformed cell lines (HaCaT, SCC-12F2 and SCC-13 cells). Compared with native or reconstructed epidermis, in epithelia reconstructed with transformed cell lines the ceramide content was low, the most polar ceramides were missing and the content of free fatty acids was low. The same holds true for covalently bound lipids that were virtually absent in these epithelia. Marked similarities were demonstrated in the overall lipid composition of free and bound stratum corneum lipids in native epidermis and in epidermis reconstructed with normal human keratinocytes. The observed imbalance in fatty acid profile may account for differences in phase behaviour of stratum corneum lipids.

Characterization and comparison of reconstructed skin models: morphological and immunohistochemical evaluation.

Reconstructed human skin equivalents are currently being investigated as in vitro models for the prediction of human skin toxicity and irritation responses. Three different industrial reconstructed skin models (EpiDerm, Episkin and SkinEthic) and one in-house equivalent were characterized and compared using light microscopy, immunohistochemistry and reduction of (3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyl tetrazolium bromide) (MTT). Their inter- and intra-batch variation was evaluated. Histological examination showed a completely stratified epithelium in all skin models, which closely resembled normal human epidermis. Low intra-batch variation in tissue architecture was observed in all skin models, but moderate to considerable inter-batch variation was noticed. Evaluation of the expression and localization of a number of differentiation-specific protein markers revealed that all skin models showed an aberrant expression of keratin 6, skin-derived antileukoproteinase, small proline rich proteins, involucrin and transglutaminase. Although variation within batches was low, in particular keratin 6, involucrin and skin-derived antileukoproteinase expression demonstrated some inter-batch variation. Reduction of MTT in vehicle-treated cultures showed high similarities between skin models, but marked differences were observed when 1.0% sodium lauryl sulfate was applied topically for 3 or 16 h. Most pronounced effects were noticed in SkinEthic cultures. Intra-batch variations were low and moderate variations were observed between batches. All skin models tested reproduced many of the characteristics of normal human epidermis and therefore provide a morphologically relevant in vitro means to assess skin irritation and other skin-related studies.