Susan Gibbs

Characterization of reconstructed skin models

The aim of the present study was to evaluate tissue architecture and lipid composition of commercially available reconstructed human skin models; EpiDerm™, SkinEthic™ and Episkin™ in comparison to in-house reconstructed epidermis on a de-epidermized dermis (RE-DED) model and native tissue. For this purpose, the tissue architecture was examined using light microscopy, electron microscopy and immunohistochemistry; epidermal lipid composition was analyzed by HPTLC. Histological examination showed a completely stratified epithelium in all skin models closely resembling 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. In the stratum corneum extracellular space, lipid lamellae consisting of multiple alternating electron-dense and electron-lucent bands were present. Lipid analyses revealed the presence of all major epidermal lipid classes. Compared with native epidermis and RE-DED in EpiDerm, SkinEthic and Episkin models, the content of polar ceramides 5 and 6 was lower, ceramide 7 was absent, and the content of free fatty acids was very low. 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. In conclusion, all 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 diminished. 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 perform other skin-related studies.

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.

Interdependent transcription control elements regulate the expression of the SPRR2A gene during keratinocyte terminal differentiation.

Expression of the SPRR2A gene, a member of the small proline-rich family of cornified cell envelope precursor proteins, is strictly linked to keratinocyte terminal differentiation both in vivo and in vitro. In this study, we explored the molecular mechanisms underlying this regulation in transiently transfected primary keratinocytes induced to differentiate in vitro. Deletion mapping and site-directed mutagenesis of SPRR2A promoter-chloramphenicol acetyltransferase constructs indicate that four transcription control elements are essential and sufficient for promoter activity. These elements were further characterized by electrophoretic mobility shift and identified as (i) an inverted octamer doublet, bound by the POU domain factor Oct-11 (Skn-1a/i, Epoc-1), (ii) an interferon-stimulated response element recognized by interferon regulatory factors 1 and 2, (iii) an Ets binding site partially overlapping the interferon-stimulated response element, and (iv) a TG box recognized by the Sp1 family of zinc finger transcription factors. Destruction of a single terminal differentiation element is sufficient to completely abolish transcription from the SPRR2A promoter, indicating that these transcription control elements function in concert in an interdependent manner. Apparently, integration of signals transmitted by the above-mentioned transcription factors is necessary and sufficient to promote gene expression during keratinocyte terminal differentiation.

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.

Intrinsic regulation of differentiation markers in human epidermis, hard palate and buccal mucosa

Different epithelia show extensive variation in differentiation. Epidermis and epithelium from the hard palate are both typical examples of orthokeratinized epithelia whereas buccal mucosa is an example of a non-keratinized epithelium. Each of these tissues can be distinguished morphologically and also by the expression of a number of structural proteins. Tissue explants derived from epidermis, hard palate or buccal mucosa were cultured at the air-liquid interface on collagen gels containing human dermal fibroblasts. Reconstructed epithelia that retained many of the morphological and immunohistochemical characteristics of the original tissue were formed. Cultures derived from epidermis and the hard palate both had a well-defined stratum basale, stratum spinosum, stratum granulosum and stratum corneum whereas cultures derived from buccal mucosa had no stratum granulosum or corneum and the cells retained their nuclei. Significantly more living cell layers were observed in both types of epithelia obtained from the mouth than in epidermis. The specific localization of proliferation and differentiation markers (Ki67, loricrin, involucrin, SPRR2, SPRR3 and keratin 10) closely resembled that of the tissue from which the cultures were derived. As identical three-dimensional culture models were used here, it is concluded that the differences observed between these epithelia were due to intrinsic properties of the keratinocytes.

Effect of skin barrier competence on SLS and water-induced IL-1α expression

Abstract: For screening of a potential irritant it is essential that an early marker for irritation should be chosen which could be detected before the physiological signs of irritation occur. Interleukin 1 alpha (IL‐1α) is widely accepted as such a marker in both in vivo and in vitro test systems. In this study, we have determined the mRNA levels of IL‐1α in the epidermis after topical application of sodium dodecyl sulphate (SLS) in both a commercially available epidermal kit (EpiDerm) and in excised skin. Furthermore, we have determined the effect of water, the vehicle for SLS, on IL‐1α mRNA levels. Topical application of water to excised skin increases IL‐1α mRNA levels sixfold in the epidermis whereas topical application of water to EpiDerm cultures did not alter IL‐1α mRNA levels. This is explained by the finding that EpiDerm cultures have a sub‐optimal barrier function when compared with excised skin – topical application of SLS was clearly toxic at much lower concentrations in EpiDerm cultures (0.2% SLS) than in excised skin (5% SLS). Also caffeine penetration was 10‐fold higher through EpiDerm cultures than through the excised skin. Therefore, incubation of control EpiDerm cultures at 100% humidity effectively mimics topical exposure to water. An additional increase in IL‐1α mRNA levels observed between topical application of water and SLS is similar (about threefold) in both experimental systems. In conclusion, in vitro reconstructed epidermis models, such as EpiDerm, can be used as a predictive model for irritancy screening. However, great care should be taken when interpreting the results due to the fact that EpiDerm cultures do not have a competent barrier function and therefore lower irritant concentrations are required than in in vivo or ex vivo studies in order to induce cytotoxic effects. Furthermore, the irritant effects of the vehicle should not be neglected. Our results show clearly that the topical application of water to excised skin results in increased levels of IL‐1α mRNA in the epidermis. This is a cytokine that is widely used as an early marker for skin irritation.

EXPRESSION OF SKIN-DERIVED ANTILEUKOPROTEINASE (SKALP) IN RECONSTRUCTED HUMAN EPIDERMIS AND ITS VALUE AS A MARKER FOR SKIN IRRITATION

For the investigation of the skin irritancy potential of chemicals in an in vitro model, it is necessary to have sensitive end-points that predict the effects on native human skin. Our aim was to investigate whether the induction of the proteinase inhibitor SKALP in reconstructed epidermis can be used as a marker. The influence of culture conditions and the effect of topical application of sodium lauryl sulfate and oleic acid on SKALP expression were evaluated using immunohistochemistry and Northern blotting. SKALP expression was induced by serum, epidermal growth factor and fibroblasts. In the presence of retinoic acid and 1,25-dihydroxyvitamin D3 SKALP expression was inhibited, whereas supplementation with ascorbic acid and a-tocopherol had no effect. Tape-stripping of excised skin and topical treatment with sodium lauryl sulfate induced SKALP protein expression. Application of sodium lauryl sulfate and oleic acid on reconstructed epidermis also induced SKALP at the protein level but no significant effects could be demonstrated at mRNA levels. In conclusion, SKALP expression, which was increased upon application of sodium lauryl sulfate and oleic acid, can be used as an in vitro end-point for skin irritancy, irrespective of the modifying effects of culture conditions.