Improved Barrier Structure Formation in Air-Exposed Human Keratinocyte Culture Systems

The epidermis (including stratum corneum) of human keratinocytes cultured at the air-liquid interface attached to an appropriate substrate shows a morphology closely mimicking that of its in vivo counterpart. In spite of the histologic similarities, the barrier function seems to be impaired. The aim of the present study was to characterize development and structure of the epidermal permeability barrier in two human skin recombinants using electron microscopy (including ruthenium tetroxide-post fixation technique) and analysis of lipid composition. The epidermis was reconstructed by growing human keratinocytes either on de-epidermized dermis or on a bovine collagen-containing matrix with active fibroblasts (Living Skin Equivalent). Ultrastructurally both culture systems showed a) an abnormal lamellar body delivery system, b) disturbance of transformation into lamellar lipid bilayers, c) an impaired structural organization and distribution of the epidermal lipids in the intercellular spaces. In either of the systems used, prolongation of the culture period did not induce any significant improvement in the stratum corneum lipid organization. Whereas the Living Skin Equivalent showed only sparse lamellar bodies, the number of lamellar bodies in the human keratinocyte culture on de-epidermized dermis grown in regular medium seemed to be comparable to native skin. Contrary to the Living Skin Equivalent, the keratinocyte culture on de-epidermized dermis contained a higher number of intracorneocytic lipid droplets correlating with a higher triglyceride content in the lipid analyses. By reconstructing the keratinocyte culture on de-epidermized dermis with the same medium as used for the Living Skin Equivalent, both lipid composition (lower triglyceride, higher ceramide contents) and structural organization were improved, and regular lamellar lipid bilayers comparable to those of native skin appeared

Acylceramide Head Group Architecture Affects Lipid Organization in Synthetic Ceramide Mixtures

The lipid organization in the upper layer of the skin, the stratum corneum (SC), is important for the skin barrier function. This lipid organization, including the characteristic 13 nm lamellar phase, can be reproduced in vitro with mixtures based on cholesterol, free fatty acids and natural as well as synthetic ceramides (CER). In human SC, nine CER classes have been identified (CER1–CER9). Detailed studies on the effect of molecular structure of individual ceramides on the SC lipid organization are only possible with synthetic lipid mixtures, as their composition can be accurately chosen and systematically modified. In the present study, small-angle X-ray diffraction was used to examine the organization in synthetic lipid mixtures of which the synthetic ceramide fraction was prepared with sphingosine-based CER1 or phytosphingosine-based CER9. The latter acylceramide contains an additional hydroxyl group at the sphingoid backbone. The results show that a gradual increase in CER1 level consistently promotes the formation of the 13 nm lamellar phase and that partial replacement of CER1 by CER9 does not affect the phase behavior. Interestingly, complete substitution of CER1 with CER9 reduces the formation of the long periodicity phase and results in phase separation of CER9

Stratum Corneum Lipid Composition and Structure in Cultured Skin Substitutes is Restored to Normal after Grafting onto Athymic Mice

Restoration of an epidermal barrier is a definitive requirement for wound closure. Cultured skin substitutes grafted onto athymic nude mice were used as a model for a long-term study of stratum corneum barrier lipid metabolism and organization. Samples of stratum corneum collected after 12 and 21 d in vitro and 6, 11, and 24 mo postgrafting were examined for their lipid and fatty acid composition, and their lipid organization and structure using electron microscopy and small angle X-ray diffraction, respectively. All of these methods confirm the impaired barrier function of cultured skin substitutes in vitro, as judged from the deviations in lipid composition and from poor organization of the stratum corneum lipids that show no lamellar structure. At 6 mo postgrafting, the total stratum corneum lipid profiles of the epidermal grafts is close to that of the human stratum corneum with the exception of the presence of mouse specific lipids. The increase of ceramides 4–7 in cultured skin substitutes after grafting indicates restored activity of processes involved in the hydroxylation of fatty acids and sphingoid bases. Conversely, the ceramide profile still reveals some abnormalities (elevated content of ceramide 2 and slightly lower content of ceramide 3) and the content of long-chain fatty acids remains below its physiologic level at 6 mo postgrafting, but normalizes by 2 y postgrafting. The ultramicroscopic observations revealed the formation of lamellar extracellular lipid domains by 4 mo postgrafting. Despite these findings, the X-ray diffraction showed differences in the diffraction pattern at 2 y after grafting, suggesting that the organization of stratum corneum lipids in all epidermal grafts differs from that of the native skin. Journal of Investigative Dermatology Symposium Proceedings 3:114–120, 199

Basement Membrane Reconstruction in Human Skin Equivalents Is Regulated by Fibroblasts and/or Exogenously Activated Keratinocytes

This study was undertaken to examine the role fibroblasts play in the formation of the basement membrane (BM) in human skin equivalents. For this purpose, keratinocytes were seeded on top of fibroblast-free or fibroblast-populated collagen matrix or de-epidermized dermis and cultured in the absence of serum and exogenous growth factors. The expression of various BM components was analyzed on the protein and mRNA level. Irrespective of the presence or absence of fibroblasts, keratin 14, hemidesmosomal proteins plectin, BP230 and BP180, and integrins α1β1, α2β1, α3β1, and α6β4 were expressed but laminin 1 was absent. Only in the presence of fibroblasts or of various growth factors, laminin 5 and laminin 10/11, nidogen, uncein, type IV and type VII collagen were decorating the dermal/epidermal junction. These findings indicate that the attachment of basal keratinocytes to the dermal matrix is most likely mediated by integrins α1β1 and α2β1, and not by laminins that bind to integrin α6β4 and that the epithelial–mesenchymal cross-talk plays an important role in synthesis and deposition of various BM components

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. (C) 2000 Elsevier Science Ltd

Expression of skin-derived antileukoproteinase (SKALP) in reconstructed human epidermis and its value as a market for skin irritation

For the investigation of the skin irritancy potential of chemicals in an 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 α-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

Modulation of IL-6 production and IL-1 activity by keratinocyte-fibroblast interaction

The present study was undertaken to investigate whether modulation of interleukin-6 and interleukin-1 production occurs owing to keratinocyte-fibroblast interaction. Normal human keratinocytes or squamous carcinoma cells were cultured either alone or in the presence of human foreskin fibroblasts or murine 3T3 cells. All cells tested produced interleukin-6, and interleukin-6 levels were markedly increased when normal or malignant keratinocytes were co-cultured with fibroblasts. The bioassay (species independent) and enzyme-linked immunosorbent assay (specific for human interleukin-6) together with use of complementary DNA probes specific for human or murine interleukin-6 revealed that fibroblasts are responsible for increased interleukin-6 levels. Moreover, interleukin-6 levels were increased when fibroblasts were cultured in conditioned media derived from normal human keratinocytes and squamous carcinoma cells-4 cultures. Interleukin-1α secreted by normal human keratinocytes and squamous carcinoma cells-4 cells was mainly responsible for the increased interleukin-6 production in fibroblasts. Although interleukin-1 activity increased linearly with the incubation time in squamous carcinoma cells-4 mono-cultures, interleukin-1 activity was low and remained unchanged in squamous carcinoma cells-4/3T3 co-cultures. Low interleukin-1 activity was most probably not due to inhibition of interleukin-1α production in squamous carcinoma cells-4/3T3 co-cultures because interleukin-lα messenger RNA expression in squamous carcinoma cells-4 cells remained unchanged in the presence of 3T3 cells. Furthermore, when 3T3 cells were incubated in conditioned medium derived from squamous carcinoma cells-4 cells, high interleukin-1 activity decreased to an undetectable level, suggesting that fibroblasts are involved in the suppression of interleukin-1 activity. The remaining interleukin-1 activity, however, was sufficient for maximal induction of interleukin-6 production in fibroblasts. These results suggest that the interaction between epithelial and mesenchymal cells is at least partly initiated by interleukin-lα secreted by the activated epithelial cell during skin injury or tumor invasion. Interleukin-1 in turn can induce modulation of the synthesis of various pro-inflammatory mediators and proteases in surrounding fibroblasts. An enhanced proteolytic activity and/or a possible induced production of an interleukin-1 inhibitor in fibroblasts and/or a receptor-mediated interleukin-1 consumption by fibroblasts will cause a decrease in interleukin-1 activity and thus exert a negative feedback

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