Our analysis of epidermal lipids revealed that (glucosyl)ceramide profiles in various human skin equivalents are different from those of native tissue. The main difference is the reduced content in skin equivalents of ceramides 4-7 and especially the very low content of the most polar ceramides 6 and 7, which contain hydroxylated sphingoid base and/or fatty acid. To facilitate hydroxylation, the culture medium was supplemented with vitamins C and E. Although in vitamin E-supplemented medium lipogenesis was not affected, in vitamin C-supplemented medium the content of glucosylceramides and of ceramides 6 and 7 was markedly increased, both in the presence and absence of serum and irrespective the substrate used (inert or natural, populated or not with fibroblasts). The improvement of the lipid profile was accompanied by a marked improvement of the barrier formation as judged from extensive production of lamellar bodies, their complete extrusion at the stratum granulosum/stratum corneum interface, and the formation of multiple broad lipid lamellar structures in the intercorneocyte space. The presence of well-ordered lipid lamellar phases was confirmed by small-angle x-ray diffraction. Some differences between native and reconstructed epidermis, however, were noticed. Although the long-range lipid lamellar phase was present in both the native and the reconstructed epidermis, the short lamellar phase was present only in native tissue. It remains to be established whether these differences can be ascribed to small differences in relative amounts of individual ceramides, to differences in fatty acid profiles, or to differences in cholesterol sulfate, pH, or calcium gradients. The results indicate the key role vitamin C plays in the formation of stratum corneum barrier lipids.

We present a study on modification of culture conditions in serially cultured human bronchial epithelial cells (HBEC), necessary to achieve bronchial epithelial cells similar to the native epithelium. Cells were obtained from bronchial biopsies and serially cultured using a previously described method (In Vitro Cell. Dev. Biol. 1993; 29A:379-387). At the air-liquid interface, the second and the subsequent passages of HBEC cultures were grown 7 to 31 days, in medium containing fetal calf serum, using de-epidermized dermis or collagen discs as substratum. Scanning and transmission electron microscopy revealed ciliogenesis after 7 days and maturation of the cilia up to 31 days, irrespective of whether de-epidermized dermis or collagen membrane was used. The transmission electron microscopy of the developing cilia showed fibrogranular masses, procentrioles, basal bodies, and in the mature cilia a normal ultrastructure of the axoneme, the nine doublets, the central pair, radial spokes, and dynein arms in the ciliary shaft. In contrast, the submerged cultures showed no signs of ciliogenesis in the same time course. Results of experiments, in which cell seeding density, the substrate used, and the manner of nutrient supplementation were modulated, revealed that the air-exposure of the cultured HBEC is a necessary requirement for the ciliogenesis. The development pathway of ciliated cells in air-exposed HBEC cultures was similar to the differentiation and maturation pattern in human fetal tracheal cells. The in vitro model of human bronchial epithelial cells derived from biopsies obtained by fiberoptic bronchoscopy offers an attractive model for future studies on the function of human bronchial epithelial cells under normal and pathologic conditions.

The re-epithelialization of the wound involves the migration of keratinocytes from the edges of the wound. During this process, keratinocyte migration and proliferation will depend on the interaction of keratinocytes with dermal fibroblasts and the extracellular matrix. The present study aimed to investigate (1) the role of fibroblasts in the re-epithelialization process and on the reconstitution of the dermal-epidermal junction (DEJ) and (2) differential protein expression during re-epithelialization. For both purposes, three-dimensional human skin equivalents (HSE) were used. A full-thickness wound in HSE was introduced by freezing with liquid nitrogen and a superficial wound by linear incision with a scalpel. The closure of the wound in the absence or presence of exogenous growth factors was followed by monitoring the rate of re-epithelialization and regeneration of the DEJ. The results obtained in this study demonstrate that fibroblasts facilitate wound closure, but they differentially affected the deposition of various basement membrane components. The deposition of laminin 5 at the DEJ was delayed in superficial wounds as compared to the full-thickness wounds. During freeze injury, some basement membrane (BM) components remain associated with the dermal compartment and probably facilitate the BM reconstitution. The re-epithelialization process in full-thickness but not in superficial wounds was accelerated by the presence of keratinocyte growth factor and especially by epidermal growth factor. In addition, we have examined the deposition of various basement membrane components and the differences in protein expression in a laterally expanding epidermis in uninjured HSE. Laminin 5, type IV and VII collagen deposition was decreased in the laterally expanding epidermis, indicating that the presence of these proteins is not required for keratinocyte migration to occur in vitro. Using two-dimensional polyacrylamide gel electrophoresis, we have identified DJ-1, a protein not earlier reported to be differently expressed during the epithelialization process of the skin.

Human skin equivalents (HSEs) show great similarities to human native skin. However, one of the key processes impaired under in vitro conditions is desquamation. Desquamation involves the degradation of the corneodesmosomes, in which various enzymes participate. Activation of these enzymes is affected by several microenvironmental factors such as pH and water level. The water level is assumed to depend on the presence of natural moisturizing factors (NMF). In this study, the levels of water and one of the prominent NMF components--pyrrolidone carboxylic acid (PCA)--were examined. In HSE generated under normal culture conditions (93% relative humidity (RH)), the water level and PCA content appeared to be much lower than in the native counterpart. To increase the water and PCA levels in HSE, a culture method was established in which HSE was reconstructed under reduced RH. Although at 40% RH the PCA levels in reconstructed and native tissue are similar, the hydration levels in reconstructed tissue remain still lower. Only topical application of water induced marked swelling of corneocytes. This clearly shows that the stratum corneum water level in HSE is regulated by other, still unknown, factors, in addition to NMF.

Calcium plays an important role in the regulation of cellular differentiation and desquamation of epidermal keratinocytes. In this study, we examined the calcium distribution in reconstructed epidermis in an attempt to understand the physiology of keratinocyte differentiation and desquamation in vitro. Ion capture cytochemistry (the potassium oxalate-pyroantimonate method) was employed to localize ionic calcium in reconstructed epidermis generated under three different culture conditions (in serum-containing medium, serum-free medium, and serum-free medium supplemented with retinoic acid), allowing a comparison of the physiology of incompletely and well-differentiated keratinocytes. The reconstructed epidermis generated in serum-containing medium showed features of incomplete differentiation, and compared with the native skin, a high calcium content within incompletely differentiated cells in the stratum corneum. Use of serum-free medium containing vitamin and lipid supplements led to a marked improvement of the stratum corneum ultrastructure and penetration pathway across the stratum corneum, indicating improved barrier formation of the reconstructed epidermis. In parallel, the calcium distribution pattern was normalized showing the highest levels of calcium in the stratum granulosum and low levels in the inner stratum corneum. Addition of retinoic acid to the serum-free medium resulted in an altered keratinocyte differentiation and re-appearance of large quantities of calcium precipitates in the stratum corneum. Proton probe X-ray microanalysis was applied to investigate the calcium distribution quantitatively in native and reconstructed epidermis generated in serum-free medium, and verified the calcium distribution demonstrated by the precipitation technique. Regardless of the presence or absence of calcium in the stratum corneum, all examined culture systems exhibited insufficient desquamation, which correlates with the finding that stratum corneum chymotryptic enzyme was present predominantly as an inactive precursor. This study demonstrates that improvement of the stratum corneum barrier properties in vitro is concurrent with the normalization of the epidermal calcium gradient, whereas deregulation of terminal differentiation correlates with an accumulation of calcium ions within incompletely differentiated corneocytes.

The limited life-span and irregularities in epidermal differentiation and barrier function that have restricted the utility of presently available skin culture models for pharmacological and toxicological studies indicate that further modifications of culture conditions are required for optimization of these models. In the present study epidermis reconstructed on de-epidermized dermis was used to investigate the effects of temperature and epidermal growth factor (EGF) on epidermal differentiation and lipogenesis. When cultured at 37 degrees C, keratinocytes formed a well-differentiated epidermis whether EGF was present or not. However, the thickness of the epidermis, particularly of the stratum corneum, was higher in the presence of EGF. Both the differentiation-specific protein markers (keratins 1 and 10, involucrin and transglutaminase) and lipid markers (ceramides) were synthesized. EGF-induced increases in triglyceride content caused accumulation of lipid droplets within the stratum corneum which is indicative of a hyperproliferative effect of EGF. In the absence of EGF, a well-differentiated epidermis was generated at 33 degrees C with a morphology showing a higher resemblance to native epidermis than cultures grown at 37 degrees C. The stratum corneum was less compact and with practically no lipid droplets, irregularly shaped keratohyalin granules were abundant in the stratum granulosum, lamellar body extrusion was improved and the number of stratum corneum layers was reduced to normal levels. However, EGF supplementation had a deleterious effect on epidermal morphogenesis and differentiation of cultures grown at 33 degrees C. The epidermis lacked a stratum granulosum and the stratum corneum contained a high number of nuclear remnants. The synthesis of the early specific protein differentiation markers (keratins 1 and 10) was suppressed on both the protein and mRNA levels without significant interference with the synthesis of late differentiation lipid markers, such as ceramides. From this observation it can be concluded that the synthesis of keratins associated with terminal differentiation is profoundly affected by the presence of EGF and is sensitive to temperature and that of ceramides is not. The finding that TGF alpha did not modulate the morphogenesis and synthesis of keratins 1 and 10 in cultures grown at 33 degrees C indicates possible differences between the postreceptor binding processes of these EGF receptor ligands.

In the present study we describe the establishment of serial cultures of human bronchial epithelial cells derived from biopsies obtained by fiberoptic bronchoscopy. The cell cultures were initiated from small amounts of material (2 mm forceps biopsies) using either explants or epithelial cell suspensions in combination with a feeder-layer technique. The rate of cell proliferation and the number of passages (up to 8 passages) achieved were similar, irrespective of whether the explants or dissociated cells were used. To modulate the extent of differentiation, the bronchial epithelial cells were cultured either under submerged, low calcium (0.06 mM) (proliferating), normal calcium (1.6 mM) (differentiation enhancing) conditions, or at the air-liquid interface. Characterization of the bronchial epithelial cell cultures was assessed on the basis of cell morphology, cytokeratin expression, and ciliary activity. The cells cultured under submerged conditions formed a multilayer consisting of maximally three layers of polygonal-shaped, small cuboidal cells, an appearance resembling the basal cells in vivo. In the air-exposed cultures, the formed multilayer consisted of three to six layers exhibiting squamous metaplasia. The cytokeratin profile in cultured bronchial epithelial cells was similar in submerged and air-exposed cultures and comparable with the profile found in vivo. In addition to cytokeratins, vimentin was co-expressed in a fraction of the subcultured cells. The ciliary activity was observed in primary culture, irrespective of whether the culture had been established from explants or from dissociated cells. This activity was lost upon subculturing and it was not regained by prolongation of the culture period. In contrast to submerged cultures and despite the squamous metaplasia appearance, the cells showed a reappearance of cilia when cultured at the air-liquid interface. Human bronchial epithelial cell cultures can be a representative model for controlling the mechanisms of regulation of bronchial epithelial cell function.

Two human skin recombinants, the epidermis reconstructed on the deepidermized dermis (RE-DED) or on fibroblast-populated collagen matrix (Living Skin Equivalent, LSE^TM), were used to study the irritating effect of sodium lauryl sulfate (SLS). The extent of cytotoxicity induced after a 24-hour exposure period to increasing concentrations of SLS (0-5%) was evaluated on the basis of (1) morphological perturbations, (2) changes in the expression of differentiation-specific protein markers (keratin 1, 10, 6, 16, involucrin and transglutaminase), (3) cell membrane integrity (LDH leakage) and (4) release of proinflammatory mediators (PGE₂, IL-1, IL-6 and IL-8). SLS induced significant changes in epidermal morphology and changes in the expression and localization of differentiation-specific protein markers when applied topically in concentrations higher than 1 % on RE-DED and higher than 0.1% on LSE. The exposure of both human skin recombinants to SLS resulted in a dose-dependent release of LDH, PGE₂ and IL-1Α and in an increase in keratinocyte intracellular IL-1 levels. Upon application of 5% SLS on RE-DED the total (intra- and extracellular) IL-1 levels remained high but due to cell damage the intracellular IL-1 level was markedly decreased and the extracellular IL-1 level increased. Similar observations have been made with LSE after application of 0.5% SLS. However, with LSE the extracellular IL-1Α levels were found to be about 100 times lower than those measured with RE-DED. Exposure of LSE to SLS induced a marked increase of IL-6 production in fibroblasts incorporated in the collagen matrix. Contrary to LSE, both intra- and extracellular levels of IL-6 were low in unexposed controls and were only marginally modulated by the exposure of the RE-DED to SLS. In addition, a dose-dependent increase in IL-8 release was observed upon application of SLS on RE-DED. The results of the present study indicate that concentrations of SLS required to induce epidermal irritancy in vitro approximate those inducing irritation in human skin in vivo. All parameters used in the present study for evaluation of toxicity can serve as useful endpoints for screening of contact skin irritancy in vitro. Compared to RE-DED, the LSE seems to be more susceptible to SLS. The differences in sensitivity between LSE and RE-DED can be ascribed to reported differences in their stratum corneum barrier function.