Quantitative nanohistology of aging dermal collagen.

The skin is the largest organ in the body and is essential for protecting us from environmental stressors such as UV radiation, pollution, and pathogens. As we age, our skin undergoes complex changes that can affect its function, appearance, and health. These changes result from intrinsic (chronological) and extrinsic (environmental) factors that can cause damage to the skin's cells and extracellular matrix. As higher-resolution microscopical techniques, such as Atomic Force Microscopy (AFM), are being deployed to support histology, it is possible to explore the biophysical properties of the dermal scaffold's constituents, such as the collagen network. In this study, we demonstrate the use of our AFM-based quantitative nanohistology, performed directly on unfixed cryosections of 30 donors (female, Caucasian), to differentiate between dermal collagen from different age groups and anatomical sites. The initial 420 (10 × 10 µm2) Atomic Force Microscopy images were segmented into 42,000 (1 × 1 µm2) images before being classified according to four pre-defined empirical collagen structural biomarkers to quantify the structural heterogeneity of the dermal collagen. These markers include interfibrillar gap formation, undefined collagen structure, and registered or unregistered dense collagen fibrillar network with evident D-banding. The structural analysis was also complemented by extensive nanoindentation (∼1,000 curves) performed on individual fibrils from each section, yielding 30,000 indentation curves for this study. Principal Component Analysis was used to reduce the complexity of high-dimensional datasets. The % prevalence of the empirical collagen structural biomarkers between the papillary and reticular dermis for each section proves determinant in differentiating between the donors as a function of their age or the anatomical site (cheek or breast). A case of abnormal biological aging validated our markers and nanohistology approach. This case also highlighted the difference between chronological and biological aging regarding dermal collagen phenotyping. However, quantifying the impact of chronic and pathological conditions on the structure and function of collagen at the sub-micron level remains challenging and lengthy. By employing tools such as the Atomic Force Microscope as presented here, it is possible to start evaluating the complexity of the dermal matrix at the nanoscale and start identifying relevant collagen morphology which could be used toward histopathology standards.

Assessing the Role of Carbonyl Adducts, Particularly Malondialdehyde Adducts, in the Development of Dermis Yellowing Occurring during Skin Photoaging.

Solar elastosis is associated with a diffuse yellow hue of the skin. Photoaging is related to lipid peroxidation leading to the formation of carbonyl groups. Protein carbonylation can occur by addition of reactive aldehydes, such as malondialdehyde (MDA), 4-hydroxy-nonenal (4-HNE), and acrolein. All the proteins concerned with this modification, and the biological consequences of adduct formation, are not completely identified. The link between yellowish skin and dermal carbonylated proteins induced by aldehyde adducts was investigated. The study was carried out on ex vivo skin samples from sun-exposed or sun-protected areas and on in vitro dermal equivalent models incubated with 5 mM MDA, 4-HNE, or acrolein. The yellow color and the level of MDA, 4-HNE, and acrolein adducts were evaluated. Yellowish color differences were detected in the dermis of sun-exposed skin compared to sun-protected skin and in in vitro models following addition of MDA, 4-HNE, or acrolein. The yellowing was correlated with the carbonyl adducts increasing in the dermis and in in vitro models incubated with aldehydes. The stronger yellowing seemed to be mediated more by MDA than 4-HNE and acrolein. These observations suggest that dermal carbonylation especially induced by MDA result in the yellow hue of dermis and is involved, in part, in the yellowing observed during skin photoaging.

A mechanistic view on the aging human skin through ex vivo layer-by-layer analysis of mechanics and microstructure of facial and mammary dermis.

Age-related changes in skin mechanics have a major impact on the aesthetic perception of skin. The link between skin microstructure and mechanics is crucial for therapeutic and cosmetic applications as it bridges the micro- and the macro-scale. While our perception is governed by visual and tactile changes at the macroscopic scale, it is the microscopic scale (molecular assemblies, cells) that is targeted by topical treatments including active compounds and energies. We report here a large dataset on freshly excised human skin, and in particular facial skin highly relevant for cosmetics and aesthetic procedures. Detailed layer-by-layer mechanical analysis revealed significant age-dependent decrease in stiffness and elastic recoil of full-thickness skin from two different anatomical areas. In mammary skin, we found that the onset of mechanical degradation was earlier in the superficial papillary layer than in the deeper, reticular dermis. These mechanical data are linked with microstructural alterations observed in the collagen and elastic networks using staining and advanced imaging approaches. Our data suggest that with ageing, the earliest microstructural and mechanical changes occur in the top-most layers of dermis/skin and then propagate deeper, providing an opportunity for preventive topical treatments acting at the level of papillary dermis.

UVA Exposure Combined with Glycation of the Dermis Are Two Catalysts for Skin Aging and Promotes a Favorable Environment to the Appearance of Elastosis.

Skin aging is the result of superimposed intrinsic (individual) and extrinsic (e.g., UV exposure or nutrition) aging. Previous works have reported a relationship between UV irradiation and glycation in the aging process, leading, for example, to modified radical species production and the appearance of AGEs (advanced glycosylation end products) in increasing quantities, particularly glycoxidation products like pentosidine. In addition, the colocalization of AGEs and elastosis has also been observed. We first investigated the combination of the glycation reaction and UVA effects on a reconstructed skin model to explain their cumulative biological effect. We found that UVA exposure combined with glycation had the ability to intensify the response for specific markers: for example, MMP1 or MMP3 mRNA, proteases involved in extracellular matrix degradation, or proinflammatory cytokine, IL1α, protein expression. Moreover, the association of glycation and UVA irradiation is believed to promote an environment that favors the onset of an elastotic-like phenomenon: mRNA coding for elastin, elastase, and tropoelastin expression is increased. Secondly, because the damaging effects of UV radiation in vivo might be more detrimental in aged skin than in young skin due to increased accumulation of pentosidine and the exacerbation of alterations related to chronological aging, we studied the biological effect of soluble pentosidine in fibroblasts grown in monolayers. We found that pentosidine induced upregulation of CXCL2, IL8, and MMP12 mRNA expression (inflammatory and elastotic markers, respectively). Tropoelastin protein expression (elastin precursor) was also increased. In conclusion, fibroblasts in monolayers cultured with soluble pentosidine and tridimensional in vitro skin constructs exposed to the combination of AGEs and UVA promote an inflammatory state and an alteration of the dermal compartment in relation to an elastosis-like environment.

Dietary Antioxidant Capacity and Skin Photoaging: A 15-Year Longitudinal Study.

The long-term effect of diet on skin aging is largely unknown, but evidence suggests that the antioxidants from foods may mitigate the main component of skin aging caused by sun exposure. We assessed the association between the total antioxidant capacity of foods people eat and the photoaging of their skin. In a community-based, prospective study among 777 Australian adults aged <55 years at baseline, we estimated the total dietary antioxidant capacity of participants' diets in 1992, 1994, and 1996 and graded photoaging severity using microtopography in 1992, 1996, and 2007. We used ordinal logistic regression and applied generalized estimating equations to estimate change in the degree of photoaging associated with increasing total antioxidant capacity compared with the group with the lowest antioxidant capacity, separately in younger (≤45 years) and older (>45 years) adults. In the 15-year study period, the overall prevalence of severe skin photoaging increased from 42% at baseline to 88%. Adults aged >45 years who consumed foods with high antioxidant capacity experienced approximately 10% less photoaging over 15 years than those who ate foods with low antioxidant capacity. No association was found among adults aged ≤45 years. Foods rich in antioxidants as measured by antioxidant capacity may retard skin aging among healthy men and women aged >45 years.

Author Correction: Reconstructed Skin Models Revealed Unexpected Differences in Epidermal African and Caucasian Skin.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Reconstructed Skin Models Revealed Unexpected Differences in Epidermal African and Caucasian Skin.

Clinical observations of both normal and pathological skin have shown that there is a heterogeneity based on the skin origin type. Beside external factors, intrinsic differences in skin cells could be a central element to determine skin types. This study aimed to understand the in vitro behaviour of epidermal cells of African and Caucasian skin types in the context of 3D reconstructed skin. Full-thickness skin models were constructed with site matched human keratinocytes and papillary fibroblasts to investigate potential skin type related differences. We report that reconstructed skin epidermis exhibited remarkable differences regarding stratification and differentiation according to skin types, as demonstrated by histological appearance, gene expression analysed by DNA microarray and quantitative proteomic analysis. Signalling pathways and processes related to terminal differentiation and lipid/ceramide metabolism were up-regulated in epidermis constructed with keratinocytes from Caucasian skin type when compared to that of keratinocytes from African skin type. Specifically, the expression of proteins involved in the processing of filaggrins was found different between skin models. Overall, we show unexpected differences in epidermal morphogenesis and differentiation between keratinocytes of Caucasian and African skin types in in vitro reconstructed skin containing papillary fibroblasts that could explain the differences in ethnic related skin behaviour.

Biological Effects Induced by Specific Advanced Glycation End Products in the Reconstructed Skin Model of Aging.

Advanced glycation end products (AGEs) accumulate in the aging skin. To understand the biological effects of individual AGEs, skin reconstructed with collagen selectively enriched with N(ɛ)-(carboxymethyl)-lysine (CML), N(ɛ)-(carboxyethyl)-lysine (CEL), methylglyoxal hydroimidazolone (MG-H1), or pentosidine was studied. Immunohistochemistry revealed increased expression of α6 integrin at the dermal epidermal junction by CEL and CML (p<0.01). Laminin 5 was diminished by CEL and MG-H1 (p<0.05). Both CML and CEL induced a robust increase (p<0.01) in procollagen I. In the culture medium, IL-6, VEGF, and MMP1 secretion were significantly decreased (p<0.05) by MG-H1. While both CEL and CML decreased MMP3, only CEL decreased IL-6 and TIMP1, while CML stimulated TIMP1 synthesis significantly (p<0.05). mRNA expression studies using qPCR in the epidermis layer showed that CEL increased type 7 collagen (COL7A1), ß1, and α6 integrin, while CML increased only COL7A1 (p<0.05). MG-H1-modified collagen had no effect. Importantly, in the dermis layer, MMP3 mRNA expression was increased by both CML and MG-H1. CML also significantly increased the mRNAs of MMP1, TIMP1, keratinocyte growth factor (KGF), IL-6, and monocyte chemoattractant protein 1 (MCP1) (p<0.05). Mixed effects were present in CEL-rich matrix. Minimally glycoxidized pentosidine-rich collagen suppressed most mRNAs of the genes studied (p<0.05) and decreased VEGF and increased MCP1 protein expression. Taken together, this model of the aging skin suggests that a combination of AGEs tends to counterbalance and thus minimizes the detrimental biological effects of individual AGEs.

The reconstructed skin model as a new tool for investigating in vitro dermal fillers: increased fibroblast activity by hyaluronic acid.

BACKGROUND: Clinical studies on dermal fillers have essentially focused upon visible improvement of skin quality and any eventual side effects, whereas very little is known about their detailed biological effects. OBJECTIVES: New skin equivalent models were created to investigate the biological impact of hyaluronic acid (HA) fillers on the dermal compartment in vitro. MATERIALS AND METHODS: Two different reconstructed skin models were developed to incorporate HA within the collagen fibers. In the mixed model, HA was distributed throughout the whole collagen gel whereas the HA was concentrated in the center of collagen gel in the inclusion model. RESULTS: A comparison of the addition of fillers in two models of reconstructed skin has permitted a better understanding of the biological impact of HA fillers. Protein profiling of supernatants from both models suggested a regulation of MMP-1 secretion by fibroblasts as a function of HA volume, distribution in the dermis and degree of cross-linking. Immunostaining of the inclusion model revealed increased production of type I and III procollagens close to the cross-linked HA. Fibroblasts located in this area showed a fusiform morphology as well as an increase in -smooth actin expression. The observed increase in collagen production may thus result in part from tension in fibroblasts surrounding the cross-linked HA. CONCLUSION: The inclusion reconstructed skin model, as compared to the mixed model, presented here, appears to be a useful tool for investigating the properties of various fillers in vitro and closer to the in vivo situation; our results show that HA fillers promote in vitro remodeling of the dermis by fibroblasts.

Skin aging by glycation: lessons from the reconstructed skin model.

BACKGROUND: Aging is the result of several mechanisms which operate simultaneously. Among them, glycation is of particular interest because it is a reaction which affects slowly renewing tissues and macromolecules with elevated half-life, like the dermis, a skin compartment highly affected by aging. Glycation produces crosslinks between macromolecules thereby providing an explanation for the increased age-related stiffness of the skin. Glycation products, also called AGEs (advanced glycation end products), accumulate primarily in extracellular matrix molecules like collagen or elastin. METHODS: In order to reproduce this phenomenon in vitro we have created a model of reconstructed skin modified by glycation of the collagen used to fabricate the dermal compartment. RESULTS: This system allowed us to uncover biological modifications of dermal markers, and more surprisingly epidermal markers, as well as an increase of metalloproteinases responsible for degradation of the dermal matrix. Consequently, the imbalance between synthesis and degradation that results from glycation, may contribute to skin aging, as shown in this model. Moreover these modifications were shown to be prevented by the addition of aminoguanidine, a well-known inhibitor of glycation. CONCLUSIONS: Using this experimental approach our results taken together stress the importance and possibly central role of glycation in skin aging and the usefulness of the reconstructed skin as a model of physiological aging.

In vivo and in vitro approaches in understanding the differences between Caucasian and African skin types: specific involvement of the papillary dermis.

BACKGROUND: Most of the identified differences between Caucasian and African skin types have been related to the superficial part of the skin, the epidermis. We investigated possible implications of the dermal compartment in cutaneous differences observed between Caucasians and Africans. METHODS: In vivo and in vitro comparative studies were carried out using normal human skin biopsies and the corresponding in vitro reconstructed skin. Skin equivalents were developed with papillary fibroblasts isolated from the superficial dermis of both Caucasian and African skin types. Expression of major components of the dermal-epidermal junction (DEJ) was examined as a function of ethnicity. RESULTS: Control histological examinations of skin biopsies showed that the African skin type had greater convoluted appearance of the DEJ than the Caucasian skin type. Immunostainings of type IV and VII collagens, laminin 5, and nidogen proteins at the DEJ were lower in African skin compared with Caucasian skin biopsies. CONCLUSIONS: This study brings together new elements on involvement of the papillary dermis in differences between Caucasian and African skin types. As fibroblasts from the superficial dermis cooperate with epidermal keratinocytes in producing protein of the membrane basal zone, present in vivo results suggest that papillary fibroblasts may play a part in the distinct features observed at the DEJ. In preliminary in vitro experiments, differences in several protein expressions contributing to the DEJ framework were found in reconstructed skin models made with papillary fibroblasts from both Caucasian and African skin types. Therefore, in vitro skin equivalents may be useful for better understanding of ethnic skin differences in the future.

Distinct and complementary roles of papillary and reticular fibroblasts in skin morphogenesis and homeostasis.

To study the biological properties of dermal fibroblast sub-populations, we used a reconstructed skin model with a dermal compartment populated with either papillary or reticular fibroblasts. The histological and immunohistological characterization of these reconstructed skins revealed distinct biological and structural differences, depending on the site-matched fibroblast population incorporated. Epidermal differentiation and maturation was favored and found optimum in the presence of papillary fibroblasts with little effect on ECM, as opposed to reticular fibroblasts, which had a significant positive effect on the production of the ECM molecules of the dermal epidermal junction and the dermis. In addition, the synthesis and release of MMPs and soluble factors like VEGF and KGF into the culture medium were influenced by the fibroblast population. MMP1 and VEGF were increased in the presence of papillary fibroblasts, whereas MMP3 and KGF levels were higher in the presence of reticular fibroblasts. Our results suggest that papillary and reticular fibroblasts exert distinct functions and activities in skin as revealed by the reconstructed skin model. These functional differences may have implications in wound healing and skin aging processes, considering the slow loss of papillary fibroblasts in human skin that occurs with age.

The Caucasian and African skin types differ morphologically and functionally in their dermal component.

In the literature, most reported differences between African and Caucasian skin properties concern pigmentation and barrier function of the stratum corneum and related photoprotective properties. However, little is known about differences in morphology and possibly related biological functions. In this study, we investigated: (i) architectural differences of Caucasian and African mammary skin biopsies using microscopy, (ii) comparative constitutive expression of cytokines, matrix metalloproteinase 1 (MMP-1) and its inhibitors in papillary dermal fibroblast (pF) and reticular dermal fibroblast (rF) cultures in order to reveal biological features. (i) Neither epidermis thickness nor superficial dermis thickness was significantly different in African versus Caucasian subjects. However, the dermal-epidermal junction (DEJ) length in African skin was about threefold that in Caucasian skin. No differences were noticed as regards elastic and collagen fibre organization. (ii) In papillary fibroblast cultures, a significantly higher level of monocyte chemotactic peptide-1 (MCP-1) protein was found in cell cultures from African donors when compared with that from Caucasians. With regard to keratinocyte growth factor (KGF), the ratio of papillary to reticular fibroblast expression was found to be twofold greater in cell cultures from African donors compared with that from Caucasian donors. The same trend was found regarding MMP-1 and tissue inhibitor metalloproteinase protein 1 (TIMP-1) protein expression. African skin displays a greater convolution of the DEJ and a higher papillary fibroblast activity. These findings reveal that differences between African and Caucasian skin do not only affect upper epidermis but also dermal functions and dermal-epidermal cellular interactions.

Aging alters functionally human dermal papillary fibroblasts but not reticular fibroblasts: a new view of skin morphogenesis and aging.

Understanding the contribution of the dermis in skin aging is a key question, since this tissue is particularly important for skin integrity, and because its properties can affect the epidermis. Characteristics of matched pairs of dermal papillary and reticular fibroblasts (Fp and Fr) were investigated throughout aging, comparing morphology, secretion of cytokines, MMPs/TIMPs, growth potential, and interaction with epidermal keratinocytes. We observed that Fp populations were characterized by a higher proportion of small cells with low granularity and a higher growth potential than Fr populations. However, these differences became less marked with increasing age of donors. Aging was also associated with changes in the secretion activity of both Fp and Fr. Using a reconstructed skin model, we evidenced that Fp and Fr cells do not possess equivalent capacities to sustain keratinopoiesis. Comparing Fp and Fr from young donors, we noticed that dermal equivalents containing Fp were more potent to promote epidermal morphogenesis than those containing Fr. These data emphasize the complexity of dermal fibroblast biology and document the specific functional properties of Fp and Fr. Our results suggest a new model of skin aging in which marked alterations of Fp may affect the histological characteristics of skin.

[Fibroblast subpopulations: a developmental approach of skin physiology and ageing]. / Etude des sous-populations de fibroblastes: une approche "développementaliste" de la physiologie et du vieillissement de la peau.

Skin is an organ whose function is far beyond a physical barrier between the inside and the outside of the body. Skin as the whole organism is subjected to ageing which concerns skin mostly in its dermal and deepest component which is also its matricial component. The dermis is a tissue rich in matricial elements and poor in cellular content and it is generally admitted that modifications occurring in the matrix are those which mostly contribute to skin ageing, by altering its biomechanical properties. Therefore it is common to address questions related to skin ageing by considering alterations in matrix molecules like collagen. Actually the dermis is a complex tissue both matricial and cellular and is divided between a superficial dermis close to epidermis and a deep dermis much thicker and histologically different. Several years ago we have undertaken investigations related to fibroblasts which are the cells responsible for the formation and maintenance of the dermis, aiming at isolation, culture and characterization of the fibroblasts from the superficial dermis also called papillary dermis and fibroblasts from the deep dermis also called reticular dermis. We were able to show that these fibroblasts in classical culture on plastic exhibit very different morphologies associated with different secretion properties and we have confirmed and expanded such observations revealing different phenotypes by incorporating these cells in reconstructed skin which allows the reproduction of a three-dimensional architecture recalling skin in vivo especially after grafting onto the nude mouse. We also raise the question of how these two dermal regions appear during the formation of the dermis and the question of their fate during ageing. Progress in solving these questions would certainly appear to be very useful for a better understanding of skin physiology and ageing and would hopefully provide new strategies in anti-ageing research.

Reconstructed skin modified by glycation of the dermal equivalent as a model for skin aging and its potential use to evaluate anti-glycation molecules.

Glycation is a slow chemical reaction which takes place between amino residues in protein and a reducing sugar. In skin this reaction creates new residues or induces the formation of cross-links (advanced glycation end products or AGEs) in the extracellular matrix of the dermis. Formation of such cross-links between macromolecules may be responsible for loss of elasticity or modification of other properties of the dermis observed during aging. We had previously developed a reconstructed skin model which enabled us to study the consequences of matrix alteration by preglycation of the collagen and have reported several modifications of interest induced by glycation in the dermal and epidermal compartments of reconstructed skin as well as at the level of the dermal-epidermal junction. For example we showed that collagen IV and laminin were increased in the basement membrane zone and that alpha6 and beta1 integrins in epidermis were expanded to suprabasal layers. The aim of this new study was to look at the biological effects of glycation inhibitors like aminoguanidine in the skin model. Aminoguanidine was mixed with collagen in the presence of ribose as reducing sugar, and immunostaining was used to visualize its effects on AGE Products and biological markers. After aminoguanidine treatment, we found a low amount of AGE products and a possible return to the normal pattern of distribution of markers in skin constructs as compared to those treated with ribose only. Interestingly similar results were also obtained, although to a lesser extent, with a blueberry extract. In conclusion the glycation inhibitory effect has been functionally demonstrated in the reconstructed skin model and it is shown that this model can be used to assess anti-glycation agents.

Collagen glycation triggers the formation of aged skin in vitro.

Glycation products accumulate during the aging of many slowly renewing tissues, including skin. We have developed an in vitro model of chronologic aging of skin based on reconstructed skin modified by artificially glycating the collagen used to prepare the dermal compartment. The morphology of the modified skin is close to the morphology usually observed except that the dermis is altered in its fibrillar structure. Moreover, the analysis of skin markers revealed several unexpected biological and morphological modifications, which reflect in vivo aging and could be related to glycation per se. These include the activation of fibroblasts, increase of matrix molecules (collagen type III and collagen type IV) and metalloproteinase production (MMP1, MMP2 and MMP9), thickening of the basement membrane zone, and more strikingly, the modification of alpha6 and beta1 integrin patterns especially in epidermis, in a way closely resembling aged skin in vivo. We also found that these effects could be related to the production of putative diffusible factors by the dermal fibroblasts activated by glycation. Finally, we show that all these effects are likely to be glycation specific since they could be inhibited by aminoguanidine, a well-known glycation inhibitor. We conclude that the reconstructed skin model modified by glycation of the collagen closely mimics chronologic aging of skin in vivo. Taken together, these results strengthen the importance of glycation reactions in skin aging.

An in vitro approach to the chronological aging of skin by glycation of the collagen: the biological effect of glycation on the reconstructed skin model.

Glycation is a slow, nonenzymatic reaction that takes place between free amino groups in proteins primarily from lysine and a reducing sugar such as glucose or ribose. In skin, this reaction creates new residues or formations of cross-links (advanced glycation end products, AGEs) in the extracellular matrix of the dermis. The formation of these bridges between dermal molecules is supposed to be responsible for loss of elasticity or other properties of the dermis observed during aging. Glycation may therefore play an important role in chronologic aging. In order to examine this hypothesis, we have developed a reconstructed skin model made of a modified dermal compartment that is a fibroblast-contracted collagen lattice prepared with preglycated collagen. The presence of AGEs (glycoxidation products) in the skin equivalents was evidenced using specific antibodies against carboxymethyllysine (CML). Several changes were observed after collagen glycation: (1) fibroblast shape and distribution (vimentin staining) were modified; (2) extracellular matrix molecules and the dermal-epidermal junction zone seemed to be enhanced (procollagen I and III, collagen IV and VII stainings); (3) stainings for beta1 and alpha6 integrins were also increased in the epidermal cell layer; and (4) collagenase activity was increased. To verify the biological effect of glycation, we used the well-known glycation inhibitor aminoguanidine. After aminoguanidine treatment, we found a low CML amount and decreased distribution of markers previously overexpressed in glycated skin constructs. These in vitro findings were at least in part related to aging in vivo and demonstrate an actual effect of glycation in skin aging.

[Natural and photo-induced aging of the skin: the three dimensional culture approach]. / Vieillissement naturel et photo-induit de la peau: approches en cultures tri-dimensionnelles.

Human skin is a complex multifunctional organ which covers and surrounds the whole body ensuring a key function of protection against external injuries. Because of this unique situation, aging of skin is the result of both extrinsic factors-mostly sun exposure leading to photoaging- and intrinsic factors assumed to represent chronological aging. Studies of such complex phenomena on human volunteers is questionable and classical cultures of skin cells are not close enough to in vivo physiological conditions. However it is possible to address these questions by reconstructing human skin in vitro with both a living dermal equivalent defined as a fibroblast-contracted collagen gel (Bell et al., 1979) and a fully differentiated epidermis characterized by horny layers (Asselineau et al., 1985).