Cell proliferation and differentiation in a model of human skin equivalent

Abstract

Recent advances in culturing technology has permitted the production of organotypic models that may be referred to as human skin equivalents (HSE). We have studied histochemical, ultrastructural, and kinetic aspects of an HSE composed by an epidermal equivalent and a dermal equivalent separated by a basement membrane. Only keratinocytes and fibroblasts were present in the epidermal and dermal equivalents, respectively; cells of other lineages were lacking. Keratinocyte stratification and differentiation seemed similar to natural skin. Evidence is shown that such an HSE may also release growth factors such as vascular endothelial growth factor that are believed to play a role in skin grafting. The distribution of cycling cells as well as the values of the growth fraction are comparable to those observed in natural skin. Although the absence of several cells populations that reside in natural skin is a remarkable feature of this HSE, the high levels of tissue organization and cell differentiation lead us to believe that such an HSE may be considered a candidate substitute of human skin in biological, pharmacologic, and clinical applications. Anat Rec 264: [261][262][263][264][265][266][267][268][269][270][271][272] 2001.

Generation of Human Epidermal Constructs on a Collagen Layer Alone

“…Our findings are in accordance with previous studies showing that the formation of basement membrane at the dermal-epidermal junction, as well as the synthesis of other matrices by dermal fibroblasts, requires the interactions between keratinocytes and dermal fibroblasts. [43][44][45]57 Although the basement membrane was absent in our constructs, basal keratinocytes expressed integrin b1 subunit and a2b1 integrin. Integrins are heterodimeric cellsurface glycoproteins that primarily mediate the attachment of basal keratinocyte to extracellular matrix proteins.…”

“…Within synthetic constructs, it was possible to identify cell layers comparable with those observed in natural skin; nevertheless, the number of the cell layers (4-6 layers, including basal cells) was lower than in natural skin and other models of bilayered skin equivalents. 43,45,51,52 The ultrastructure of the synthetic construct showed a typical structure in the basal stratum with the natural epidermis. 53 However, electron microscopy analysis revealed that the overall organization of these layers, as well as keratinocyte differentiation, was poorer than with natural epidermis, including the formation of junctional complexes, basement membrane, and keratinization.…”

“…1,41,42 Previous studies have shown that epithelial-mesenchymal interactions (epidermal-dermal signaling) are required for epidermal histogenesis in vitro. 3,4,[43][44][45] Type I collagen is the major matrix components of dermis (the mesenchymal counterpart of the epidermis in natural skin). We assumed that it could be the most representative matrix that could mimic the connective tissue underlying the differentiation of keratinocytes in vitro.…”

Epidermal growth factor improves the migration and contractility of aged fibroblasts cultured on 3D collagen matrices

“…Due to this characteristic of skin, monolayer cell culture models cannot fully recapitulate the functional complexity of human skin. Thus, tissue-engineered skin substitutes utilizing alternative skin scaffold approaches, such as 3-dimensional (3D) collagen matrices may represent more effective systems for skin research (24,25). In addition, tissue-engineered skin substitutes provide a novel platform for testing the effects and biological reactions of pharmacological agents on the assembly of tissuelike structures, and may be suitable for replacing some in vivo experiments and preliminary real human skin studies (26,27).…”

A mathematical model for the design of fibrin microcapsules with skin cells

“…The cells with their characteristic round morphology are in active proliferation, as revealed by the presence of mitotic figures, until they organize in three-dimensional compact clusters. At this stage they were immuno-stained with anti-Cytokeratin, clon AE1/AE3, which reacts with most cytokeratins [36,37] allowing positive identification of keratinocytes inside the microcapsule. Moreover, when histological sections were immuno-stained with polyclonal anti-Vimentin, looking for the presence of fibroblasts, scarce reaction was verified, thereby indicating a predominant population of keratinocytes with meaningless presence of fibroblasts.…”