Physical characterization of the stratum corneum of an in vitro human skin equivalent produced by tissue engineering and its comparison with normal human skin by ATR-FTIR spectroscopy and thermal analysis (DSC).

An in vitro human skin equivalent may be obtained by culturing human keratinocytes on a collagen gel containing fibroblasts. The anchored skin equivalent cultured at the air-liquid interface closely resembles human skin and is acceptable for in vitro percutaneous absorption. However, it is still more permeable than human skin. Since intercellular lipids have been recognized to play an important role in skin permeability, infrared spectroscopy and differential scanning calorimetry were performed on the stratum corneum of bovine or human skin equivalents grown at different days of air-liquid culture. The symmetric and asymmetric CH(2) stretching vibrations suggested that for all days observed, the intercellular lipids were less organized than those in human skin, irrespective of whether bovine or human collagen was used. Different culture conditions were also tested and the medium without serum and no epidermal growth factor at the air-liquid culture showed results significantly more comparable to human skin. Actually, the thermal behavior of in vitro stratum corneum showed transitions at lower temperatures than human skin. The transition around 80 degrees C, in the form of a lipid-protein complex, was absent. These results showed that the structural arrangement of intercellular lipids and their thermodynamic properties hold a crucial role in the barrier function of the stratum corneum.     

Establishment of 3D organotypic cultures using human neonatal epidermal cells

This protocol describes an ex vivo three-dimensional coculture system optimized to study the skin regenerative ability of primary human keratinocytes grown at the air-liquid interface on collagen matrices embedded with human dermal fibroblasts. An option for enrichment of keratinocyte stem cells and their progeny using fluorescence-activated cell sorting is also provided. Initially, dermal equivalents, comprising human passaged fibroblasts seeded in a collagen matrix, are grown on porous filters (3 mum) placed in transwells. After 1 week, primary human keratinocytes are seeded on this base. One week later, an air-lift transition is performed, leading to the differentiation of the keratinocytes, which are macroscopically visible as artificial skin after a couple of days. The cultures can be harvested 1 week after the air-lift and processed for immunohistochemistry or gene expression analysis. The overall procedure can be completed in 3 weeks, including the preparation of the dermal equivalent and the seeding of the primary keratinocytes.     

Identification of functional markers in a self-assembled skin substitute in vitro

Some functional parameters were identified and assessed in a tissue-engineered self-assembled skin substitute. This skin substitute was produced using fibroblasts and keratinocytes isolated from adult human skin. Keratinocytes were seeded on a dermal layer, composed of two fibroblast sheets cultured for 35 d. The epidermal cells formed a stratified and cornified epidermis and expressed differentiation markers, notably involucrin and transglutaminase. Interestingly and for the first time, the receptor for vitamin D3 was detected in all of the epidermal cell layers of the skin substitute, as it is reported for normal human skin. This observation suggests that keratinocytes retain key receptors during their differentiation in the skin model. A network of collagen fibers was observed by electron microscopy in the dermal layer of the model. In the dermis, collagen fibers remodeling and assembly is dependent on enzymes, notably prolyl-4-hydroxylase. For the first time in a skin construct, the expression of prolyl-4-hydroxylase was detected in dermal fibroblasts by in situ hybridization. The secretion of collagenases by the cells seeded in our skin substitute was confirmed by zymography. We conclude that the self-assembly approach allows the maintenance of several functional activities of human skin cells in a skin model in vitro.     

Development of a bilayered living skin construct for clinical applications

An in vitro construct of human skin (living skin equivalent, LSE) has been engineered using serially passaged human epidermal keratinocytes and human dermal fibroblasts with a matrix of type I collagen. Cells are obtained from neonatal foreskin. LSE is cast, cultured, and shipped in a single culture insert. The size and shape of theinsert determines thesize and shape of the LSE. The dermal matrix consists of dermal fibroblasts within a condensed collagen lattice. The overlying epidermis is developed at the air-liquid interface to generate a protective cornified layer. Serum was not necessaryfor development of the epidermis. LSE for graft (Graftskin) has handling characteristics similar to split-thickness skin allowing it to be meshed, stapled, and sutured. LSE was cryopreserved using 65% glycerol an rapid freezing. Viability and in vivo performance on athymic mice were similar to fresh LSE. Cells derived from human eccrine gland were able to invade and form tubules rudimentary appendages may be possible.     

Influence of dermal equivalent maturation on the development of a cultured skin equivalent.

Histologic and immunofluorescence methods were used to analyse the presence of fibronectin, chondroitin-4-sulphate and chondroitin-6-sulphate, type III and IV collagens, laminin, and keratins to assess the maturation level of cultured dermal and skin equivalents. In a first phase, fibroblasts in monolayer culture were compared with dermal equivalents in which fibroblasts are embedded in a type I collagen gel. Different fluorescent patterns were observed depending on the culture system used. A sequential appearance of macromolecules was noticed in dermal equivalents. Fibronectin was first detected after 4 days of culture, whereas chondroitin-4-sulphate and chondroitin-6-sulphate and type III collagen were present after 7 days. In contrast, all three macromolecules were detected at 24 h of culture in fibroblastic monolayer cultures. In a second phase, the quality of our skin equivalents was evaluated according to the seeding time of epidermal cells upon dermal equivalents (1, 4, or 7 days). A satisfactory stratification was obtained when keratinocytes were seeded after 4 and 7 days of dermal equivalent culture. Laminin and fibronectin were detected at the dermo-epidermal junction, but type IV collagen was absent. Various keratins, as detected by the AE1, AE2, and AE3 antibodies, were present in the epidermal layer. Following keratinocyte confluence, a change in the organization pattern of type III collagen in the dermal fraction of the skin equivalent was also noticed. Our comparative results show that seeding of epidermal cells on a more mature dermal equivalent leads to improved differentiation status of the epidermal layer.     

Characterization of a new tissue-engineered human skin equivalent with hair

Summary We designed a new tissue-engineered skin equivalent in which complete pilosebaceous units were integrated. This model was produced exclusively from human fibroblasts and keratinocytes and did not contain any synthetic material. Fibroblasts were cultured for 35 d with ascorbic acid and formed a thick fibrous sheet in the culture dish. The dermal equivalent was composed of stacked fibroblast sheets and exhibited some ultrastructural organization found in normal connective tissues. Keratinocytes seeded on this tissue formed a stratified and cornified epidermis and expressed typical markers of differentiation (keratin 10, filaggrin, and transglutaminase). After 4 wk of culture, a continuous and ultrastructurally organized basement membrane was observed and associated with the expression of laminin and collagen IV and VII. Complete pilosebaceous units were obtained by thermolysin digestion and inserted in this skin equivalent in order to assess the role of the transfollicular route in percutaneous absorption. The presence of hair follicles abolished the lag-time observed during hydrocortisone diffusion and increased significantly its rate of penetration in comparison to the control (skin equivalent with sham hair insertion). Therefore, this new hairy human skin equivalent model allowed an experimental design in which the only variable was the presence of pilosebaceous units and provided new data confirming the importance of hair follicles in percutaneous absorption.     

Ultrastructural and immunocytochemical detection of keratins and extracellular matrix proteins in lizard skin cultured in vitro

The present study shows the localization of epidermal and dermal proteins produced in lizard skin cultivated in vitro. Cells from the skin have been cultured for up to one month to detect the expression of keratins, actin, vimentin and extracellular matrix proteins (fibronectin, chondroitin sulphate proteoglycan, elastin and collagen I). Keratinocytes and dermal cells weakly immunoreact for Pan-Cytokeratin but not with the K17-antibody at the beginning of the cell culture when numerous keratin bundles are present in keratinocyte cytoplasm. The dense keratin network disappears after 7-12 days in culture, and K17 becomes detectable in both keratinocytes and mesenchymal cells isolated from the dermis. While most epidermal cells are lost after 2 weeks of in vitro cultivation dermal cells proliferate and form a pellicle of variable thickness made of 3-8 cell layers. The fibroblasts of this dermal equivalent produces an extracellular matrix containing chondroitin sulphate proteoglycan, collagen I, elastic fibers and fibronectin, explaining the attachment of the pellicle to the substratum. The study indicates that after improving keratinocyte survival a skin equivalent for lizard epidermis would be feasible as a useful tool to analyze the influence of the dermis on the process of epidermal differentiation and the control of the shedding cycle in squamates.     

Testing of promising dressing materials in cultures of fibroblasts and keratinocytes of human skin]

Cell cultures of epidermal keratinocytes and dermal fibroblasts were used to test collagen dressings in vitro. It was shown that effects of the dressings on skin major cell types might be differentially evaluated.     

Reconstituted skin from murine embryonic stem cells

Embryonic stem (ES) cell lines can be expanded indefinitely in culture while maintaining their potential to differentiate into any cell type. During embryonic development, the skin forms as a result of reciprocal interactions between mesoderm and ectoderm. Here, we report the in vitro differentiation and enrichment of keratinocytes from murine ES cells seeded on extracellular matrix (ECM) in the presence of Bone Morphogenic Protein-4 (BMP-4) or ascorbate. The enriched preparation of keratinocytes was able to form an epidermal equivalent composed of a stratified epithelium when cultured at the air-liquid interface on a collagen-coated acellular substratum. Interestingly, an underlying cellular compartment that belongs to the fibroblast lineage was systematically formed between the reconstituted epidermis and the inert membrane. The resulting tissue displayed morphological patterns similar to normal embryonic skin, as evidenced by light and transmission electron microscopy. Immunohistochemical studies revealed expression patterns of cytokeratins, basement membrane (BM) proteins and late differentiation markers of epidermis, as well as fibroblast markers, similar to native skin. The results demonstrate the capacity of ES cells to reconstitute in vitro a fully differentiated skin. This ES-derived bioengineered skin provides a powerful tool for studying the molecular mechanisms controlling epidermal and dermal commitments.     

The interaction of human papillary and reticular fibroblasts and human keratinocytes in the contraction of three-dimensional floating collagen lattices

Fibroblasts derived from the papillary and reticular dermis of human skin and human keratinocytes show differences in their abilities to contract floating three-dimensional gels constructed from type I collagen. Reticular fibroblasts produce greater gel contraction than papillary fibroblasts. When equal numbers of papillary and reticular fibroblasts are mixed in the gels, papillary fibroblasts consistently inhibit gel contraction by reticular fibroblasts indicating interaction between these cell types in the contraction process. Surprisingly, keratinocytes alone produce greater gel contraction than that produced by either fibroblast type. Cooperativity in the gel contraction process is observed when fibroblasts are incorporated into the collagen matrix and keratinocytes are seeded onto the gel surface. Keratinocytes and dermal fibroblasts adhere to the collagen fibril to induce gel contraction by different mechanisms. Fibroblast contraction of collagen gels does not require fibronectin but is a serum-dependent reaction. In contrast, keratinocyte contraction of collagen gels occurs in a serum-free environment. Polyclonal, affinity-purified antibodies to human plasma fibronectin at high concentrations do not inhibit gel contraction by keratinocytes, making unlikely the possibility that fibronectin synthesized by the keratinocyte is a significant factor in the gel contraction process. We are currently examining the possibilities either that keratinocytes are synthesizing other adhesion proteins or that receptors on the cell surface can interact directly with the collagen fiber.     

Comparison of hair dermal cells and skin fibroblasts in a collagen sponge for use in wound repair

The adult hair follicle has well-defined dermal and epithelial populations that display distinct developmental properties. The follicular dermal cells, namely the dermal papilla and dermal sheath, are derived from the same mesenchymal cells as dermal fibroblasts and therefore, we believed that follicular cells could be useful sources of interfollicular keratinocytes and fibroblast for skin wound repair. In this study, we evaluated the relative effect of various mesenchymal-derived cells on wound healing following skin injury. Human dermal cells, including two different follicular dermal cells and skin fibroblasts were cultured in collagen sponges and compared with respect to wound healing. Results indicated that there was no significant difference in wound contraction and angiogenesis among the cell types. Further, dermal sheath cells exhibited relatively poor results compared with other cells in new collagen synthesis. Finally, basement membrane reformation and new collagen synthesis for the dermal papilla cell grafts was superior to those of the dermal sheath cells or fibroblasts.     

Microfabrication of an analog of the basal lamina: biocompatible membranes with complex topographies.

A microfabrication approach was used to produce novel analogs of the basal lamina with complex topographic features. A test pattern of ridges and channels with length scales (40 to 310 micrometer) similar to the invaginations found in a native basal lamina was laser machined into the surface of a polyimide master chip. Negative replicates of the chip were produced using polydimethylsiloxane silicone elastomer and these replicates were used as templates for the production of thin ( approximately 21 micrometer) membranes of collagen or gelatin. The resulting membranes had a complex topography of ridges and channels that recapitulated the features of the master chip. To demonstrate their utility, these complex membranes were laminated to type I collagen sponges and their surfaces were seeded with cultured human epidermal keratinocytes to form a skin equivalent. The keratinocytes formed a differentiated and stratified epidermis that conformed to the features of the microfabricated membrane. The topography of the membrane influenced the differentiation of the keratinocytes because stratification was enhanced in the deeper channels. Membrane topography also controlled the gross surface features of the skin equivalent; infolds of the epidermis increased as channel depth increased. These novel microfabricated analogs of the basal lamina will help to elucidate the influence of topography on epithelial cell proliferation and differentiation and should have applications in the tissue engineering of skin equivalents as well as other basal lamina-containing tissues.     

Novel Biodegradable Porous Scaffold Applied to Skin Regeneration

Skin wound healing is an important lifesaving issue for massive lesions. A novel porous scaffold with collagen, hyaluronic acid and gelatin was developed for skin wound repair. The swelling ratio of this developed scaffold was assayed by water absorption capacity and showed a value of over 20 g water/g dried scaffold. The scaffold was then degraded in time- and dose-dependent manners by three enzymes: lysozyme, hyaluronidase and collagenase I. The average pore diameter of the scaffold was 132.5±8.4 µm measured from SEM images. With human skin cells growing for 7 days, the SEM images showed surface fractures on the scaffold due to enzymatic digestion, indicating the biodegradable properties of this scaffold. To simulate skin distribution, the human epidermal keratinocytes, melanocytes and dermal fibroblasts were seeded on the porous scaffold and the cross-section immunofluorescent staining demonstrated normal human skin layer distributions. The collagen amount was also quantified after skin cells seeding and presented an amount 50% higher than those seeded on culture wells. The in vivo histological results showed that the scaffold ameliorated wound healing, including decreasing neutrophil infiltrates and thickening newly generated skin compared to the group without treatments.     

Expression of high molecular weight (67K) keratin in human keratinocytes cultured on dead de-epidermized dermis

The influence of living dermal tissue upon epidermal differentiation during embryonic development as well as in vitro culture has been documented. Living dermal tissue contains both cellular and matricial elements. In the present study, third-passage subcultured adult human keratinocytes were either seeded on plastic dishes or recombined with dead de-epidermized dermis and further cultured for 3 weeks. After this time, keratins were extracted and analysed by one- and two-dimensional gel electrophoresis. The 67K keratin subunit, which is thought to be involved in the process of in vivo type skin differentiation, was absent in ordinary cultures; however, it was expressed in air-exposed cultures on dead de-epidermized dermis. Quantitatively, however, it did not reach the in vivo level. This suggests that in principle, the induction of the expression of this protein does not require the presence of living dermal cells.     

Induction of human microvascular endothelial tubular morphogenesis by human keratinocytes: involvement of transforming growth factor-alpha.

Transforming growth factor-alpha(TGF-alpha), homologous to epidermal growth factor(EGF), is closely involved in hyperproliferation of human keratinocytes. Psoriasis is a common hyperproliferative skin disease characterized by hyperproliferation of keratinocytes and abnormal development of dermal capillary networks. In this study, we have examined whether keratinocytes could enhance angiogenesis. TGF-alpha or EGF efficiently stimulated formation of tubular-like structures of human omental microvascular endothelial(HOME) cells in type I collagen gels. Human keratinocytes produced TGF-alpha. To examine whether co-cultured keratinocytes could induce tubulogenesis of HOME cells in collagen gel, we have developed a co-culture system with human keratinocytes. Surprisingly, there appeared new development of many tubular-like structures of HOME cells in collagen gels when co-cultured with keratinocytes. This keratinocytes-dependent tubulogenesis was almost completely blocked when anti-TGF-alpha-antibody was present. The TGF-alpha molecules derived from keratinocytes appeared to enhance tubulogenesis of human microvascular endothelial cells. We propose the hypothesis that secretory TGF-alpha from human keratinocytes may promote an autocrine loop to proliferate the skin keratinocytes and also a paracrine loop to induce the skin angiogenesis.     

Influence of initial collagen and cellular concentrations on the final surface area of dermal and skin equivalents: A box-behnken analysis

Summary Our laboratory has been involved in finding optimal conditions for producing dermal and skin equivalents. As an original approach, a Box-Behnken experimental design was used to study the effects of the initial collagen and fibroblast concentrations and the initial gel thickness on the contraction of dermal and skin equivalents. The final surface area of dermal equivalent varied significantly with the initial concentration of collagen and fibroblast, whereas the initial thickness of gel had no appreciable effect on the contraction of the dermal equivalent. When keratinocytes were grown on these dermal equivalents they produced a very severe contraction, to an extent that all skin equivalents had a similar final surface area. This severe contraction was independent of collagen and fibroblast concentrations. Models for the prediction of the final percentage contraction of dermal and skin equivalents as a function of the initial concentration of collagen, the logarithm of fibroblast concentration, and the initial gel thickness were obtained and analyzed. Keratinocytes grown at the lowest seeding density did not contract the equivalents. However, histologic analysis has shown an incomplete coverage by these cells of the equivalents. The extensive contraction of the skin equivalent presenting adequate morphology is a major drawback toward its clinical utilization for burn wound coverage. The financial supports for this project were received from Canadian NSERC postgraduate scholarship (P. Rompré), Québec FCAR postgraduate scholarship (C.A. López Valle), France-Québec research grant in Biotechnology (F.A. Auger), Canadian MRC grant (F.A. Auger), and NSERC grants (A. LeDuy and J. Thibault).     

A co-cultured skin model based on cell support membranes

Tissue engineering of skin based on collagen:PCL biocomposites using a designed co-culture system is reported. The collagen:PCL biocomposites having collagen:PCL (w/w) ratios of 1:4, 1:8, and 1:20 have been proven to be biocompatible materials to support both adult normal human epidermal Keratinocyte (NHEK) and mouse 3T3 fibroblast growth in cell culture, respectively, by Dai, Coombes, et al. in 2004. Films of collagen:PCL biocomposites were prepared using non-crosslinking method by impregnation of lyophilized collagen mats with PCL/dichloromethane solutions followed by solvent evaporation. To mimic the dermal/epidermal structure of skin, the 1:20 collagen:PCL biocomposites were selected for a feasibility study of a designed co-culture technique that would subsequently be used for preparing fibroblast/biocomposite/keratinocyte skin models. A 55.3% increase in cell number was measured in the designed co-culture system when fibroblasts were seeded on both sides of a biocomposite film compared with cell culture on one surface of the biocomposite in the feasibility study. The co-culture of human keratinocytes and 3T3 fibroblasts on each side of the membrane was therefore studied using the same co-culture system by growing keratinocytes on the top surface of membrane for 3 days and 3T3 fibroblasts underneath the membrane for 6 days. Scanning electron microscopy (SEM) and immunohistochemistry assay revealed good cell attachment and proliferation of both human keratinocytes and 3T3 fibroblasts with these two types of cells isolated well on each side of the membrane. Using a modified co-culture technique, a co-cultured skin model presenting a confluent epidermal sheet on one side of the biocomposite film and fibroblasts populated on the other side of the film was developed successfully in co-culture system for 28 days under investigations by SEM and immunohistochemistry assay. Thus, the design of a co-culture system based on 1:20 (w/w) collagen:PCL biocomposite membranes for preparation of a bi-layered skin model with differentiated epidermal sheet was proven in principle. The approach to skin modeling reported here may find application in tissue engineering and screening of new pharmaceuticals.