Christian Collombel

Collagen synthesis by fibroblasts cultured within a collagen sponge

We prepared a collagen sponge made of type I and III bovine collagen, glycosaminoglycans (GAG) and chitosan. Fibroblasts grown within the collagen sponge express a sixfold increase of their collagen synthesis, compared with fibroblasts embedded in a collagen gel. Moreover, collagen synthesis is twice as high in the collagen sponge than in a monolayer culture. The collagen sponge culture system promotes a dynamic model for us to perform studies on the regulations of collagen synthesis. Increased collagen production within the collagen sponge leads fibroblasts to reconstitute their own extracellular matrix, which should be more physiological than a bovine collagen gel.

Characterization of Skin Reconstructed on a Chitosan-Cross-Linked Collagen-Glycosaminoglycan Matrix

Reconstruction of skin requires both the dermal and epidermal equivalent of the skin. We have developed a reconstructed skin composed of two compartments: (1) a dermal equivalent comprising an acellular dermal substrate populated by foreskin fibroblasts and (2) an epidermis regenerated from normal human keratinocytes seeded onto the dermal equivalent. The dermal substrate contains type I and III collagen and glycosaminoglycans (GAGs) cross-linked by chitosan. Fibroblasts seeded into the porous structure of the dermal substrate provide a dermal equivalent suitable to support epidermal cells. Keratinocytes attach quickly, exhibit mitotic activity and form a continuous and stratified epidermis. After 2 weeks of culture, histological sections show a basal layer with cuboidal cells attached to the dermal equivalent and several suprabasal cell layers including the stratum corneum. Transmission electron microscopy revealed the cell membrane densification (hemidesmosomes) at the dermoepidermal junction; however, the lamina densa was found discontinuous at this stage. We noted the presence of lipid vesicles in spinous layer and keratohyalin granules in granular layer. The epidermal differentiation was complete terminal with the stratum corneum containing several layers of corneocytes filled with tonofilaments. Reconstructed skin, based on our chitosan-cross-linked collagen-GAG matrix is morphologically equivalent to normal human skin and should thus provide a useful tool for in vitro toxicological studies as well as a suitable wound covering for the treatment of patients with severe burns.

Deposition of collagen fibril bundles by long-term culture of fibroblasts in a collagen sponge.

Human fibroblasts cultured for 10 days in a collagen sponge migrated through the pores of the sponge and expressed a moderate mitotic activity, which stabilized after 10 days, and a high collagen and protein synthesis. Between 10 and 27 days, the newly synthesized collagen filled the pores of the sponge. This matrix accumulation induced a delayed decrease of collagen and protein synthesis. Finally, after 27 days of culture, the fibroblasts expressed low biosynthetic activities similar to the ones exhibited in vivo. The newly synthesized matrix was highly differentiated, as shown by the presence of a dense network of quarter-staggered collagen fibrils (42 nm +/- 6 nm in diameter) surrounding the cells. The size and the shape of these fibrils demonstrated that the newly synthesized procollagen was fully processed in collagen by removal of their N- and C-terminal propeptides. Moreover, these fibrils were packed in bundles organized into an interwoven network that mimics the pattern of the papillary dermis. These findings show that fibroblasts cultured for one month in a collagen sponge construct large amounts of a highly differentiated connective tissue.

Use of dermal equivalent and skin equivalent models for identifying phototoxic compounds in vitro

Phototoxicity inducing in vivo photoirritation, a reversible inflammatory reaction of the skin after chemical contact and UVA radiation exposure, is increasingly observed as a side effect associated with the use of both cosmetics and systemic drugs. In order to systematically screen for the phototoxic potential of new compounds, we propose two three‐dimensional models suitable for in vitro testing: a dermal equivalent (DE) and a skin equivalent (SE) model. The DE model includes a collagen‐glycosaminoglycans‐chitosan porous matrix populated by normal human fibroblasts. The SE model is made by seeding normal human keratinocytes onto the DE, leading to a fully differentiated epidermis. The objectives of this pilot study are: 1) to compare the deleterious effects of UVA radiation on the two models and 2) to evaluate to what extent the in vitro results can predict the in vivo phototoxicity caused by well‐known photoirritant compounds, included in the COLIPA validation phototoxicity reference chemical list. Dilutions of thiourea, sulisobenzone, promethazine, chlorpromazine and tetracycline were applied (20 μl) onto DEs and SEs (n = 6) and incubated for 1 h (or 15 h) at 37°C. Irradiated samples received 3 J/cm2 UVA. The 24 h post‐irradiation residual cellular viability was measured using the MTT test on treated and untreated tissues and IL‐1α release measurement in collected SE culture media. A concordance in terms of photoirritant/non‐photoirritant was obtained between the in vivo data and the in vitro results, suggesting that the DE and the SE models could be integrated, after a complete validation study, into a protocol for in vitro testing of the photoirritant potential of new molecules.

Mesenchymal-epithelial interactions regulate gene expression of type VII collagen and kalinin in keratinocytes and dermal-epidermal junction formation in a skin equivalent model.

Anchoring fibrils constituted primarily of type VII collagen and anchoring filaments composed of kalinin are essential structural elements of the dermal‐epidermal junction and critical for its stability. The role of fibroblasts in the production of these structural elements and the formation of the dermal‐epidermal junction was studied by using a living skin equivalent model. This model had been modified such that keratinocytes and fibroblasts were allowed direct contact. After 2 weeks, immunohistochemical studies showed the linear deposition of type VII collagen and kalinin, as well as type IV collagen and α6 integrin at the dermal‐epidermal junction. By electron microscopy, anchoring fibrils, a continuous lamina densa, and numerous hemidesmosomes were noted. Reverse transcriptase‐polymerase chain reaction analysis showed an increased expression of both type VII collagen and kalinin genes in keratinocytes when they were in direct contact with fibroblasts. These results suggest that fibroblasts synthesize an extracellular matrix which favors keratinocyte adhesion and the formation of a dermal‐epidermal junction by increasing the production and the further arrangement of dermal‐epidermal junction components.

Measurements of the Protective Effect of Topically Applied Sunscreens using in vitro Three-dimensional Dermal and Skin Equivalents

Abstract— For preventing or minimizing acute and chronic skin damage caused by UV radiation, the use of sunscreens is probably the most important measure. To screen the protective efficacy of new sunscreen molecules or formulations against UV rays, we evaluated as in vitro testing methods the use of two three‐dimensional models, a dermal equivalent (DE) and a skin equivalent (SE). The DE is composed of a porous collagen‐glycosaminoglycans‐chitosan matrix populated by normal human fibroblasts. The SE is comprised of a fully differentiated epidermis realized by seeding keratinocytes onto the DE. In this study, we demonstrated that the DE and SE models react to the deleterious effects of UVA and UVB. Then, we extended our research to the evaluation of their usefulness for photoprotection trials. Sunscreen agents (Euso‐lex 8020 and 6300) and commercially available sunscreens (chemical and physical filter formulations) that protect the skin against either UVA or UVB were evaluated. The tested products were applied (n = 6) topically (10 μL) and incubated for 30 min prior to irradiation over a range of UVA (0‐50 J/cm2) or UVB (0‐5 J/cm2). The photoprotection provided by the tested sunscreen molecules and formulations was evaluated by measurement of residual cellular viability 24 h postirradiation using the 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetra‐zolium bromide (MTT) test and assessment of the inflammation response by interleukin‐la release assay. When sunscreens were applied prior to UV exposure, a higher residual cellular viability versus control was obtained, demonstrating the photoprotective effects of the tested products. These in vitro models could be used for screening tests to evaluate the protective effects of sunscreen molecules and formulations, especially for UVA trials because there is a lack of consensus for an in vivo method.

Pigmented human skin equivalent—as a model of the mechanisms of control of cell-cell and cell-matrix interactions

The melanin pigment system in human skin is extraordinarly well developed and assures the photoprotection of the skin against harmful solar radiation. Specific cell-cell interactions between one melanocytes and keratinocytes play a fundamental role in the regulation of melanogenesis and melanin pigementation, the two key elements of this system, giving rise to the concept of a structural, functional collaborative ‘epidermal melanin unit,’ Early experiments strongly suggested that melanocyte growth and differentiation are regulated by paracrine factors from keratinocytes and other skin cells. In addition, co-culture studies with keratinocytes has shown that the extracellular matrix acts as a local environmental signal for dendrite formation and melanogenesis. Attempts to reconstruct pigmented human skin in vitro have made great progress over the last decade. The behavior of cells in these pigmented human skin equivalents closely resembles that in vivo, and the cells can still respond to appropriate extrinsic regulatory stimuli such as ultraviolet radiation. Keratinocytes and fibroblasts have been shown to be active partners in the regulation of melanocyte distribution, viability and other differentiation functions, presumably by direct contact and the effects of various soluble paracrine factors. By reproducing cell-cell and cell-matrix interactions, these culture systems provide a promising experimental model for investigating regulation of the skin pigmentary system and the role of photoprotection against harmful solar radiation.

Use of Dermal Equivalent and Skin Equivalent Models for in Vitro Cutaneous Irritation Testing of Cosmetic Products: Comparison with in Vivo Human Data

AbstractThe development of new cosmetic formulations requires precise assessment of their safety and efficacy. Today, legislation imposes increasing measures of safety as well as the limitation of animal use for such testing (European Community directive 93/35/CEE). Subsequently, safety assessment protocols now focus on in vivo human volunteer tests and in vitro methods. In this study, in vivo testing consisted of 48 h patch tests on human volunteers followed by a clinical evaluation of irritation based on a visual scoring system including evaluation of erythema, edema, dryness, and vesicles. For in vitro testing to substantiate the safety of cosmetic products, we propose two three-dimensional models, a dermal equivalent (DE) and a skin equivalent (SE). The DE is composed of a porous collagen-glycosaminoglycans-chitosan dermal substrate populated by normal human fibroblasts. The SE is completed by a fully differentiated epidermis made by seeding normal human keratinocytes onto the DE.To evaluate the usefu...

35 From monolayered culture to pigmented skin equivalent: complexification of the skin cell culture and contributions in pharmaco-toxicology

We present the evolution of the cellular models developed in our laboratory f r o m monolayered cultures of normal human fibroblasts, keratinocytes and melanocytes to three-dimensional (3-D) models: Dermal Equivalent (DE) obtained by fibroblasts cultiare on a collagen-GAG-chitosan porous matrix S k i n Equivalent (SE) by seeding keratinocytes on this DE P i g m e n t e d S k i n E q u i v a l e n t ( P S E ) b y incorporating melanocytes to the SE.