Rapid developments in tissue engineering have renewed interest in biodegradable three-dimensional structures such as collagen-based biomaterials. Collagen matrices seeded in vitro with fibroblasts, osteoblasts, and chondrocytes can form tissues resembling skin, bone, and cartilage that could be used as functional substitutes for damaged tissues. Collagen is associated with both dystrophic calcification of collagenous implants and bone mineralization. We report here the calcification properties of collagen sponges incubated in cell-free media. Mineral deposited in sponges was identified by X-ray and electron diffraction, Fourier transform infrared spectroscopy, and the molar ratio of calcium:phosphorus (Ca:P) as a poorly crystalline apatite similar to bone. The degree of calcification increased with length of incubation and the Ca and P content of the media, with 10-15% Ca (dry weight) after 21 days' incubation in media containing 1.6-3 mM Ca and a Ca x P molar product of 2-3 mM(2), but only 2% Ca after incubation in medium with 1.33 mM Ca and a 1.7 mM(2) Ca x P molar product. Mineral deposition was completely inhibited in sponges that were washed extensively and initially contained less than 0.01% P. Readdition of phosphate in these sponges and subsequent freeze drying and sterilization restore their mineralization capacity, suggesting that collagen per se cannot initiate calcification and that the inorganic phosphate content associated with the collagen preparation process is in the solid state a potential nucleator. Addition of chondroitin 4-sulfate to the sponges partially or totally inhibited mineral deposition, even though 80-90% of the compound was released within 24 hours. These results indicate that acellular calcification of collagen-based biomaterials can occur under the culture conditions currently used in tissue engineering.

The epidermis is continuously renewed by stem cell proliferation and differentiation. Basal keratinocytes append the dermal‐epidermal junction, a cell surface‐associated, extracellular matrix that provides structural support and influences their behaviour. It consists of laminins, type IV collagen, nidogens, and perlecan, which are necessary for tissue organization and structural integrity. Perlecan is a heparan sulfate proteoglycan known to be involved in keratinocyte survival and differentiation. Aging affects the dermal epidermal junction resulting in decreased contact with keratinocytes, thus impacting epidermal renewal and homeostasis. We found that perlecan expression decreased during chronological skin aging. Our in vitro studies revealed reduced perlecan transcript levels in aged keratinocytes. The production of in vitro skin models revealed that aged keratinocytes formed a thin and poorly organized epidermis. Supplementing these models with purified perlecan reversed the phenomenon allowing restoration of a well‐differentiated multi‐layered epithelium. Perlecan down‐regulation in cultured keratinocytes caused depletion of the cell population that expressed keratin 15. This phenomenon depended on the perlecan heparan sulphate moieties, which suggested the involvement of a growth factor. Finally, we found defects in keratin 15 expression in the epidermis of aging skin. This study highlighted a new role for perlecan in maintaining the self‐renewal capacity of basal keratinocytes.

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Human skin equivalents (SEs) are popular threedimensional (D) cell culture systems in fundamental and applied dermatology. They have been made to contain dendritic cells, but so far no study on the incorporation of potentially antiinflammatory dermal macrophages has been performed. Here, we show that monocyte-derived dermal-type macrophages can be introduced into a rigid scaffold with dermal fibroblasts. They maintain their cell surface markers CD163, DC-SIGN ⁄ CD209 and HLA-DR, which discriminate them from monocytes and dendritic cells. They retain the ability to produce the anti-inflammatory cytokine IL-10 in response to lipopolysaccharide (LPS) and to phagocytose latex beads. We thus demonstrate the feasibility of creating macrophage-fibroblast 3D cultures as a first step towards generating SEs with dermal macrophages.

Proopiomelanocortin is a precursor peptide that gives rise to several neuropeptides including adrenocorticotrophic hormone (ACTH) and β-endorphin. POMC-derived peptides have been shown to be synthesized in human epidermis where they modulate numerous skin functions. Because we previously observed that melanocortin receptor-2 and μ-opioid receptor 1, the respective receptors for ACTH and β-endorphin decreased with ageing in human epidermis, we have selected an active ingredient (INCI name: Achillea millefolium extract) able to upregulate receptor expressions. The aim of the present work was first to evaluate the effect of A. millefolium extract on the expression pattern of various epidermal differentiation markers ex vivo in normal human skin biopsies using quantitative image analysis and second to evaluate its capacity to rejuvenate the appearance of skin surface in vivo. Results show an improved expression profile of cytokeratin 10, transglutaminase-1 and filaggrin in cultured skin biopsies as well as an increased epidermal thickness. In vivo, a 2-month treatment with A. millefolium extract at 2% significantly improved the appearance of wrinkles and pores compared with placebo. Results were also directionally better than those of glycolic acid that was chosen as reference resurfacing molecule.

During aging, the epidermis and dermis become thin and an efficient anti-aging product should be able to stimulate the metabolism of senescent fibroblast and keratinocytes, in order to increase the quantity of extra-cellular matrix components such as collagen and glycosaminoglycans. A study performed in parallel on an in vitro skin equivalent model, and in vivo, with human volunteers, demonstrated the efficacy of one specific soya biopeptide for anti-aging properties. Such a biopeptide induces a significant increase of glycosaminoglycans synthesis in vitro and in vivo after a one-month treatment. We also showed that this new cosmetic ingredient is able to stimulate favourably the collagen synthesis in vitro and in vivo. This study provided the proof for anti-aging properties of a new soya biopeptide and also validated the skin equivalent model developed for this experimentation as an alternative method to animal or human testing for some cosmetic efficacy evaluations.

Human skin melanin pigmentation is regulated by systemic and local factors. According to the type of melanin produced by melanocytes, the transfer and degradation of melanosomes differ, thus accounting for most variations between ethnicities. We made the surprising observation that in a drastically changed environment, white and black phenotypes are reversible since Caucasian skin grafted onto nude mice can become black with all black phenotypic characteristics. Black xenografts differed essentially from other grafts by the levels of epidermal FGF‐2 and keratin 5. In vitro analysis confirmed that FGF‐2 directly regulates keratin 5. Interestingly, this phenomenon may be involved in human pathology. Keratin 5 mutations in Dowling–Degos Disease (DDD) have already been associated with the pheomelanosome–eumelanosome transition. In a DDD patient, keratin 5 was expressed in the basal and spinous layers, as observed in black xenografts. Furthermore, in a common age‐related hyperpigmentation disorder like senile lentigo (SL), keratin 5 distribution is also altered. In conclusion, modulation of keratin 5 expression and distribution either due to mutations or factors may account for the development of pigmentary disorders.

Atopic dermatitis (AD), the most common inflammatory skin disorder, is a multifactorial disease characterized by a genetic predisposition, epidermal barrier disruption, a strong T helper (Th) type 2 immune reaction to environmental antigens and an altered cutaneous microbiome. Microbial dysbiosis characterized by the prevalence of Staphylococcus aureus (S. aureus) has been shown to exacerbate AD. In recent years, in vitro models of AD have been developed, but none of them reproduce all of the pathophysiological features. To better mimic AD, we developed reconstructed human epidermis (RHE) exposed to a Th2 pro-inflammatory cytokine cocktail and S. aureus. This model well reproduced some of the vicious loops involved in AD, with alterations at the physical, microbial and immune levels. Our results strongly suggest that S. aureus acquired a higher virulence potential when the epidermis was challenged with inflammatory cytokines, thus later contributing to the chronic inflammatory status. Furthermore, a topical application of a Castanea sativa extract was shown to prevent the apparition of the AD-like phenotype. It increased filaggrin, claudin-1 and loricrin expressions and controlled S. aureus by impairing its biofilm formation, enzymatic activities and inflammatory potential.