Being exposed to ground-level ozone (O3), as it is often the case in polluted cities, is known to have a detrimental impact on skin. O3 induces antioxidant depletion and lipid peroxidation in the upper skin layers and this effect has repercussions on deeper cellular layers, triggering a cascade of cellular stress and inflammatory responses. Repetitive exposure to high levels of O3 may lead to chronic damages of the cutaneous tissue, cause premature skin aging and aggravate skin diseases such as contact dermatitis and urticaria. This review paper debates about the most relevant experimental approaches that must be considered to gather deeper insights about the complex biological processes that are activated when the skin is exposed to O3. Having a better understanding of O3 effects on skin barrier properties and stress responses could help the whole dermato-cosmetic industry to design innovative protective solutions and develop specific cosmetic regime to protect the skin of every citizen, especially those living in areas where exposure to high levels of O3 is of concern to human health.
A three-dimensional human epidermis model reconstructed from neonatal primary keratinocytes is presented. Herein, a protocol for the cultivation process and the characterization of the model is described. Neonatal primary keratinocytes are grown submerged on permeable polycarbonate inserts and lifted to the air-liquid interface three days after seeding. After fourteen days of stimulation with defined growth factors and ascorbic acid in high calcium culture medium, the model is fully differentiated.Histological analysis revealed a completely stratified epidermis, mimicking the morphology of native human skin. To characterize the model and its barrier functions, protein levels and localization specific for early-stage keratinocyte differentiation (i.e., keratin 10), late-stage differentiation (i.e., involucrin, loricrin, and filaggrin) and tissue adhesion (i.e., desmoglein 1), were assessed by immunofluorescence. The tissue barrier integrity was further evaluated by measuring transepithelial electrical resistance. Reconstructed human epidermis was responsive to proinflammatory stimuli (i.e., lipopolysaccharide and tumor necrosis factor alpha), leading to increased cytokine release (i.e., interleukin 1 alpha and interleukin 8). This protocol represents a straightforward and reproducible in vitro method to cultivate reconstructed human epidermis as a tool to assess environmental effects and a broad range of skin-related studies.
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