Development of an in vitro model of menopause using primary human dermal fibroblasts.

OBJECTIVE: To overcome the current lack of in vitro models to specifically reproduce hormonal skin ageing in women, and in search of active ingredients with innovative efficacy claim for cosmetic skin care, we developed a cell culture-based model by simulating menopause's hormonal decline and assessed several parameters of collagen metabolism. METHODS: Human dermal fibroblasts were incubated with media containing 17ß-oestradiol, progesterone, dehydroepiandrosterone, growth hormone and insulin-like growth factor-1 at concentrations corresponding to those of non-menopausal women's sera and then of menopausal women's sera. We measured cell proliferation [by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT)], matrix metalloproteinase-1 and metalloproteinase-3 (MMPs) release (by enzyme-linked immunosorbent assay - ELISA), total collagen deposition (by Sirius red staining), types I and III collagen deposition (by ELISA), and types I and III procollagen gene expression (by real-time q-RT-PCR). RESULTS: Our results showed a significant decrease over time in cell proliferation, collagen deposition and type III/type I collagen ratio, together with an increase in MMP release, when cells were incubated in media containing sex hormones at menopausal levels. This is consistent with in vivo data from menopausal women available in the literature. Surprisingly, procollagen gene expression was only reduced within the first hours and increased afterwards when compared with non-menopausal culture conditions. CONCLUSION: Our results demonstrated that the increased procollagen synthesis with menopausal conditions was not sufficient to compensate for the MMPs' catabolic effects and/or the impaired procollagen protein maturation, resulting in a decrease in extracellular collagen content. These findings add to the overall understanding of hormone-dependent skin behaviour and highlight the suitability of this in vitro model for cosmetic actives testing aiming to underpin claims of anti-ageing efficacy, specifically for menopausal women, regarding collagen metabolism and balance of types, for maintenance of dermal mechanical properties.

A new potent natural antioxidant mixture provides global protection against oxidative skin cell damage.

Oxidative stress occurs when there is an over production of free radicals and cells are not able to neutralize them by their own antioxidant mechanisms. These excess of free radicals will attack cellular macromolecules leading to cell damage, function impairment or death. Because of that, antioxidant substances have been largely used in products to offer complementary protection. In this study a new mixture of three known antioxidants (cocoa, green tea and alpha-tocopherol) was evaluated and its antioxidant protection was assessed focusing on its capacity to protect main cell macromolecules. Results have shown that it has a high antioxidant capacity by protecting lipids, DNA and proteins against oxidative damage. The antioxidant effect of the mixture on cells was also investigated and it was able to reduce oxidative stress generated by lipopolisacharide in human fibroblasts. Finally, as the mixture has proved to be highly antioxidant, its effect on cell senescence was evaluated, and it was demonstrated that fibroblasts in culture had delayed senescence when treated with these actives on a mixture. All results together provide important data about a new antioxidant mixture that uses a small amount of actives and is able to protect cell against oxidative damages in a global way.