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 17β-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.