Development of an ex vivo human skin model and evaluation of biological responses to subcutaneously injected hyaluronic acid formulations

Abstract

We have previously described a model using porcine ear skin ex vivo for longitudinal studies into the disposition of macromolecules after subcutaneous injection. Since porcine skin cannot fully mimic biological responses in human skin, we now describe an ex vivo system using “full thickness” human skin. Spongiosis and epidermal detachment were the primary endpoints to evaluate skin structural integrity over a 9-day culture period. Epidermal barrier function and basal cell proliferation were monitored using expression of claudin-1 and Ki-67, respectively. Immunofluorescent staining of type I and type III procollagens and elastin after subcutaneous injection of TGF-β3, a cross-linked hyaluronic acid hydrogel, and saline solution and “no treatment” controls, showed that the model enabled visualization of changes in extracellular matrix proteins. Semi-quantitative, automated image analysis methods using multiple ROIs were evaluated to assess signal intensity and expression area of type I procollagen but displayed high inter-regional variability due to skin sample heterogeneity. Absolute quantitative methods, e.g. RT-qPCR or ELISA, which enable determination of biomarkers at either the mRNA level or the amounts of protein expressed in the sample, could be a better reporting tool. In conclusion, we successfully developed an ex vivo “full thickness” human skin model that retained viability over 9 days and which could be deployed in combination with qualitative/quantitative methods to evaluate local biological effects of subcutaneously injected biomacromolecules.