Microscopic examination and measurement of pigmentation in the <i>in vitro</i> pigmented skin model reconstructed in the presence or absence of fibroblasts in the dermal compartment.

<p><b>A</b>) Microscopic examination of HES staining of cross-sections showed a correct epidermal architecture and differentiation process leading to the development of a stratum granulosum and stratum corneum in both 3D systems. The CD13 staining (green) of cross-sections confirmed the presence or absence of fibroblasts (from adult 21 yr-old donor) in the dermal equivalent. Macroscopic pictures of PRS illustrate the resulting drastic difference in pigmentation between the two conditions which was confirmed by <b>B</b>) Luminance measurement (mean of 3 experiments). Fontana-Masson staining for melanin granules detection (black points), and <b>C</b>) graph of melanin quantification (mean of 3 experiments) performed by image analysis of Fontana-Masson (FM) staining on cross-sections. To compare the two conditions, results on melanin content were normalized to the control condition (with fibroblasts). TRP-1 staining (red) of cross-sections shows melanocytes positioned at the basal layer of the epidermis in both 3D systems. TRP-1 staining (green) of PRS epidermal sheets and <b>D</b>) corresponding graph of melanocyte quantification (mean of 3 experiments) performed by image analysis. The values represent means ± standard deviation (SD). Statistical significance was evaluated by the Student's <i>t</i>-test; *** p-value <0.001. Magnification: A HES, FM = x400, CD13, TRP1/section = x200, TRP1/sheet = x50.