Fig. 4. Fibroblast pre-selection fosters prevascularization of the bilayer construct. (a) Schematic representation of the reticular and papillary fibroblasts, endothelial cells and keratinocyte seeding strategy on the bilayer structure. (b) Representative immunohistochemistry images of the expression of podoplanin (PDPN) and transglutaminase-2 (TGM2) at days 8 and 28. Scale bar = 200 μm. (c) Representative immunohistochemistry images of the organization of hDMECs (CD31+, orange arrowheads) in the bilayer skin constructs prepared with reticular and papillary fibroblasts unsorted fibroblasts. (i–ix) Higher magnification of the areas outlined in the respective left panel. Nuclei were counterstained with DAPI. Scale bar = 200 μm, 50 μm. (d) Quantification of the amount of capillary-like structures at days 21 and 28. Quantitative results are expressed as the mean ± standard deviation, *p < 0.05, two-way ANOVA with Tukey multiple comparison post-test. E – epidermis, P – papillary-like dermis, R – reticular-like dermis.