Fig. 4

Softening of corneal tissue with collagenase increases expression of limbal markers during ex vivo re-epithelialisation. a Representative whole-cornea X–Z scan of Brillouin frequency shifts measured at a sample spacing of 20 µm from healthy intact human corneas after collagenase treatment (n = 3). Insets correspond to (b) the regions of the central cornea softened with collagenase (treated) or left untreated (control) analysed at very high-resolution scanning, using a sampling distance of 2.5 µm, showing a distinct epithelium (Ep), sub-epithelial layer (Sub), and stroma (St), as well as the location of the Bowman’s layer (white arrowhead). This detailed analysis evidenced the loss of the stiffer Bowman’s layer (black arrowhead) and reduced Brillouin frequency shifts in both epithelium and stroma resulting from collagenase treatment. c Brillouin frequency shifts from the anterior part of both treated (white) and control regions (grey bars) of the central cornea and limbus. The plot represents average ± S.D. of measurements taken from the epithelium, sub-epithelium, and stroma from three independent experiments (100 individual measurements per each individual area, per experiment; n = 3; ns corresponds to p > 0.05 and *, **, and *** to p < 0.05, 0.01, and 0.001 after two-way ANOVA, respectively). Source data are provided as a Source Data file. d Representative confocal immunofluorescence micrographs (3D reconstruction) of laminin-1 (green) and collagen-VII distribution (red staining) in the central cornea, before (upper) and after collagenase treatment (central panel), and after re-epithelialisation (lower panel). e Representative confocal immunofluorescence micrographs (3D reconstruction) of collagenase-softened central cornea after re-epithelialisation. Epithelial cells repopulating softened corneas expressed ABCG2 and CK15 (limbal markers; green) while showing lower CK3+12 and β-catenin expression (differentiation markers; red staining) compared to cells growing on the stiffer, untreated corneas (control). f Epithelial cells expressed integrin-α9 and deposited laminin-γ3 when grown on collagenase-softened corneal substrates, but not on untreated central cornea (control). Cell nuclei were detected using DAPI in all three independent experiments. Scale bars, 50 µm