Fig 1.
(a) Schematic drawings of different approaches to the transplantation of corneal tissue. PK = Penetrating keratoplasty, DSAEK = Descemet stripping automated endothelial keratoplasty, DMEK = Descemet membrane endothelial keratoplasty.). (b) Side view and (c) top view of human DM in buffer solution. DM tissue is transparent and tends to coil up, posing intraoperative challenges to unfolding during DMEK surgery. Representative grey-scale images of coiled up 8 mm DM scroll. Scale bar = 1 mm.
Fig 2.
Assessment of changes in DM elastic moduli using AFM.
(a) Schematic drawing of the experimental setup. Each cornea was cut into four pieces. AFM measurements were performed before exposure to a vital dye (providing a baseline K value) and one or four minutes after exposure to TB (TB1, TB4) or MB (MB1, MB4). (b) Boxplots showing apparent elastic moduli K of the four different groups. All treatments led to a significant increase in corneal stiffness (n = 3, P < 0.001, Kruskal-Wallis Anova). Treatment with MB led to a slightly larger but non-significant increase in tissue stiffness compared to TB. Dark lines indicate median values, grey boxes the Q1-Q3 percentiles. (* (P < 0.05); ** (P < 0.01); *** (P< 0.001), Wilcoxon rank sum test). DM = Descemet’s Membrane, TB = RS Trypan Blue, MB = Membrane Blue Dual.
Fig 3.
Assessment of TB and MB toxicity on human corneal endothelium in human corneas.
(a) Images of corneal tissue after one and four minutes of exposure to the two dyes (RS- Blue,TB and Membrane blue Dual MB). (b) Boxplot showing the relative increase in staining intensity after exposure to TB and MB for one and four minutes (n = 5; P = 0.2115, Kruskal-Wallis ANOVA). Dark lines indicate median values, grey boxes the Q1-Q3 percentiles.
Fig 4.
Retention of dye after 25 min treatment.
(a) Image of Descemet membranes stained with Trypan Blue (TB, left) and Membrane Blue (MB, right). Dye retention was assessed 25 min after staining. (Scale bar = 1 mm).
Fig 5.
Possible mechanisms of corneal stiffening by TB and MB.
(a) The two possible modes of crosslinking of the ECM. Dye intercalates the protein structure of the ECM in the non-covalent mode, increasing the number of attractive supra-molecular interactions (dashed lines). Photochemical excitation of the dye, on the other hand, could lead to the generation of covalent crosslinks (solid lines) between nearby amino acid residues on the proteins. (b) Trypan Blue C) Coomassie Brilliant Blue G 250. Both dyes contain a variety of polar and non-polar functional groups, allowing for multiple kinds of non-covalent interactions.