Functional assessment and characterization of the tissue-engineered corneal endothelial graft in a rabbit model of bullous keratopathy. (A) Graph summarizing the corneal thickness of the 3 groups of rabbits used in this study, including 2 control groups, post-operatively until 28 days following the surgeries. Controls were ‘DM stripped’ (n = 4; Control group 1) control where only the rabbit’s DM was removed and ‘DM stripped with carrier only’ (n = 4; Control group 2) control where the rabbit’s DM was removed, followed by transplantation of an empty carrier (without cells). Rabbits in the treatment group received TE-EK graft following removal of DM (n = 4). Corneal thickness of rabbits was comparable in all three groups as shown pre-operatively, and at the first, fourth and seventh day following surgery. The corneas of rabbits receiving the TE-EK graft were significantly thinner when assessed on Day 14 compared to control group 1 (*p < 0.05) and control group 2 (**p < 0.05). The statistical significance were maintained for assessments on Day 21, as well as on Day 28, where corneal thickness of rabbits with TE-EK grafts recovered completely to pre-operative corneal thickness of 459.3 µm ± 65.4 µm at Day 21, and 517.3 µm ± 151.3 µm at Day 28. (B) Representative slit-lamp image of corneal clarity were taken pre-operatively. Slit-lamp image and AS-OCT scan taken at Day 28 of (C) a rabbit in control group 2, showing an opaque cornea with a corneal thickness of approximately 1.21mm, and (D) a rabbit which received TE-EK graft with a clear central cornea and a corneal thickness of 447 µm. Distinctive areas with different degree of corneal clarity can be detected (see insert; dotted line). Staining of a human-specific nuclei antibody on (E) a human donor cornea as a positive control; (F) the rabbit cornea as a negative control; and on (G) an section of an excised cornea of rabbit receiving TE-EK graft Scale bar: 50 µm. Flat-mount Trypan Blue and Alizarin Red staining of (H) cornea of a normal rabbit; (I) stromal layer of cornea of rabbit from control group 1; and (J) cornea of rabbit receiving the TE-EK graft. Dotted lines demarcate the edge of the TE-EK graft and the underlying stroma layer Scale bar: 100 µm. (K) A panoramic SEM image of rabbit cornea showing the TE-EK graft, a zone of bare stroma and the rabbit own corneal endothelium. Scale bar: 400 µm. Magnified within (K): is (L) the TE-EK graft†; (M) the boundary between the TE-EK graft and the underlying stroma‡; and (N) the rabbit’s native corneal endothelium§. Scale bar: 50 µm.