Table 1.
Cornea´s donor data, including age, sex, days stored in Optisol until culture, and endothelial cell count.
Fig 1.
hCEC culture characterization.
(A) Microscopic image of haematoxylin and eosin staining confirmed that the corneal stroma structure of decellularized corneal sheets was well maintained. (B) Lack of DAPI staining demonstrated the absence of cells in the scaffold. (C) Macroscopic image of decellularized lamina over a grid showing 90% transparency. (D) Phase contrast image of cultured hCECs isolated from donor corneas, showing the hexagonal morphology at passage 0. (E) Demonstration of cultured hCEC pumping function by Na+/K+-ATPase pump immunohistochemistry and confocal microscopy. Nuclei are stained with DAPI.
Fig 2.
hCEC colonized laminas characterization.
(A) Phase contrast images of corneal laminas with various coatings and repopulated with hCECs. Sheets were subjected to 10 minutes or 1 hour of centrifugation. (B) Electron microscopy images of various coated laminas after 1 hour of centrifugation. Note the perfectly aligned collagen bundle fibres in the decellularized lamina (Decell). Also, note the flat endothelial morphology and filopodia development between parallel collagen fibres in the FNC-coated sheet in comparison with double-layered cells in uncoated and Matrigel-coated sheets, and the poor adhesion in the gelatine-coated and uncoated sheets. C: Collagen fibres. Arrows: endothelial cells.
Fig 3.
Haematoxylin-eosin histological images of control and experimental (TEEK) corneas both at a peripheral section and at a central section of the cornea. Note the increased thickness of the control due to increased oedema. Graft thickness is also increased in both groups (G), and the experimental corneas shows endothelial cells. Insets show higher magnification demonstrating endothelial cells in the TEEK group (arrows). Also note the absence of leukocyte infiltration in every cornea. (B) Corneal thickness measurement graph showing decreased corneal thickness in the TEEK group at 4 weeks after the transplant. Data are given as mean±SD Asterisk indicates statistical significance at P ≤.05.
Fig 4.
Human CEC corneal colonization.
A: Immunofluorescence for anti human-specific ribonucleoprotein and anti human-specific mitochondria in hCEC transplanted rabbit´s corneas at 4 weeks. Note merge of green (anti-human nuclei) and DAPI in every cell of the endothelium. B: PCR amplification of the housekeeping gene β-actin at 122 bp and the human specific gene β2-microglobulin in experimental rabbits’ corneas. Every experimental cornea shows 235 bp β2-microglobulin amplification.