Kinetic study of cell proliferation in a new wound healing model using tissue cultured corneal endothelial cells
Miyata, K.; Murao, M.; Sawa, M.; Tanishima, T.
Nippon Ganka Gakkai Zasshi 93(3): 287-293
1989
ISSN/ISBN: 0029-0203 PMID: 2773713 Document Number: 329477
We studied cell kinetics in the wound healing process using cultured bovine corneal endothlium. We cultured bovine endothelial cells on coverslips coated by hydroxethyl metacrylate (HEMA) which had on 8 mm HEMA-free zone in diameter, and produced wounds at the center of the monolayer cell sheets using a rotating silicone tip. At various time periods after injury, we added bromodeoxyuridine (BrdU) to the medium and incubated for 12 hours. Then, the specimens with fixed 10% phosphate-buffered formalin and were incubated with a monoclonal antibody against BrdU. The cells incorporating BrdU into DNA were stained by the Avidin Biotin Peroxidase Complex (ABC) method. At 12 hours, no labeled cells were observed. At 24 hours, 14.6 .+-. 4.0 cells were stained. The maximum labeling occurred during 48-60 hours after the wound. Seventy two hours after the wound, labeled cells decreased rapidly. Labeled cells were localized within 0.6 mm from the wound edge throughout the wound healing process. It can be thought that the wound healing process comprised four phases, i.e., latent, migration plus mitosis, and contact inhibition phases. The first is the latent phase observed during the first six hours after wound infliction. The cells respond to external expansion. The second phase in the migration which is last until 24 hours after wound infliction, and occurs mainly through cellular migration, while proliferation has only minor contribution in this phase. The third is the migration plus mitosis phase. The cell proliferation shows a rapid increase after 72 hours after wound infliction. The wound healing model of corneal endothlium using cultured bovine endothelial cells is useful for quantitative studies of wound healing area and kinetic studies of endothelial cell proliferation.