We studied cell kinetics in the wound healing process using cultured bovine corneal endothelium. We cultured bovine endothelial cells on coverslips coated by
hydroxyethyl methacrylate (
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 0.6mm 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, 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 phase which is last until 24 hours after
wound infliction, and occurs mainly through cellular migration, while proliferation has only a 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.(ABSTRACT TRUNCATED AT 250 WORDS)