Effects of growth factors (EGF, PDGF-BB and TGF-beta 1) on cultured equine epithelial cells and keratocytes: implications for wound healing.

OBJECTIVE

The physiologic mechanisms involving growth factors, including PDGF-BB, EGF, and TGF-beta 1, as potent mediators of fibroblasts and epithelial cells in corneal wound healing remain unknown. The goal of this study was to determine culture methods for equine epithelial cells and keratocytes and to investigate how exogenous growth factors influence proliferation of both cell types.

PROCEDURES

Cell cultures were established from healthy corneas harvested from horses immediately following euthanasia and maintained using standard tissue culture protocols. To determine the effects of PDGF-BB, EGF, TGF-beta 1, keratocytes (1 x 10(5)/well) and epithelial cells (2 x 10(5)/well) were each cultured in 12 well plates and exposed separately to the growth factors. The cells were exposed to concentrations of EGF between 0 and 50 ng/mL; PDGF-BB between 0 and 75 ng/mL; and TGF-beta 1 between 0 and 10 ng/mL. Cell proliferation was measured using 3H-thymidine assay and differences in growth determined using anova and Tukey's HSD test (P < 0.05).

RESULTS

Epithelial cell and keratocyte cultures were successfully established. EGF maximally stimulated keratocyte and epithelial cells at 25 ng/mL and 5 ng/mL, respectively. PDGF-BB maximally stimulated keratocytes and epithelial cells at 50 ng/mL and 5 ng/mL, respectively. TGF-beta 1 inhibited keratocytes at 5 ng/mL and 10 ng/mL, and epithelial cells at 1 ng/mL and 2 ng/mL.

CONCLUSIONS

Methods were established to maintain epithelial cells and keratocytes in vitro. PDGF-BB and EGF stimulate, while TGF-beta 1 inhibits the proliferation of epithelial cells and keratocytes. These growth factors may play a role in maintenance and repair of the equine cornea.