Temporal stimulation of corneal fibroblast wound healing activity by differentiating epithelium in vitro

PURPOSE. TO determine whether differentiating corneal epithelium can tempor ally stimulate fibroblast activity. METHODS. Corneal epithelial cells were cultured to confluence and then stim ulated to mature into multilayered epithelia with addition of serum-contain ing medium. Differentiation was assessed morphologically and immunocytochem ically using a monoclonal antibody to cytokeratin 3. At intervals after ons et of differentiation serum-free conditioned medium was collected up to 28 days. Preliminary experiments deduced the optimum concentration of conditio ned medium to be used for assessing fibroblast activity. Conditioned medium (25% vol/vol) was added to donor-matched corneal fibroblasts in migration chambers, WST-1 reagent proliferation assays, and fibroblast-populated coll agen gel contraction assays. Platelet-derived growth factor (PGDF)-AB and i nterleukin (IL)-1 beta in conditioned media were quantified by enzyme-linke d immunosorbent assay (ELISA) Fibroblast migration and collagen contraction assays were performed with concentrations of PDGF-AB. RESULTS. Conditioned medium collected from differentiating epithelium stimu lated fibroblast migration and collagen gel contraction, with activity peak s occurring with medium collected on day 14 (P < 0.05). No significant diff erence was detected between fibroblast growth rates in each of the conditio ned media. Levels of PDGF-AB increased during epithelial culture up to 22 d ays (up to approximately 360 pg/ml) with a subsequent decrease by 28 days. IL-1<beta> inversely correlated with fibroblast activity induced by conditi oned medium, with a trough in concentration (2 pg/ml) occurring at 14 days. Both fibroblast migration and collagen contraction were stimulated in a do se-dependent manner by PDGF-AB. CONCLUSIONS. Corneal epithelium is capable of temporally stimulating fibrob last wound-healing characteristics during its differentiation. One of the g rowth factors potentially involved in this epithelial-stromal interaction i s PDGF. This work demonstrated that developing epithelium (possibly similar to repairing epithelium in vivo) regulated fibroblast behavior and may ind icate a mechanism of fibroblast recruitment to a wound after epithelial clo sure.