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Julie T. Daniels, Peng T. Khaw; Temporal Stimulation of Corneal Fibroblast Wound Healing Activity by Differentiating Epithelium In Vitro. Invest. Ophthalmol. Vis. Sci. 2000;41(12):3754-3762.
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purpose. To determine whether differentiating corneal epithelium can temporally
stimulate fibroblast activity.
methods. Corneal epithelial cells were cultured to confluence and then
stimulated to mature into multilayered epithelia with addition of
serum-containing medium. Differentiation was assessed morphologically
and immunocytochemically using a monoclonal antibody to cytokeratin 3.
At intervals after onset of differentiation serum-free conditioned
medium was collected up to 28 days. Preliminary experiments deduced the
optimum concentration of conditioned 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 collagen gel contraction
assays. Platelet-derived growth factor (PGDF)-AB and interleukin
(IL)-1β in conditioned media were quantified by enzyme-linked
immunosorbent assay (ELISA). Fibroblast migration and collagen
contraction assays were performed with concentrations of PDGF-AB.
results. Conditioned medium collected from differentiating epithelium stimulated
fibroblast migration and collagen gel contraction, with activity peaks
occurring with medium collected on day 14 (P < 0.05).
No significant difference was detected between fibroblast growth rates
in each of the conditioned media. Levels of PDGF-AB increased during
epithelial culture up to 22 days (up to approximately 360 pg/ml) with a
subsequent decrease by 28 days. IL-1β inversely correlated with
fibroblast activity induced by conditioned medium, with a trough in
concentration (2 pg/ml) occurring at 14 days. Both fibroblast migration
and collagen contraction were stimulated in a dose-dependent manner by
conclusions. Corneal epithelium is capable of temporally stimulating fibroblast
wound-healing characteristics during its differentiation. One of the
growth factors potentially involved in this epithelial–stromal
interaction is PDGF. This work demonstrated that developing epithelium
(possibly similar to repairing epithelium in vivo) regulated fibroblast
behavior and may indicate a mechanism of fibroblast recruitment to a
wound after epithelial closure.
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