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Invest Ophthalmol Vis Sci. 2010 Feb;51(2):864-75. doi: 10.1167/iovs.09-4200. Epub 2009 Oct 8.

Growth factor regulation of corneal keratocyte differentiation and migration in compressed collagen matrices.

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  • 1Department of Ophthalmology, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9057, USA.

Abstract

PURPOSE:

To evaluate a novel 3D culture model of the corneal stroma and apply it to investigate how key wound-healing growth factors regulate the mechanics of corneal keratocyte migration.

METHODS:

Rabbit corneal keratocytes were seeded within collagen matrices that were compacted using external compression. Six-millimeter-diameter buttons were then incubated in media supplemented with 10% FBS, TGFbeta1, TGFbeta2, platelet-derived growth factor (PDGF), or no growth factor (control). After 1, 3, or 7 days, matrices were labeled with phalloidin and a nucleic acid dye, and were imaged using laser confocal microscopy. To study cell migration, buttons were nested within acellular uncompressed outer collagen matrices before growth factor stimulation.

RESULTS:

Corneal keratocytes in basal media within compressed matrices had a broad, convoluted cell body and thin dendritic processes. In contrast, cells in 10% FBS developed a bipolar fibroblastic morphology. Treatment with TGFbeta induced the formation of stress fibers expressing alpha-smooth muscle actin, suggesting myofibroblast transformation. PDGF induced keratocyte elongation without inducing stress fiber formation. Both 10% FBS and PDGF stimulated significant keratocyte migration through the uncompressed outer matrix, but 10% FBS produced more cell-induced collagen matrix reorganization. TGFbeta induced the smallest increase in migration and the greatest matrix reorganization.

CONCLUSIONS:

Corneal keratocytes are able to differentiate normally and respond to growth factors within compressed collagen matrices, which provide a high-stiffness, 3D environment, similar to native stromal tissue. In addition, nesting these matrices provides a unique platform for investigating the mechanics of keratocyte migration after exposure to specific wound-healing cytokines.

PMID:
19815729
[PubMed - indexed for MEDLINE]
PMCID:
PMC2819331
Free PMC Article
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