Abstract
Following laser vision correction, corneal keratocytes must repopulate areas of cell loss by migrating through the intact corneal stroma, and this can impact corneal shape and transparency. In this study, we evaluate 3D culture models for simulating this process in vitro. Buttons (8 mm diameter) were first punched out of keratocyte populated compressed collagen matrices, exposed to a 3 mm diameter freeze injury, and cultured in serum-free media (basal media) or media supplemented with 10% FBS, TGFβ1 or PDGF BB. Following freeze injury, a region of cell death was observed in the center of the constructs. Although cells readily migrated on top of the matrices to cover the wound area, a limited amount of cell migration was observed within the constructs. We next developed a novel “sandwich” model, which better mimics the native lamellar architecture of the cornea. Using this model, significant migration was observed under all conditions studied. In both models, cells in TGFβ and 10% FBS developed stress fibers; whereas cells in PDGF were more dendritic. PDGF stimulated the most inter-lamellar migration in the sandwich construct. Overall, these models provide insights into the complex interplay between growth factors, cell mechanical phenotypes and the structural properties of the ECM.
Highlights
The cornea is an optically clear tissue that forms the front surface of the eye, and accounts for nearly two-thirds of its refractive power
Since quiescent cells can be used in the inner matrix, the effects of specific growth factors can be assessed without pre-exposure to serum or other factors which may permanently alter these responses [20]
PDGF appeared more elongated with branching, dendritic cell processes. These results provide new insights into the interplay between growth factor signaling, cell mechanics and matrix structure
Summary
The cornea is an optically clear tissue that forms the front surface of the eye, and accounts for nearly two-thirds of its refractive power. Modern laser vision correction procedures, such as photorefractive keratectomy (PRK) and laser-assisted in situ keratomileusis (LASIK), reshape the corneal stroma using photoablation in order to achieve a desired change in refractive power Both of these procedures induce keratocyte death in stromal tissue surrounding the area of photoablation [8]. While repopulation of these regions is desirable, the normal wound healing response can lead to fibrosis and scarring along the visual axis in a subset of patients, especially following PRK [9]. We investigate whether compressed collagen matrices can be used as a novel platform for investigating keratocyte behavior during intrastromal wound healing
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