Graduation Year

2009

Document Type

Dissertation

Degree

Ph.D.

Degree Granting Department

Pathology and Cell Biology

Major Professor

John R. Hassell, Ph.D.

Committee Member

David E. Birk, Ph.D.

Committee Member

Patricia A. Kruk, Ph.D.

Committee Member

Santo V. Nicosia, M.D.

Committee Member

Hormuz Wadia, M.D.

Keywords

cornea, proteoglycans, extracellular matrix, wound healing, collagen

Abstract

The corneal wound healing process is a complex process, which often leads to the development of scar tissue with loss of transparency. When the cornea is wounded, some of the viable keratocytes are activated by growth factors to proliferate, and repair the wound by the production of a new extracellular matrix (ECM) that is either normal or is disorganized (fibrotic). The first part of this dissertation aims to show that the growth factors IGF-I, TGF-ß, FGF-2, and PDGF stimulate keratocytes to synthesize different levels of collagens and proteoglycans and are therefore responsible for initiating the wound healing repair process. FGF-2 stimulated keratocytes to proliferate but did not stimulate collagen production; IGF-I and PDGF stimulated keratocytes to proliferate and produce a collagenous ECM that could restore transparency; while, TGF-ß stimulated keratocytes produce a fibrocollagenous ECM that is opaque.

The second part of this dissertation aims to evaluate collagen fibril content, distribution, and orientation in the ECM deposited by keratocytes cultured in IGF-I, TGF-ß, FGF-2, and PDGF under a layer of agarose: a culture modification that enhances the formation of ECM in vitro. FGF-2 agarose cultures had little ECM and the keratocytes were in close cell contact while IGF-I, TGF-ß, and PDGF agarose cultures had the least cell contact with an extensive fibrillar ECM. This newly developed agarose overlay cell culture system increases ECM formation with the cell layer only when the synthesis of collagen is stimulated and that the ECM morphology is growth factor specific.

Cell culture has proven to be a reliable technique to study the keratocytes response to trauma and disease; however, limitations exist. Primary keratocytes that possess quiescent phenotype are unable to be rapidly expanded or subcultured without the addition of a mitogen(s). Most commonly, keratocytes are cultured and passaged in the presence of fetal bovine serum (FBS), which activate the cells to proliferate and differentiate into fibroblasts or myofibroblasts as well as lose expression of their unique transparency enabling gene products. The third part of this dissertation aims to develop a defined culture media that can be used to expand and subculture keratocytes that express keratocyte specific markers. Culture medium supplemented with FGF-2 combined with ITS was used to: expand and subculture primary bovine keratocytes while maintaining their expression of keratocan and to restore keratocan expression to bovine keratocytes expanded and subcultured with media containing 2.5% FBS.

This dissertation shows the significance of signaling molecules in vitro to produce keratocyte cultures useful for understanding normal stromal biology and its repair process.

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