Characterization of a New Tissue-engineered Wound Healing Model for Human Corneal Reepithelialization

Abstract

Abstract: : Purpose: In order to get a representative in vitro wound healing model to study reepithelialization of the human cornea, we adapted a new skin wound healing model (Laplante, A. et al, (2002), FASEB J., 12: 47-56). This in vitro construct allows the migration of corneal human cells on a natural extracellular matrix. In the present work, we validated and characterized this corneal wound healing model by studying the expression of extracellular matrix components that play an important role during the epithelial cell migration process that characterize corneal wound healing. Methods: The auto-assembly approach was used to produce the corneal epithelial cells reepithelialization model. First, a human reconstructed tissue was built by culturing fibroblasts in the presence of ascorbic acid to produce sheets on which corneal epithelial cells were then seeded and cultured. This reconstructed tissue was wounded with a punch biopsy. It was then placed on an intact fibroblast sheet to allow reepithelialization by corneal cells. Biopsies of the reepithelialized surface were harvested three days after wounding. Histological and immunohistological (type IV and VII collagen, laminin, fibronectin) studies were performed on this model. Results: The human corneal epithelial cells seeded on the sheets differentiated and formed a 4-6 cell-layer-thick epithelium histologically resembling that of a human cornea. After wounding, epithelial cells migrated over the natural tissue containing fibroblasts and extracellular matrix components. All the basement membrane components studied were present at the epithelial-stroma junction and were progressively reexpressed under the regenerating epithelium. Work is in progress to study the distribution of integrin subunits (α5ß1 and α6ß4) expressed by corneal epithelial cells during the reepithelialization of the wound. Conlusions: The similarity observed with the in vivo wound healing process supports the use of this tissue-engineered model for investigating the basic mechanisms involved in the reepithelialization process of corneal epithelial cells. Moreover, this model could also be used as a tool for the screening of agents affecting reepithelialization or to evaluate the effect of growth factors before animal testing.

Keywords: wound healing • cornea: epithelium • extracellular matrix