INTRODUCTION The cornea is the tough, transparent anterior surface of the eye. It is the primary refractive element in the ocular optical pathway. The cornea’s simple microanatomy, avascularity, and relative immune privilege make it the most successfully transplanted human tissue. Its superficial location, however, makes it susceptible to accidental exposures to potentially toxic exogenous substances. With these considerations in mind, we developed a prototype engineered human cornea that comprises a hydrated extracellular matrix (ECM)-based scaffold with human corneal cell lines for use as a potential animal alternative for toxicology testing and wound healing research (1). The unique properties of ECM macromolecules affect the growth and differentiation of cells. In the human cornea, ECM comprises up to 85% of the stroma. Given the structural and physiological importance of ECM in the human cornea, it is critical to understand its role in matrix- cell interactions within in vitro engineered corneas. The objective of this study was to examine and characterize the physical properties of 3-dimensional, stabilized collagen-glycosaminoglycan (GAG) matrices as scaffolds for tissue engineered human corneas. Desired properties for the matrix include high tensile strength for easy handling, longevity during use, and biocompatibility to support cell differentiation and maintenance as in native human corneas. We examined interactions of keratocytes with the scaffolds and overall effects of cell-matrix Tissue Engineering The International Journal of Artificial Organs / Vol. 26 / no. 8, 2003 / pp. 764-773 A collagen-based scaffold for a tissue engineered human cornea: Physical and physiological properties C.J. DOILLON 1 , M.A. WATSKY 2 , M. HAKIM 3 , J. WANG 2 , R. MUNGER 3 , N. LAYCOCK 3 , R. OSBORNE 4 , M. GRIFFITH 3 1 CHUL Research Centre, Laval University, Quebec City, Quebec - Canada 2 Department of Physiology, University of Tennessee Health Science Center, Memphis, TN - USA 3 University of Ottawa Eye Institute, University of Ottawa, Ottawa - Canada 4 The Procter & Gamble Company, Miami Valley Laboratories, Cincinnati, OH - USA © Wichtig Editore, 2003 0391-3988/764-10 $15.00/0 ABSTRACT: Stabilized collagen-glycosaminoglycan scaffolds for tissue engineered human corneas were characterized. Hydrated matrices were constructed by blending type I collagen with chondroitin sulphates (CS), with glutaraldehyde crosslinking. A corneal keratocyte cell line was added to the scaffolds with or without corneal epithelial and endothelial cells. Constructs were grown with or without ascorbic acid. Wound-healing was evaluated in chemical-treated constructs. Native, noncrosslinked gels were soft with limited longevity. Crosslinking strengthened the matrix yet permitted cell growth. CS addition increased transparency. Keratocytes grown within the matrix had higher frequencies of K + channel expression than keratocytes grown on plastic. Ascorbic acid increased uncrosslinked matrix degradation in the presence of keratocytes, while it enhanced keratocyte growth and endogenous collagen synthesis in crosslinked matrices. Wounded constructs showed recovery from exposure to chemical irritants. In conclusion, this study demonstrates that our engineered, stabilized matrix is well-suited to function as an in vitro corneal stroma. (Int J Artif Organs 2003; 26: 764-73) KEY WORDS: Artificial cornea, Extracellular matrix, Keratocyte, Wound healing, Tissue engineering, Ascorbic acid