Tissue Engineered Human Corneas for In Vitro TestingMay Griffith1, Rosemarie Osborne2, Eric J. Suuronen1, Christopher R. McLaughlin1 and Mitchell A. Watsky31Dept. of Cellular and Molecular Medicine, University of Ottawa and University of Ottawa Eye Institute, Ottawa, Ontario, Canada; 2The Procter & Gamble Company, Cincinnati, OH; 3University of Tennessee Health Science Center, Memphis, TN. OBJECTIVEMETHODSRESULTS and DISCUSSIONNERVE MORPHOLOGY

All corneal nerve fiber types were observed in our model. Using immunofluorescence with a nerve specific marker (anti-NF200), we were able to identify: stromal nerves (fig. 3A) a nerve plexus below the epithelium (Fig. 3B) beaded (arrow) and smooth (arrowhead) fibers in the epithelium (Fig. 3C).These nerves were similar to their human counterparts (insets).

TEM showed nerve fibers invaginated epithelial cells (Fig. 3D; arrow). This suggests that cells receive direct innervation, as previously reported for human corneas7.Red = surface, green = -5 m, blue = -15 m SUMMARY TE corneas were constructed using immortalized human corneal cell lines as described by Griffith et al.6 (1999). Dorsal root ganglia (DRG) derived cells (E8.0) served as the nerve source. DRG cells were embedded within the corneal matrix or in a collagen ring (sclera) within which a cornea was constructed (Fig. 1, 2). Constructs were maintained for up to 10 days until used for functional testing.

To innervate the tissue engineered (TE) cornea developed in our lab, and to determine if this tissue is representative of the native human cornea in terms of both morphology and function.

Hypothesis: a functional, innervated TE cornea can be constructed, if provided with an appropriate environment. Proper innervation can be confirmed by functional analyses.

Nerves were introduced into a TE cornea and it was shown that these nerves: (a) were morphologically equivalent to natural corneal nerves; (b) made appropriate contacts with target cells; (c) generated action potentials; (d) responded to chemical and physical stimuli; and (e) played an important role in the overall functioning of the tissue. This model also provided the basis for the development of a polymer composite for transplantation, which was successfully innervated both in vitro and after implantation into corneas.

This innervated functional TE cornea can be used as:

1. An alternative to animals for ocular toxicity testing

2. A research model for studying the role of nerves in corneal function

3. The basis for the development of artificial corneas for transplantation

that require the regeneration of nerves