Studies of corneal development and tissue engineering

The overall objective this work is to contribute to the understanding of how the precise structure of the corneal stroma is achieved during development, and to apply this knowledge to the latest attempts at engineering effective stromal constructs for use in transplantation. The cornea is the major refractive element of the human eye, accounting for two-thirds of total focusing power. Representing around 85% of corneal thickness, the stroma possesses the mechanical strength needed to protect intraocular tissues, whilst still achieving the high level of transparency necessary for light transmission. This is chiefly due to the small, uniform diameter collagen fibrils arranged into a precisely ordered series of orthogonal lamellae. Proteoglycans in the stroma are thought to regulate the arrangement and diameter of the collagen fibrils, although the mechanism by which this occurs is not fully understood. The deceptively complex organisation of the stroma may be responsible for the relatively little progress that has been made in engineering constructs that can reproduce the structural and functional characteristics of the cornea. Further study into the embryonic development of the cornea may aid attempts to recapitulate in vivo mechanisms for corneal construction. Of particular relevance would be the method of collagen organisation and deposition in the developing avian corneal stroma and the interactions that occur within the collagen fibril bundles as development progresses. Initially, en face sections were used to study the organisation and arrangement of collagen fibrils in the developing stroma. It is hypothesized that in tendon, the formation of parallel arrays of collagen fibrils occurs via fibroblast surface recesses and invaginations. It was evident through transmission electron microscopy that this process also occurs in the developing corneal stroma via surface recesses on stromal keratocytes. Analysis of the interactions between the collagen and proteoglycans within fibril bundles demonstrated that the developing cornea is less well structured than often considered and is possibly a much more fluid and dynamic system than originally thought. Proteoglycan size and orientation show a degree of variety and disorder and appear to follow no set organisation or positioning. The data suggests that proteoglycans were seen forming aggregates that were capable of bridging the gap between more distant neighbouring fibrils. Following the study of the developing corneal stroma, collagen gel based constructs were engineered and their structural and functional characteristics were analysed to assess their potential as stromal equivalents for use in tissue engineering. Manipulating the assembly of collagen fibrils by varying the pH and cross-linker concentration had a dramatic effect on the structure and functionality of the final gel construct. A range of collagen gels were then implanted into intra-stromal pockets to determine their biocompatibility and in vivo properties