The assembly of the extracellular matrix produced by corneal fibroblasts into a precisely organized stroma necessary for optical transparency will be investigated. Monoclonal antibodies to type V collagen will be used to study the distribution and interaction of this minor collagen with respect to the major corneal type I collagen. These morphological studies will be coupled with in vitro studies on type I and V interactions during heat induced fibril formation and an analysis of fibril structure. Immunoelectron microscopy with antibodies to the carboxy and amino terminal propeptides of type I collagen will be used to determine the role of these peptides and their processing in the control of fibril formation and fibril diameter. The influence of proteoglycans in fibril formation, control of fibril diameter and interfibrillar spacing will be studied morphologically using histochemical methods coupled with enzymatic digestions in normal corneal stroma at different stages in development both before and after inhibition of glycosaminoglycan synthesis with inhibitors. The role of the corneal fibroblast cell surface in the control of collagen fibrillogenesis, fibril bundle formation and development of tissue specific architecture will be studied by transmission, and scanning electron microscopy and by high voltage electron microscopy. The surface topography of these cells and their compartmentalization of the extracellular space will be characterized after three dimensional reconstruction from 0.5 Mum serial sections examined by high voltage electron microscopy. These studies on the regulation of corneal fibril formation contribute to our understanding of the mechanisms by which the precise collagen architecture associated with transparency is controlled during corneal development and growth. This knowledge is fundamental to the possible manipulation of fibril formation during injury and repair.
