Abstract
Purpose: :
To determine how the three-dimensional (3D) morphology of proteoglycans and their interactions with collagen fibrils in the chick corneal stroma change during embryonic development.
Methods: :
Chick corneas at embryonic days 12, 14 and 16 were studied by transmission electron microscopy. Cuprolinic blue staining during primary fixation was used to enhance proteoglycan contrast. A series of images tilted from -60° to +60° were captured at x20,000 magnification. Alignment correction, back projection and tomograph generation was then carried out on these images using EM3D software. Segmentation of this reconstruction then allowed us to create three-dimensional models of the extracellular matrix components.
Results: :
The 3D reconstructions showed that collagen fibrils often associated with sulphated proteoglycans at distinct binding regions along their length. However, the proteoglycans showed no discernible pattern or regularity in their spatial orientation. Collagen interfibrillar spacing became more uniform and regular as development progressed. Proteoglycans exhibited various lengths, bridging gaps between neighbouring fibrils or passing around them to interconnect a third adjacent collagen fibril. The proteoglycans fell into sub-populations determined by size, probably indicative of the different proteoglycan subtypes that are present in the corneal stroma. Total proteoglycan number increased from day 12 to 16 as the 3D organisation of the collagen array became more refined, with longer and thicker proteoglycans present in higher numbers towards the later developmental stage.
Conclusions: :
Based on these 3D reconstructions, we propose that the interactions between collagen fibrils and proteoglycans are complex and subject to change during development in the chick corneal stroma, indicative of the multi-functional role that these matrix components fulfil.
Keywords: cornea: stroma and keratocytes • extracellular matrix