Abstract
Purpose: :
During development, keratocytes deposit collagen fibrils into bundles which ultimately they organise into an orthogonal lamellar architecture with cornea-specific function. We used serial block face scanning electron microscopy to examine keratocyte-collagen interactions at early stages of lamella formation and, over large tissue volumes, to determine if discrete events in matrix assembly occur at the cell-matrix interface.
Methods: :
Corneas were isolated from chick embryos at 12 days of incubation (E12), at which time cells secrete orientated collagen fibril bundles that later coalesce into a regular lamellar arrangement. Corneas were fixed in glutaraldehyde, then passed through multiple solutions to intensify backscatter electron (BSE) contrast, including osmium ferricyanide, tannic acid, osmium tetroxide and uranyl acetate, prior to dehydration and embedding in Araldite resin. Polymerised blocks were transferred to a FEI Quanta FEG ESEM equipped with a Gatan 3View system, where data sets of up to 1000 consecutive BSE images were acquired of the block face, refreshed by alternate sectioning at 100 nm. 3D reconstructions of selected serial image sequences extracted from the image stacks were made using ImageJ and IMOD software.
Results: :
Keratocytes at E12 exhibited complex morphologies with extensive lamellipodia, and filopodia extending up to 100 microns from the cell surface. Primary cilia were conspicuous processes of the majority of cells at this stage. Small numbers of collagen fibrils appeared in membrane-bound intracellular compartments reminiscent of fibripositors, which are abundant in embryonic tendon. Larger numbers of fibrils appeared within cell involutions, eventually opening into fibril bundles that dispersed from the cell surface into the extracellular space. Occasionally, spiralling of individual fibril bundles was evident, perhaps derived from changes in orientation of membranous cellular protrusions with which they were associated.
Conclusions: :
In the developing cornea, keratocyte membrane activity appears closely associated with collagen fibrillogenesis and extracellular aggregation of fibrils into higher order structures. These events lead ultimately to formation of the transparent lamellar matrix.
Keywords: cornea: stroma and keratocytes • development • microscopy: electron microscopy