Abstract
Purpose :
Macromolecular crowding as a mechanism for collagen alignment and fibrillogenesis has been shown in acellular experiments. We visualize the juxtacellular region of primary human corneal fibroblasts (HCF) in vitro as they develop a corneal stroma-like construct by quick-freeze/deep-etch (QFDE) electron microscopy to test for crowding.
Methods :
HCF are grown for 4 weeks in the presence of ascorbic acid, developing a construct approximately 35 µm in thickness comprising of lamella collagen layers between two confluent layers of cells. Constructs are prepared for TEM imaging by the QFDE method where they are impact frozen without chemical fixation, and rotary shadowed. Gold nanoparticles are used to identify type I collagen (the main collagen type) and hyaluronic acid (a critical crowding agent).
Results :
In the juxtacellular region, we observed fully developed collagen fibrils embedded in the fibroblast membrane (fig 1), extending into the greater collagenous ECM, and fibrils in development surrounded by small filamentous structures (2-3 nm diameter), abut filapodia/keratopodia (fig. 2). Across multiple constructs, extracellular collagen fibril diameters range from 20-60 nm where fully formed (i.e. banded) fibrils range from 40-60 nm and unbanded fibrils range from 20-30 nm. Unbanded fibrils appear to be in the fibrillogenesis process as part of the fibril is banded, with a larger diameter, and the smaller unbanded regions comprise of smaller filaments wound together.
Conclusions :
The micrographs obtained from the corneal stroma-like construct suggests that in the juxtacellular region where collagen fibrils are assembling, confinement of monomer-like filaments appears to be occurring.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.