Modulation of keratocyte phenotype by collagen fibril nanoarchitecture in membranes for corneal repair

Abstract

Type I collagen membranes with tailored fibril nanoarchitectures were fabricated through a vitrification processing, which mimicked, to a degree, the collagen maturation process of corneal stromal extracellular matrix in vivo. Vitrification was performed at a controlled temperature of either 5 °C or 39 °C at a constant relative humidity of 40% for various time periods from 0.5 wk up to 8 wk. During vitrification, the vitrified collagen membranes (collagen vitrigels, CVs) exhibited a rapid growth in fibrillar density through the evaporation of water and an increase in fibrillar stiffness due to the formation of new and/or more-stable interactions. On the other hand, the collagen fibrils in CVs maintained their D-periodicity and showed no significant difference in fibrillar diameter, indicating preservation of the native states of the collagen fibrils during vitrification. Keratocyte phenotype was maintained on CVs to varying degrees that were strongly influenced by the collagen fibril nanoarchitectures. Specifically, the vitrification time of CVs mainly governed the keratocyte morphology, showing significant increases in the cell protrusion number, protrusion length, and cell size along with CV vitrification time. The CV vitrification temperature affected the regulation of keratocyte fibroblasts' gene expressions, including keratocan and aldehyde dehydrogenase (ALDH), demonstrating a unique way to control the expression of specific genes in vitro.

Physical limits to biomechanical sensing in disordered fibre networks

“…However, certain types of cellular protrusions have also been observed to oscillate with a constant period on the order of tens of seconds47, which suggests that the dynamics of local stiffness are a promising direction for future study. Furthermore, while we have only considered isotropic networks, tissues can have aligned fibres due to remodelling and prestrain1348. Our results for isotropic prestrain suggest that aligned networks should yield a narrower stiffness distribution when probed along a fixed direction.…”

“…Eukaryotic cells, including fibroblasts, mesenchymal stem cells and cancer cells, attach to substrates via transmembrane protein complexes called focal adhesions, allowing the cell to sense stiffness3891011. Knockdown studies have established that this mechanosensing contributes both to motility and to the regulation of cell shape in three-dimensional in vitro systems that closely resemble in vivo cellular environments11121314151617, where cells are surrounded by a loosely connected, disordered network of protein fibres, such as collagen or fibrin18. These biopolymers form a major structural component of the extracellular matrix (ECM), which serves as the physical scaffolding within which cells live and move.…”

The Mechanobiology of Aging

“…By exerting tangential stretching forces on the collagen, the fibroblasts under the physiological conditions of the tissues can organize the collagen fibrils into sheets and cables, which in turn drastically changes the alignment of the collagen-fiber network (1). This dynamic change in the alignment of collagen fibers by the fibroblasts results in reciprocal changes through feed-forward signaling that further dictates the orientation, morphology, and alignment of neighboring cells (e.g., keratinocytes and epithelial cells) (15). Collagen molecules and fibers typically bind to other components of the ECM, such as elastins, whose fibers provide the elastic and recoil properties of tissues that undergo repetitive stretching.…”

Influence of collagen source on fibrillar architecture and properties of vitrified collagen membranes

“…On a cellular level, mechanical properties by crosslinking cause an increase in density of collagen fibers in the material. It has been previously shown that collagen fiber density correlates with proliferation and morphology of keratocytes . Optical properties do not directly relate to keratocyte function or proliferation but are critical for maintaining vision after application.…”

“…It has been previously noted that collagen substrates readily support cellular proliferation . Thus, this collagen scaffold would initially serve to hold corneal cells and would be remodeled over time to more organized structures.…”