Abstract
Purpose :
Corneal wound healing depends on extracellular matrix (ECM) and topography
cues that allow migration and proliferation of regenerating cells. Here we try to accelerate
corneal wound healing by combining silk film topography with various ECM.
Methods :
We used silk films with distinct topographies: micro-and nano-parallel ridge
widths of 2000 nm, 1000 nm, 800 nm and flat surface. The films were left untreated or
coated with different ECM; type I collagen, fibronectin, laminin, and poly-D-lysine.
Primary mouse corneal epithelial cells (MCEC) and primary rabbit corneal epithelial
cells (RCEC) were grown on the silk films. Phase contrast images of cells and
immunofluorescence staining images of vinculin were taken for cell numbers counting
and focal adhesion observation. The effects of silk film topography and ECM on corneal
wound recovery were studied by in vitro RCEC scratch assay. The signaling pathways
involved in this process were elucidated using ingenuity pathway analysis (IPA) and
inhibitors ML141 and ZCL278 that block these pathways were used to test their
involvement in wound healing assays.
Results :
We demonstrated that silk films with 800 nm ridge width provided better
MCEC and RCEC cellular growth on the first five days of culture. Cell number increased
6 to10 folds when films were coated with 50g/ml type I collagen regardless of
topography, whereas the other ECM have no significant effects. Immunofluorescence
staining showed that nano-topographies influenced vinculin redistribution, which was
more abundant on 800 nm topography. Wound healing recovery was faster (13-28%,
n=3) in 800 and 1000 nm ridge width topographies. Addition of collagen accelerated
wound healing in all topographies by around 20% (n=3). IPA indicated that topography
and type I collagen regulates cell behaviors via integrin receptor, focal adhesion complex,
CDC42, PAK, WASP, ARP2/3, and regulate filopodia formation. We further used
CDC42 inhibitors ML141 and ZCL278 on scratch assay, and found that wound healing
on 1000 and 800 nm was delayed.
Conclusions :
Silk film nano-topography in combination with type I collagen increased
cell number, focal adhesion, and wound healing, and constitute a better substrate for
cornea wound repair. The mechanisms involved require activation of integrin receptor
and focal adhesion complex via the CDC42 pathway.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.