Abstract
Purpose :
During corneal wound healing, keratocytes within the corneal stroma are activated into a repair phenotype when soluble growth factors, such as transforming growth factor-beta 1 (TGF-β1) and platelet-derived growth factor-BB (PDGF-BB), are released into the stromal space. This process is often accompanied by changes in tissue stiffness, and previous work has shown that the TGF-β1-mediated myofibroblastic activation of these cells is stiffness-dependent. Still, it is unclear if stiffness can also regulate keratocyte behavior in response to other growth factors, such as PDGF-BB. Here, we used a polyacrylamide (PA) gel system to determine whether changes in substratum stiffness can modulate the proliferation and motility of primary corneal keratocytes treated with PDGF-BB.
Methods :
Functionalized PA substrata with an elastic modulus of either 1 kPa (soft) or 10 kPa (stiff) were fabricated to mimic the mechanical properties of either normal or fibrotic corneal tissue. The substrates were then plated with primary rabbit corneal keratocytes (NRKs) and cultured in defined serum-free media in either the presence or absence of PDGF-BB. Fixed cells were then stained with either F-actin, to visualize cell morphology, or EdU incorporation, to quantify proliferation. In other experiments, a circular freeze injury was introduced to mimic wound healing, and time-lapse phase-contrast images were captured to quantify keratocyte motility.
Results :
In the presence of PDGF-BB, NRKs plated on stiffer substrates exhibited a more elongated morphology, an increased cell area, and a higher rate of proliferation than their counterparts cultured on soft PA gels. To assay changes in keratocyte motility, we also quantified NRK migration into decellularized wounds created on both soft and stiff PA substrata. On all substrata, the NRKs migrated from the densely populated region surrounding the wound into the decellularized area. Keratocytes treated with PDGF-BB traveled further into the wound and exhibited faster migration speeds than cells maintained in serum-free conditions, but we did not observe any stiffness-dependent differences in the PDGF-BB-driven motility of these cells.
Conclusions :
Taken together, these results suggest that changes in ECM stiffness can modulate the proliferation and morphology of corneal keratocytes, but not their motility, in response to PDGF-BB.
This is a 2021 ARVO Annual Meeting abstract.