The Role of the Primary Cilium in Substrate Stiffness Sensing in Chondrocytes

Abstract

The mechanical properties of the extracellular matrix (ECM) in determining cell fate and behaviour are now recognised as fundamental biological processes. Varying tissue elasticity or substrate stiffness alone can guide stem cells towards neurons, muscle cells or bone. Articular cartilage is a mechanically responsive tissue that undergoes dynamic ECM changes during normal daily activity, and it is the primary tissue affected in osteoarthritis (OA). However, few studies have examined how chondrocytes respond to different local substrate stiffness and how these changes are signalled to the cell. The aim of this study was to examine how chondrocytes, and its own nanoscale mechanosensor, the primary cilium, interact with and respond to varying substrate stiffness, and how this feeds back into phenotypic changes in cell shape, cell area and chondrocyte gene expression. Wild-type (WT) and ORPKift88 cilia-deficient mouse chondrocytes were seeded on collagen type II-coated polyacrylamide (PA) hydrogels with stiffnesses that mimic osteoarthritic (3.9 kPa - soft) and healthy (50.2 kPa - hard) pericellular matrix, as on glass coverslips (>GPa), for 48 hours in low-serum ciliogenic culture medium. Primary cilia and the actin cytoskeleton were fluorescently labelled and primary cilia frequency, cilia length, cell area and cell aspect ratio were assessed using widefield fluorescent microscopy and image analysis. The effect of substrate stiffness on the chondrocyte transcriptome was also assessed in wild-type and ORPKift88 cells following culture on soft and hard substrates using Clariom™ S arrays and Affymetrix® Transcriptome Analysis Console to perform hierarchial clustering. On all substrates, WT chondrocytes had greater number of ciliated cells and longer cilia than ORPKift88 cells. However, substrate stiffness did not have a major effect on ciliogenesis or cilia length in both WT and ORPKift88, although WT cilia frequency was greater on soft substrates compared to glass coverslips. Increasing stiffness did increase cell area, but this did not correlate with the presence of cilia or cilia length, suggesting a limited relationship between cell spreading and ciliogenesis in chondrocytes. However, transcriptome analysis revealed that pathways associated with inflammation and Wnt signalling pathways with differentially expressed on soft substrates suggesting that softer PA hydrogels promote an OA genotype in chondrocytes. The chondrocyte primary cilium does not appear to influence cell changes in response to substrate stiffness, signifying that the cilium is not a key organelle essential for cartilage mechanotransduction in 2D culture. However, the soft substrate effectively supported OA chondrocyte phenotype and provides a promising cell model for examining OA.