Stiffness controls primary cilia position and length: mechanisms and potential in reversing osteoarthritis phenotypes

Abstract

The focus of this thesis was to determine how the stiffness of the extracellular microenvironment influences cellular mechanosensitivity through the action of, the primary cilium. Mechanical stress and the stiffness of the extracellular matrix are key drivers of tissue development and homeostasis. Aberrant mechanosensation is associated with a wide range of pathologies, including diseases such as osteoarthritis (OA). Substrate stiffness is one of the well-known mechanical properties of the matrix that enabled establishing the central dogma of an integrin-mediated mechanotransduction using stem cells. However, how specific cells ‘feel’ or sense substrate stiffness requires further study. The primary cilium is an essential cellular organelle that senses and integrates mechanical and chemical signals from the extracellular environment. This study hypothesised that the primary cilium dynamically alters its length and position to fine-tune cell mechanosignalling based on substrate stiffness alone. A polyacrylamide hydrogel system of varying substrate stiffness was used to examine the role of substrate stiffness on cilia frequency, length and centriole position as well as cell and nuclei area over time in mouse articular chondrocytes. Contrary to other cell types, this study shows that chondrocyte primary cilia shorten on softer substrates demonstrating tissue-specific mechanosensing which is aligned with the tissue stiffness the cells originate from. This study further shows that stiffness alone determine centriole positioning to either the basal or apical membranes during attachment and spreading, with centriole positioned towards the basal membrane on stiffer substrates. These phenomena are mediated by force generation actin-myosin stress fibres in a time-dependent manner. The investigation continues to understand how OA chondrocytes respond to changes in stiffness and whether OA chondrocyte behaviour can be mechanically reversed and the role of primary cilia in mediating the process. OA arises from multiple factors which ultimately leads to the failure of the joint tissues. As OA progresses, the extracellular matrix directly surrounding the chondrocytes begins to soften and degrade. It remains unclear if and how OA cells sense the changes to local tissue stiffness as it softens but translational evidence for the Wnt pathway, one of the well known ciliary signalling pathways, involvement in OA progression is mounting. Using chondrocytes enzymatically isolated from articular cartilage obtained from total knee arthroplasty, the effect of stiffness on cilia characteristics and morphological changes were examined. Gene expression of OA, Wnt signalling, Hedgehog signalling markers and PIEZO1 mechanosensitive ion channel were then compared to the expression in fresh tissue, whose RNA was directly isolated by pulverisation. This study shows that OA phenotypes were ameliorated when chondrocytes isolated from diseased regions of cartilage was exposed to healthy stiffness. Centrioles were positioned differently when comparing between chondrocytes from macroscopically normal and diseased cartilage, and the positioning was stiffness sensitive. This study further suggests that the non-canonical Wnt signalling is potentially involved in mediating stiffness sensing. This study shows that substrate stiffness dynamically regulates primary cilium length and position through integrin-mediated traction forces and non-canonical Wnt signalling. This study supports the promising potential of primary cilia as a novel target in mechanotherapy for improved clinical outcomes in osteoarthritis treatment.