Microscale frictional strains determine chondrocyte fate in loaded cartilage

Abstract

Mounting evidence suggests that altered lubricant levels within synovial fluid have acute biological consequences on chondrocyte homeostasis. While these responses have been connected to increased friction, the mechanisms behind this response remain unknown. Here, we combine a frictional bioreactor with confocal elastography and image-based cellular assays to establish the link between cartilage friction, microscale shear strain, and acute, adverse cellular responses. Our incorporation of cell-scale strain measurements reveals that elevated friction generates high shear strains localized near the tissue surface, and that these elevated strains are closely associated with mitochondrial dysfunction, apoptosis, and cell death. Collectively, our data establish two pathways by which chondrocytes negatively respond to friction: an immediate necrotic response and a longer term pathway involving mitochondrial dysfunction and apoptosis. Specifically, in the surface region, where shear strains can exceed 0.07, cells are predisposed to acute death; however, below this surface region, cells exhibit a pathway consistent with apoptosis in a manner predicted by local shear strains. These data reveal a mechanism through which cellular damage in cartilage arises from compromised lubrication and show that in addition to boundary lubricants, there are opportunities upstream of apoptosis to preserve chondrocyte health in arthritis therapy.

Introduction

Under healthy conditions, articular cartilage provides joints with the most efficient bearing surface found in nature. However, the failure of this tissue in osteoarthritis (OA) is the leading cause of severe disability in the United States (Murphy et al., 2008). Despite its widespread prevalence, early stages of OA and its progression are not well understood (Anderson et al., 2011, Buckwalter et al., 2013). Even in cases with a known initiating event such as a traumatic injury, symptomatic OA can take decades to manifest (Brown et al., 2006) obscuring mechanisms leading to the initiation of dysfunction. This knowledge gap arises from an incomplete understanding of how mechanical perturbations dictate biological responses in the early stages of disease (Buckwalter et al., 2013).

There is mounting experimental evidence that friction acutely affects chondrocyte homeostasis (Waller et al., 2013). Cartilage exposed to high friction exhibits significantly more chondrocyte apoptosis compared to cartilage lubricated by healthy synovial fluid (Waller et al., 2013). Such findings indicate that elevated cartilage friction coefficients increase shear strains (Wong et al., 2008), a known correlate to cell death. From these previous findings, a picture emerges where factors such as aging (Temple-Wong et al., 2016), traumatic injury (Elsaid et al., 2008), and disease (Kosinska et al., 2015) affect levels of lubricants such as lubricin and hyaluronic acid in synovial fluid, which in turn increases the friction coefficient at the cartilage surface (Bonnevie et al., 2015, Schmidt et al., 2007). However, the relationship between elevated friction, frictional shear strains, and adverse cellular responses such as apoptosis is not clear.

Mechanically-mediated apoptosis often progresses through mitochondria-driven processes (Wang and Youle, 2009). In these cascades, excessive intracellular calcium leads to mitochondrial depolarization, and mitochondrial depolarization can initiate caspase activation though cytochrome C release (Huser and Davies, 2007). Recent evidence suggests that excessive friction can play a role in initiating this cascade, as lubricin knock-out (i.e., lubrication inhibition) leads to mitochondrial dysregulation (Waller et al., 2017). However, it is unclear whether this cellular dysregulation is dependent on friction-mediated mechanics. In part, this knowledge gap arises due to the difficulty of measuring friction coefficients, the resulting microscale mechanics and cellular response in a single tissue source. To address this missing link regarding the role of friction and the resulting mechanics that stimulate chondrocyte dysfunction, we combined a frictional bioreactor with confocal elastography and image-based cellular assays to establish the link between cartilage friction, micro-scale shear strain, and acute, adverse cellular responses.