The Role of the Actin Cytoskeleton in the Response of Chondrocytes to Mechanical Loading: A Computational and Experimental Investigation

Abstract

The biomechanisms which govern the response of chondrocytes to mechanical stimuli are poorly understood. This thesis aims to provide a more in depth understanding of chondrocyte biomechanics by focusing on the role of actin cytoskeleton in the response of chondrocytes to mechanical loading. Novel single cell in vitro experiments in tandem with finite element simulations using a 3D active modelling formulation, incorporating actin cytoskeleton remodelling and contractility, are performed. Experimentally, single chondrocytes are subjected to shear deformation by a horizontally moving probe. Untreated chondrocytes containing a contractile actin cytoskeleton exhibit a distinctive force-indentation curve whereby force increases rapidly upon initial probe contact, followed by a yield point and a reduced rate of force increase. In contrast, cells in which the actin cytoskeleton has been disrupted exhibit a linear force-indentation curve, with measured forces being significantly lower that for untreated cells. Simulations using the active 3D framework reveal that the distinctive response of untreated cells to applied shear is due to the yielding of the actin cytoskeleton in tensile regions of the cell and dissociation of the actin cytoskeleton in compressive regions of the cell. In contrast, a simple passive hyperelastic model is sufficient to predict the linear force-indentation curve for cells in which the actin cytoskeleton has been disrupted. Disruption of intermediate filaments and microtubules did not alter the distinctive force-indentation curve observed for untreated cells, further highlighting the critical role of the actin cytoskeleton in chondrocyte biomechanics. The 3D active modelling framework is also implemented to investigate the increased probe force required to detach spread chondrocytes form a flat substrate. Simulations reveal that spread cells with a flattened morphology have a more highly developed actin cytoskeleton than rounded cells. Rounded cells provide less support for tension generated by the actin cytoskeleton, hence a high level of dissociation is predicted. As a result of the higher level of actin cytoskeleton in the cytoplasm of spread cells, significantly higher detachment forces are computed than for round cells, as observed in the experimental study of Huang et al. (2003). Furthermore, the biomechanical response of chondrocytes in situ, and in particular, the response of the actin cytoskeleton to physiological and abnormal strain loading is investigated. Simulations predict that the presence of a focal defect significantly affects cellular deformation, increases the stress experienced by the nucleus, and alters the distribution of the actin cytoskeleton. It is also demonstrated that during dynamic loading, cyclic tension reduction in the cytoplasm leads to continuous dissociation of the actin cytoskeleton. In contrast, significant changes in cytoplasm tension are not predicted during static loading, and hence the rate of dissociation of the actin cytoskeleton is reduced. The combined modelling-experimental approach presented in this thesis provides new insight into the role of the active contractility and remodelling of the actin cytoskeleton in the response of chondrocytes to mechanical loading. The findings of this thesis may have important implications for understanding the mechanisms involved in the pathogenesis of cartilage tissue and for tissue engineering of cartilage.