Single cell active force generation under dynamic loading - Part II: Active modelling insights.

In Part I of this two-part study a novel single cell AFM experimental investigation reveals a complex force-strain response of cells to cyclic loading. The biomechanisms underlying such complex behaviour cannot be fully understood without a detailed mechanistic analysis incorporating the key features of active stress generation and remodelling of the actin cytoskeleton. In order to simulate untreated contractile cells an active bio-chemo-mechanical model is developed, incorporating the key features of stress fibre (SF) remodelling and active tension generation. It is demonstrated that a fading memory SF contractility model accurately captures the transient response of cells to dynamic loading. Simulations reveal that high stretching forces during unloading half-cycles (probe retraction) occur due to tension actively generated by axially oriented SFs. On the other hand, hoop oriented SFs generate tension during loading half-cycles, providing a coherent explanation for the elevated compression resistance of contractile cells. Finally, it is also demonstrated that passive non-linear visco-hyperelastic material laws, traditionally used to simulate cell mechanical behaviour, are not appropriate for untreated contractile cells, and their use should be limited to the simulation of cells in which the active force generation machinery of the actin cytoskeleton has been chemically disrupted. In summary, our active modelling framework provides a coherent understanding of the biomechanisms underlying the complex patterns of experimentally observed single cell force generation presented in the first part of this study. STATEMENT OF SIGNIFICANCE: A novel computational investigation into the active and passive response of cells to dynamic loading is performed. An active formulation that considers key features of actin cytoskeleton active contractility and remodelling throughout the cytoplasm is implemented. Simulations provide new insights into the sub-cellular biomechanical response, providing a coherent explanation for the complex patterns of cell force uncovered experimentally in the first part of this study. Our computational models also reveal that passive non-linear visco-hyperelastic material laws, traditionally used to simulate cell mechanical behaviour, are not appropriate for untreated contractile cells, and their use should be limited to the simulation of cells in which the active force generation machinery of the actin cytoskeleton has been chemically disrupted.

Single cell active force generation under dynamic loading - Part I: AFM experiments.

A novel series of experiments are performed on single cells using a bespoke AFM system where the response of cells to dynamic loading at physiologically relevant frequencies is uncovered. Measured forces for the untreated cells are dramatically different to cytochalasin-D (cyto-D) treated cells, indicating that the contractile actin cytoskeleton plays a critical role in the response of cells to dynamic loading. Following a change in applied strain magnitude, while maintaining a constant applied strain rate, the compression force for contractile cells recovers to 88.9±7.8% of the steady state force. In contrast, cyto-D cell compression forces recover to only 38.0±6.7% of the steady state force. Additionally, untreated cells exhibit strongly negative (pulling) forces during unloading half-cycles when the probe is retracted. In comparison, negligible pulling forces are measured for cyto-D cells during probe retraction. The current study demonstrates that active contractile forces, generated by actin-myosin cross-bridge cycling, dominate the response of single cells to dynamic loading. Such active force generation is shown to be independent of applied strain magnitude. Passive forces generated by the applied deformation are shown to be of secondary importance, exhibiting a high dependence on applied strain magnitude, in contrast to the active forces in untreated cells. STATEMENT OF SIGNIFICANCE: A novel series of experiments are performed on single cells using a bespoke AFM system where the response of cells to dynamic loading at physiologically relevant frequencies is uncovered. Contractile cells, which contain the active force generation machinery of the actin cytoskeleton, are shown to be insensitive to applied strain magnitude, exhibiting high resistance to dynamic compression and stretching. Such trends are not observed for cells in which the actin cytoskeleton has been chemically disrupted. These biomechanical insights have not been previously reported. This detailed characterisation of single cell active and passive stress during dynamic loading has important implications for tissue engineering strategies, where applied deformation has been reported to significantly affect cell mechanotransduction and matrix synthesis.