Image-derived modeling of nucleus strain amplification associated with chromatin heterogeneity.

Beyond the critical role of cell nuclei in gene expression and DNA replication, they also have a significant influence on cell mechanosensation and migration. Nuclear stiffness can impact force transmission and, furthermore, act as a physical barrier to translocation across tight spaces. As such, it is of wide interest to accurately characterize nucleus mechanical behavior. In this study, we present a computational investigation of the in situ deformation of a heterogeneous chondrocyte nucleus. A methodology is developed to accurately reconstruct a three-dimensional finite-element model of a cell nucleus from confocal microscopy. By incorporating the reconstructed nucleus into a chondrocyte model embedded in pericellular and extracellular matrix, we explore the relationship between spatially heterogeneous nuclear DNA content, shear stiffness, and resultant shear strain. We simulate an externally applied extracellular matrix shear deformation and compute intranuclear strain distributions, which are directly compared with corresponding experimentally measured distributions. Simulations suggest that the mechanical behavior of the nucleus is highly heterogeneous, with a nonlinear relationship between experimentally measured grayscale values and corresponding local shear moduli (µn). Three distinct phases are identified within the nucleus: a low-stiffness mRNA-rich interchromatin phase (0.17 kPa ≤ µn ≤ 0.63 kPa), an intermediate-stiffness euchromatin phase (1.48 kPa ≤ µn ≤ 2.7 kPa), and a high-stiffness heterochromatin phase (3.58 kPa ≤ µn ≤ 4.0 kPa). Our simulations also indicate that disruption of the nuclear envelope associated with lamin A/C depletion significantly increases nuclear strain in regions of low DNA concentration. We further investigate a phenotypic shift of chondrocytes to fibroblast-like cells, a signature for osteoarthritic cartilage, by increasing the contractility of the actin cytoskeleton to a level associated with fibroblasts. Peak nucleus strains increase by 35% compared to control, with the nucleus becoming more ellipsoidal. Our findings may have broad implications for current understanding of how local DNA concentrations and associated strain amplification can impact cell mechanotransduction and drive cell behavior in development, migration, and tumorigenesis.

Influence of multi-axial dynamic constraint on cell alignment and contractility in engineered tissues.

In this study an experimental rig is developed to investigate the influence of tissue constraint and cyclic loading on cell alignment and active cell force generation in uniaxial and biaxial engineered tissues constructs. Addition of contractile cells to collagen hydrogels dramatically increases the measured forces in uniaxial and biaxial constructs under dynamic loading. This increase in measured force is due to active cell contractility, as is evident from the decreased force after treatment with cytochalasin D. Prior to dynamic loading, cells are highly aligned in uniaxially constrained tissues but are uniformly distributed in biaxially constrained tissues, demonstrating the importance of tissue constraints on cell alignment. Dynamic uniaxial stretching resulted in a slight increase in cell alignment in the centre of the tissue, whereas dynamic biaxial stretching had no significant effect on cell alignment. Our active modelling framework accurately predicts our experimental trends and suggests that a slightly higher (3%) total SF formation occurs at the centre of a biaxial tissue compared to the uniaxial tissue. However, high alignment of SFs and lateral compaction in the case of the uniaxially constrained tissue results in a significantly higher (75%) actively generated cell contractile stress, compared to the biaxially constrained tissue. These findings have significant implications for engineering of contractile tissue constructs.

On the role of the actin cytoskeleton and nucleus in the biomechanical response of spread cells.

Micropipette aspiration (MA) has been used extensively in biomechanical investigations of un-adhered cells suspended in media. In the current study, a custom MA system is developed to aspirate substrate adhered spread cells. Additionally, the system facilitates immuno-fluorescent staining of aspirated cells to investigate stress fibre redistribution and nucleus deformation during MA. In response to an applied pressure, significantly lower aspiration length is observed for untreated contractile cells compared to cells in which actin polymerisation is chemically inhibited, demonstrating the important contribution of stress fibres in the biomechanical behaviour of spread cells. Additional experiments are performed in which untreated contractile cells are subjected to a range of applied pressures. Computational finite element simulations reveal that a viscoelastic material model for the cell cytoplasm is incapable of accurately predicting the observed aspiration length over the range of applied pressures. It is demonstrated that an active computational framework that incorporates stress fibre remodelling and contractility must be used in order to accurately simulate MA of untreated spread cells. Additionally, the stress fibre distribution observed in immuno-fluorescent experimental images of aspirated cells is accurately predicted using the active stress fibre modelling framework. Finally, a detailed experimental-computational investigation of the nucleus mechanical behaviour demonstrates that the nucleus is highly deformable in cyto, reaching strain levels in excess of 100% during MA. The characterisation of stress fibres and nucleus biomechanics in spread cells presented in the current study can potentially be used to guide tissue engineering strategies to control cell behaviour and gene expression.