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.

A Novel 3D Osteoblast and Osteocyte Model Revealing Changes in Mineralization and Pro-osteoclastogenic Paracrine Signaling During Estrogen Deficiency.

Recent in vitro studies have revealed that the mechanobiological responses of osteoblasts and osteocytes are fundamentally impaired during estrogen deficiency. However, these two-dimensional (2D) cell culture studies do not account for in vivo biophysical cues. Thus, the objectives of this study are to (1) develop a three-dimensional (3D) osteoblast and osteocyte model integrated into a bioreactor and (2) apply this model to investigate whether estrogen deficiency leads to changes in osteoblast to osteocyte transition, mechanosensation, mineralization, and paracrine signaling associated with bone resorption by osteoclasts. MC3T3-E1s were expanded in media supplemented with estrogen (17ß-estradiol). These cells were encapsulated in gelatin-mtgase before culture in (1) continued estrogen (E) or (2) no further estrogen supplementation. Constructs were placed in gas permeable and water impermeable cell culture bags and maintained at 5% CO2 and 37°C. These bags were either mechanically stimulated in a custom hydrostatic pressure (HP) bioreactor or maintained under static conditions (control). We report that osteocyte differentiation, characterized by the presence of dendrites and staining for osteocyte marker dentin matrix acidic phosphoprotein 1 (DMP1), was significantly greater under estrogen withdrawal (EW) compared to under continuous estrogen treatment (day 21). Mineralization [bone sialoprotein (BSP), osteopontin (OPN), alkaline phosphatase (ALP), calcium] and gene expression associated with paracrine signaling for osteoclastogenesis [receptor activator of nuclear factor kappa-ß ligand (RANKL)/osteoprotegerin OPG ratio] were significantly increased in estrogen deficient and mechanically stimulated cells. Interestingly, BSP and DMP-1 were also increased at day 1 and day 21, respectively, which play a role in regulation of biomineralization. Furthermore, the increase in pro-osteoclastogenic signaling may be explained by altered mechanoresponsiveness of osteoblasts or osteocytes during EW. These findings highlight the impact of estrogen deficiency on bone cell function and provide a novel in vitro model to investigate the mechanisms underpinning changes in bone cells after estrogen deficiency.