Stiffening symphony of aging: Biophysical changes in senescent osteocytes

Abstract

Senescent osteocytes are key contributors to age-related bone loss and fragility; however, the impact of mechanobiological changes in these cells remains poorly understood. This study provides a novel analysis of these changes in primary osteocytes following irradiation-induced senescence. By integrating subcellular mechanical measurements with gene expression analyses, we identified significant, time-dependent alterations in the mechanical properties of senescent bone cells. Increases in classical markers such as SA-β-Gal activity and p16^Ink4a expression levels confirmed the senescence status post-irradiation. Our key findings include a time-dependent increase in cytoskeletal Young's modulus and altered viscoelastic properties of the plasma membrane, affecting the contractility of primary osteocytes. Additionally, we observed a significant increase in Sclerostin (Sost) expression 21 days post-irradiation. These biophysical changes may impair osteocyte mechanosensation and mechanotransduction, contributing to bone fragility. This is the first study to time-map senescence-associated mechanical changes in the osteocyte cytoskeleton. Our findings highlight the potential of biophysical markers as indicators of cellular senescence, providing more specificity than traditional, variable biomolecular markers. We believe these results may support biomechanical stimulation as a potential therapeutic strategy to rejuvenate aging osteocytes and enhance bone health.

Keywords: cellular senescence; cytoskeleton mechanics; osteocyte mechanobiology; sub cellular structure.

FIGURE 1

Overview of the study and gene expression changes in senescent primary osteocytes. (a) Schematic representation of primary osteocyte isolation, culture, and induction of senescence by irradiation. (b) Bright field microscopy of irradiated primary cell culture showing increased in SA‐β‐Gal activity with peaking to over 70% by day 21 [scale bar = 50 μm]. (c, d) Normalized mRNA expression levels of key osteocyte and senescence‐associated genes. Increases in Sost and Mepe, along with a decrease in Mmp9, were observed in senescent osteocytes compared to non‐senescent control cells. Elevated p16 expression, along with increased SA‐β‐Gal activity (b), confirmed the senescence status. As illustrated in representative immunofluorescence staining images (F‐actin/DAPI) [scale bar = 25 μm], (e) changes in cell size, dendritic network (arrows in Sn‐D7), and morphology (dashed boundary in Sn‐D21) were observed. We have provided additional IF images in Appendix S1. These micrographs, from different regions of the CTRL and Sn‐D21 cultures, allow for better observation of the morphological changes due to senescence condition across different regions. Data are presented as means ± SD, with statistical significance denoted by *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

FIGURE 2

Mechanobiological Changes in Senescent Osteocytes. (a) Representative images of our mechanobiology experimental setup (b) Mechanical measurements showing increased Young's modulus and potentially altered viscoelastic properties in senescent osteocytes. A minimum of N = 90 single cells per study group (i.e., CTRL, Sn‐D7, Sn‐D14, and Sn‐D21) were mechanically tested at every time point. Young's modulus was obtained using the Hertzian contact model with Poisson's ratio $\nu =0.5$. (c) Mean unloading portions of indentation curves and (d) load‐time curves, demonstrating significantly higher minimum adhesion force and delayed recovery in senescent cells (p = 0.0084). In plot (c), all indentation data from control and Sn‐D21 were used, while plot (d) includes experimental data from cells exhibiting osteocyte‐like morphology. (e) Schematic drawing connecting these findings to the impaired mechanosensation and mechanotransduction capabilities of senescent osteocytes. These changes contribute to age‐related bone loss and fragility. Data are presented as means ± SD, with statistical significance denoted by *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.