Mechanosensitive Ca2+ signaling and coordination is diminished in osteocytes of aged mice during ex vivo tibial loading

Abstract

Purpose: The osteocyte is considered the major mechanosensor in bone, capable of detecting forces at a cellular level to coordinate bone formation and resorption. The pathology of age-related bone loss, a hallmark of osteoporosis, is attributed in part to impaired osteocyte mechanosensing. However, real-time evidence of the effect of aging on osteocyte responses to mechanical load is lacking. Intracellular calcium (Ca²⁺) oscillations have been characterized as an early mechanosensitive response in osteocytes in systems of multiple scales and thus can serve as a real-time measure of osteocyte mechanosensitivity. Our objective was to utilize an ex vivo model to investigate potentially altered mechanosensing in the osteocyte network with aging.Methods: Tibiae were explanted from young-adult (5 mo) and aged (22 mo) female mice and incubated with Fluo-8 AM to visualize osteocyte intracellular Ca²⁺. Whole tibiae were cyclically loaded while in situ osteocyte Ca²⁺ dynamics were simultaneously imaged with confocal microscopy. Responsive osteocyte percentage and Ca²⁺ peak characteristics were quantified, as well as signaling synchrony between paired cells in the field of view.Results: Fewer osteocytes responded to mechanical loading in aged mice compared to young-adult and did so in a delayed manner. Osteocytes from aged mice also lacked the well-correlated relationship between Ca²⁺ signaling synchrony and cell-cell distance exhibited by young-adult osteocytes.Conclusions: We have demonstrated, for the first time, real-time evidence of the diminished mechanosensing and lack of signaling coordination in aged osteocyte networks in tibial explants, which may contribute to pathology of age-induced bone loss.

Keywords: Ca2+ signaling; Osteocyte; aging; explant; mechanotransduction.

Figure 1.

Ex vivo model of osteocyte Ca²⁺ signaling. A. Mouse tibiae are dissected and transferred to a custom mechanical loading system, where cyclic compressive loads are applied axially to the distal end of the whole bone while live osteocytes are imaged using confocal microscopy. B. The intracellular Ca²⁺ indicator Fluo-8 AM is incubated with tibia samples to allow for imaging of Ca²⁺ dynamics in live osteocytes over time in response to mechanical and biochemical stimuli. C. Fluo-8 intensity is normalized to baseline intensity and plotted over time for individual osteocytes (B, red oval). Peaks in intracellular Ca²⁺ are identified and characterized in response to cyclic mechanical loading.

Figure 2.

Age-induced changes in cortical bone. A. Representative ex vivo micro-CT cross sections of tibial cortical bone in 5-month-old and 22-month-old female mice show decreased cortical thickness (Ct.Th) in the aged mice compared to young-adult. B. Average Ct.Th quantified for aged (n=4) and young-adult (n=5) tibia samples. Data are mean ± SD. **p<0.01 (Student’s t-test).

Figure 3.

Age-induced changes in osteocyte network morphology. Fixed ex vivo osteocyte membranes are stained with DiI and imaged with confocal microscopy. Representative images are provided for cell networks from tibiae of 5-month-old (A, C) and 22-month old (B, D) mice.

Figure 4.

Representative Ca²⁺ traces for young-adult and aged ex vivo osteocytes. Average Fluo-8 AM intensity normalized by average baseline intensity prior to load is plotted over time for all osteocytes in the field of view. A. Traces of a representative 5-month-old mouse tibia (n=53 cells) and B. 22-month-old mouse tibia (n=32 cells). Cyclic mechanical loading was initiated at t=25 s (arrows). Ca²⁺ peaks in each individual cell are defined as spikes in intensity that are greater than 3 times the standard deviation of the baseline intensity for that cell.

Figure 5.

Load-induced Ca²⁺ signaling parameters in young-adult and aged osteocytes. Based on Ca²⁺ peak identification from all Fluo-8 dyed osteocytes in the field of view (FOV) from 5-month-old (n=8 tibiae) and 22-month-old mice (n=6 tibiae). A. Percentage of responsive osteocytes (those that exhibit > 1 Ca²⁺ peak) of all cells in the FOV. B. Number of intracellular Ca²⁺ peaks exhibited by responsive osteocytes. C. Magnitude of Ca²⁺ peaks relative to baseline Fluo-8 AM intensity. D. Time between initiation of mechanical loading and Ca²⁺ signaling. Data are mean ± SD. **p<0.01, ***p<0.001 (Student’s t-test).

Figure 6.

Ca²⁺ peak synchrony in osteocyte networks of young-adult and aged mice. Synchronous cell pairs, in which the time between the any of the Ca²⁺ peaks from each cell in the pair is less than 15 seconds, are totaled and normalized to the total number of cell pairs in the FOV. Data points represent normalized synchronous cell pair numbers according to cell-cell distance for each tibia sample (n=6-8/group). Trend line represents negative correlation between average synchronous cell pairs and cell-cell separation in 5-month-old mice. Data are mean ± SD.