In situ intracellular calcium oscillations in osteocytes in intact mouse long bones under dynamic mechanical loading

Abstract

Osteocytes have been hypothesized to be the major mechanosensors in bone. How in situ osteocytes respond to mechanical stimuli is still unclear because of technical difficulties. In vitro studies have shown that osteocytes exhibited unique calcium (Ca(2+)) oscillations to fluid shear. However, whether this mechanotransduction phenomenon holds for in situ osteocytes embedded within a mineralized bone matrix under dynamic loading remains unknown. Using a novel synchronized loading/imaging technique, we successfully visualized in real time and quantified Ca(2+) responses in osteocytes and bone surface cells in situ under controlled dynamic loading on intact mouse tibia. The resultant fluid-induced shear stress on the osteocyte in the lacunocanalicular system (LCS) was also quantified. Osteocytes, but not surface cells, displayed repetitive Ca(2+) spikes in response to dynamic loading, with spike frequency and magnitude dependent on load magnitude, tissue strain, and shear stress in the LCS. The Ca(2+) oscillations were significantly reduced by endoplasmic reticulum (ER) depletion and P2 purinergic receptor (P2R)/phospholipase C (PLC) inhibition. This study provides direct evidence that osteocytes respond to in situ mechanical loading by Ca(2+) oscillations, which are dependent on the P2R/PLC/inositol trisphosphate/ER pathway. This study develops a novel approach in skeletal mechanobiology and also advances our fundamental knowledge of bone mechanotransduction.

Experimental studies of bone mechanoadaptation: bridging in vitro and in vivo studies with multiscale systems

“…We recently modified the ex vivo system described earlier to observe Ca 2þ responses in live osteocytes in a mouse long bone subjected to dynamic, deformational loading (figure 2a). Osteocytes exhibited robust oscillations in Ca 2þ in response to load in a load-dependent manner (figure 2b) [79]. This pattern was more pronounced than the autonomous Ca 2þ responses of osteocytes, which were undetectable, as well as the loadinginduced and autonomous responses of cells on the bone surface (figure 2b) [76,79].…”

Dynamic fluid flow induced mechanobiological modulation of in situ osteocyte calcium oscillations

“…Recent research has also shown the real-time Ca 2+ oscillations in response to a direct mechanical stimulation on an intact ex vivo mouse tibia [31]. However, the Ca 2+ oscillation pattern has not been identified through fluid flow alone mechanism, in the absence of mechanical strain, until this study, which can be achieved with a direct DFFS into the marrow cavity of an intact ex vivo mouse long bone to generate fluid shear.…”

“…For Ca 2+ fluorescent labeling, the bone samples were incubated for 2 hours in 15 μM Fluo-8 AM (Santa Cruz Biotechnology, Inc., Dallas, Texas, U.S.A.) dissolved in dimethyl sulfoxide (DMSO) and culture medium before confocal imaging [31]. …”

“…Furthermore, in response to in vitro and in situ mechanical stimulations, osteocytes seem to be more sensitive than osteoblasts, in terms of Ca 2+ oscillations. Recent studies have observed the real-time Ca 2+ oscillations in response to fluid shear on ex vivo bone segments, as well as a direct mechanical stimulation on an intact ex vivo mouse tibia [31–33]. However, these studies either investigated the effect of fluid shear on osteocytic Ca 2+ oscillations only on ex vivo bone segment surfaces that were not yet adapted for dynamic mechanical events within bone, or studied the effect of osteocytic Ca 2+ oscillations of intact mouse long bones deformable bone loading that the recording was done only at the resting period with time delay.…”

Bone Homeostasis and Repair: Forced Into Shape

“…Osteocytes subjected to mechanical stimulation respond rapidly by mobilizing a series of second messengers, including calcium [59,60], nitric oxide [61,62], and prostaglandins [63]; by activating kinase signaling cascades, including the MAP kinase and PKC pathways [64]; and by exhibiting alterations in gene expression [65]. In addition, osteocytes respond to biophysical cues by releasing soluble factors important for cellular proliferation and differentiation and for recruitment of osteoblasts and osteoclasts [66][67][68][69][70].…”