Fig. 4

Osteocytes in their native bone matrix environment exhibit Ca²⁺-dependent actin network contractions. a Simultaneous loading and imaging of ex vivo osteocytes tagged with Lifeact mRFP and dyed with Fluo-8 AM. b Time-course of intracellular Ca²⁺ (Fluo-8 AM intensity), the osteocyte actin network (RFP-tagged F-actin), and whole-cell normal strain contours. c Representative traces of actin cytoskeleton strains and Ca²⁺ dynamics in an ex vivo osteocyte in response to cyclic mechanical loading of the tibia at an 8 N load magnitude. Decreasing normal strains develop in both axes of the cell following a Ca²⁺ peak. d A Ca²⁺ transient induced by ATP results in a phasic contractile response in normal strains in both axes of the osteocyte. e Ionomycin induces a step-increase in intracellular Ca²⁺ and overall decreases in normal strains in both axes of the cell. f Quantification of osteocyte Ca²⁺ oscillations and contractile dynamics reveals an average Ca²⁺ spike frequency of (0.012 ± 0.004) Hz and average contraction frequency of (0.009 3 ± 0.003) Hz, which were not found to be significantly different (n = 9 cells). Osteocyte contractions exhibit a percent synchrony with Ca²⁺ peaks of (52.4 ± 16.8) %. Arrowheads indicate initiation of contractile events. *P < 0.05. Error bars are standard deviations.