Matrix-dependent adhesion mediates network responses to physiological stimulation of the osteocyte cell process

Osteocytes are bone cells that form cellular networks that sense mechanical loads distributed throughout the bone tissue. Interstitial fluid flow in the lacunar canalicular system produces focal strains at localized attachment sites around the osteocyte cell process. These regions of periodic attachment between the osteocyte cell membrane and its canalicular wall are sites where pN-level fluid-flow induced forces are generated in vivo. In this study, we show that focally applied forces of this magnitude using a newly developed Stokesian fluid stimulus probe initiate rapid and transient intercellular electrical signals in vitro. Our experiments demonstrate both direct gap junction coupling and extracellular purinergic P2 receptor signaling between MLO-Y4 cells in a connected bone cell network. Intercellular signaling was initiated by pN-level forces applied at integrin attachment sites along both appositional and distal unapposed cell processes, but not initiated at their cell bodies with equivalent forces. Electrical coupling was evident in 58% of all cell pairs tested with appositional connections; coupling strength increased with the increasing number of junctional connections. Apyrase, a nucleotidedegrading enzyme, suppressed and abolished force-induced effector responses, indicating a contribution from ATP released by the stimulated cell. This work extends the understanding of how osteocytes modulate their microenvironment in response to mechanical signals and highlights mechanisms of intercellular relay of mechanoresponsive signals in the bone network.