In situ imaging of the autonomous intracellular Ca ²⁺ oscillations of osteoblasts and osteocytes in bone

Bone cells form a complex three-dimensional network consisting of osteoblasts and osteocytes embedded in a mineralized extracellular matrix. Ca ²⁺ acts as a ubiquitous secondary messenger in various physiological cellular processes and transduces numerous signals to the cell interior and between cells. However, the intracellular Ca ²⁺ dynamics of bone cells have not been evaluated in living bone. In the present study, we developed a novel ex-vivo live Ca ²⁺ imaging system that allows the dynamic intracellular Ca ²⁺ concentration ([Ca ²⁺] _i) responses of intact chick calvaria explants to be observed without damaging the bone network. Our live imaging analysis revealed for the first time that both osteoblasts and osteocytes display repetitive and autonomic [Ca ²⁺] _i oscillations ex vivo. Thapsigargin, an inhibitor of the endoplasmic reticulum that induces the emptying of intracellular Ca ²⁺ stores, abolished these [Ca ²⁺] _i responses in both osteoblasts and osteocytes, indicating that Ca ²⁺ release from intracellular stores plays a key role in the [Ca ²⁺] _i oscillations of these bone cells in intact bone explants. Another possible [Ca ²⁺] _i transient system to be considered is gap junctional communication through which Ca ²⁺ and other messenger molecules move, at least in part, across cell-cell junctions; therefore, we also investigated the role of gap junctions in the maintenance of the autonomic [Ca ²⁺] _i oscillations observed in the intact bone. Treatment with three distinct gap junction inhibitors, 18α-glycyrrhetinic acid, oleamide, and octanol, significantly reduced the proportion of responsive osteocytes, indicating that gap junctions are important for the maintenance of [Ca ²⁺] _i oscillations in osteocytes, but less in osteoblasts. Taken together, we found that the bone cells in intact bone explants showed autonomous [Ca ²⁺] _i oscillations that required the release of intracellular Ca ²⁺ stores. In addition, osteocytes specifically modulated these oscillations via cell-cell communication through gap junctions, which maintains the observed [Ca ²⁺] _i oscillations of bone cells.