They have also shown that osteocytes may shed membrane-bound vesicle-like structures from their cell body and dendrites [46]. The function of these vesicles is currently unclear. They may provide a mechanism for reduction of osteocyte cytoplasmic volume. Alternatively, they may PF-562271 ic50 regulate mineral deposition and/or may provide a mechanism for intercellular communication through delivery of messenger RNA and proteins to the target cells, as has been described for microvesicles in other cell systems (reviewed in [47] and [48]).

A surprising finding from live imaging studies of osteocytes in neonatal calvarial organ cultures was the observation of a subpopulation of motile cells on the bone surface that express the Dmp1-GFP transgene but exhibit a polygonal non-dendritic morphology [43] and [46]. It was shown that these surface motile Dmp1-GFP positive cells also express DNA Synthesis inhibitor the early osteocyte marker E11/gp38, suggesting that they may represent a precursor

that is already committed to becoming an osteocyte [43]. Time-lapse imaging studies in mineralizing osteoblast cultures have revealed that the kinetics of Dmp1-GFP expression and mineralization are integrated, with clusters of motile cells first switching on Dmp1-GFP expression followed by mineral deposition [42] and [44] (Fig. 5). These Dmp1-GFP positive cells also express E11/gp38, suggesting that they are transitioning towards the osteocyte phenotype. Deposition of mineral was found to be associated with an arrest in motility of the Dmp1-GFP positive cells and a change in morphology from a polygonal to a highly dendritic morphology, characteristic of osteocytes. The data suggest that the processes of osteocyte differentiation and mineralization are tightly integrated and that the cell type responsible for mineralization is a cell that is already transitioning towards the osteocyte phenotype. Recently, Ishihara et al. have used time-lapse imaging approaches to image calcium signaling oscillations in living

osteocytes in embryonic Methocarbamol chick calvaria [49] (Fig. 6). Their studies showed that osteoblasts and osteocytes show oscillations in intracellular calcium concentrations and that calcium release from intracellular stores plays a key role in these calcium oscillations. In osteocytes but not osteoblasts, gap junctional communication appeared to be important for maintenance of the calcium oscillations. Such studies are an important advance, as prior to this work, intracellular calcium signaling has been reported from in vitro studies of osteocytes and was thought to be important in mechanotransduction [50], [51] and [52]. However, it was not known whether these phenomena actually occur in osteocytes in situ within their mineralized lacunae. Live cell imaging studies as applied to investigating osteocyte biology are still in their infancy.