Calcium ions released from mineral trioxide aggregate are taken up by C2C12 cells via the L-type voltage-dependent calcium channel

Mineral trioxide aggregate (MTA) is a commonly used endodontic repair material reported to exhibit calcified-tissue conductive activity. The effect of MTA on osteoblast differentiation has been well-defined; however, its effect on mesenchymal cells is not fully understood. Recently, we reported that MTA converts the differentiation pathway of pluripotent mesenchymal cells (C2C12 cells) into osteoblast lineages, possibly through the influx of elution components, such as calcium ions released from MTA. The purpose of this study was to investigate whether calcium ions released from MTA flow directly into C2C12 cells and whether this influx increases intracellular calcium levels. C2C12 cells were loaded with fluo-3 acetoxymethyl ester, which acts as a calcium-sensitive fluorescent probe. The intracellular calcium levels of C2C12 cells were assessed by confocal laser scanning microscopy. MTA induced a rapid increase in fluorescence intensity (FI) in C2C12 cells; however, application of Dulbecco's modified Eagle's medium (DMEM) as the growth medium had no such effect. To clarify the specific effects of MTA-derived calcium ions, we used calcium chloride as an exogenous calcium ion source and ethylene glycol tetraacetic acid (EGTA) as a calcium chelator. DMEM containing calcium chloride elicited a rapid rise in FI, whereas MTA plus EGTA did not affect FI. Moreover, MTA did not affect FI in C2C12 cells pre-incubated with verapamil, an L-type voltage-dependent calcium channel (VDCC_L) blocker. Our data suggest that calcium ions released from MTA flow directly into C2C12 cells via VDCC_L.