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Osteoblasts change their intracellular calcium ion concentration in response to mechanical stimuli. Although it has been reported that osteoblasts sense and respond to stretching of a substrate on which osteoblastic cells have adhered, the details of the dynamic characteristics of their calcium signaling response remain unclear. Motion artifacts such as loss of focus during stretch application make it difficult to conduct precise time-course observations of calcium signaling responses. Therefore, in this study, we observed intracellular calcium signaling responses to stretch in a single osteoblastic cell by video rate temporal resolution. Our originally developed cell-stretching microdevice enables in situ observation of a stretched cell without excessive motion artifacts such as focus drift. Residual minor effects of motion artifacts were corrected by the fluorescence ratiometric method with fluorescent calcium indicator Fluo 8H and fluorescent cytoplasm dye calcein red-orange. We succeeded to detect the intracellular calcium signaling response to stretch by video rate temporal resolution. The results revealed a time lag from stretch application to initiation of the intracellular calcium signaling response. We compared two time lags measured at two different cell areas: central and peripheral regions of the cell. The time lag in the central region of the cell was shorter than that in the peripheral region. This result suggests that the osteoblastic calcium signaling response to stretching stimuli initiates around the central region of the cell.

Evaluation of initiating characteristics of osteoblastic calcium signaling responses to stretch by video rate time-course observation