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In bone cells, intracellular Ca2+ (iCa2+) functions as a second messenger in the mechanotransduction pathway. Its responses to mechanical stimulation can be observed microscopically on a rigid flat surface with a Ca2+ fluorescent indicator, generally, under fluid flow. However, bone cells are physiologically exposed to dynamic loading accompanied by bone matrix deformation. In this situation, microscopic methods of observing iCa2+ responses cannot be used because of the loss of focus or movement of cells out of the observation area. The purpose of this study was to develop a compact optical device for the observation of iCa2+ responses of osteoblasts to dynamic loadings accompanied by substrate deformation. This system comprised four light emitting diodes (LEDs) and a photodetector (PD) placed underneath a culture chamber, specifically designed for tissue-level iCa2+ observations. This device was used to study the frequency dependence of iCa2+ responses of osteoblasts to dynamic loading. MC3T3-E1 osteoblasts were cultured three-dimensionally in a collagen sponge scaffold with the fluorescent Ca2+ indicator Fluo-4 AM and mechanically stimulated by a 0.2% deformation of the sponge at 0.2, 2, or 20 Hz for 150 s. Our device succeeded in detecting temporal changes in the intensity of emitted fluorescent light, showing a frequency-dependent increase in fluorescence intensity. This device may contribute to further understanding of the mechanosensing and mechanotransduction mechanisms in bone under near-physiological conditions

Intracellular Ca<SUP>2+</SUP> Responses of 3D-Cultured Osteoblasts to Dynamic Loading