Figure 1.

Direct mechanical stimulation transiently increased [Ca²⁺]_i in dentin matrix protein 1 (DMP-1)-, nestin-, and dentin sialophosphoprotein (DSPP)-immunopositive human odontoblasts. (A–D) Human odontoblasts showed positive immunoreactivity to the DMP-1 (red in A), nestin (red in B), DSPP (red in C), and Piezo1 channels (green in D). Nuclei were represented in blue. Scale bar: 100μm. No fluorescence was detected in the negative controls (not shown). (E) Experimental setup for the measurement of Ca²⁺-sensitive dye fluorescence. Standard extracellular solution (standard ECS) with or without pharmacological Piezo1 channel activator/inhibitors was applied from inlet by a rapid gravity-fed perfusion system and aspirated using a vacuum pump. Diagram also shows mechanical stimulation induced by vertical micropipette displacement downward by 8.0μm. (F) Representative trace of transient increases in [Ca²⁺]_i during repeated mechanical stimulations (black boxes). (G) Bar graph showing the F/F₀ values as a function of numbers of mechanical stimuli by vertical micropipette displacement (8μm). Each bar indicates the mean±SD. Numbers in parentheses indicate the number of experimented cells. N.S., not significant.