Extracellular pH is a critical regulator of osteoclast fusion, size and activation.

Osteoclast activity is regulated by extracellular pH, whereby bone resorption is near-maximally activated at pH 7.0 but limited at ≥pH 7.4. This study examined the effects of low pH on osteoclast fusion, multi-nucleation, resorption and cell transcriptome. Osteoclasts were cultured on dentine discs at pH 7.4 (control) or pH 7.0 (acidified) for 5-7 days. Osteoclast number and resorptive activity were 1.9-fold and 6.7-fold higher, respectively, in acidified cultures. However, acidified osteoclasts were smaller, with fewer nuclei than controls (53 μm diameter with 9 ± 1 nuclei/cell versus 100 μm with 24 ± 3 nuclei/cell). mRNA expression analysis revealed that osteoclast formation and resorption-associated genes were increased in acidified osteoclasts. Switching mature osteoclasts formed for 5 days at pH 7.4 to acidified conditions decreased cell size 30 % within 4 h, resulting in a 2-fold increase in osteoclast numbers after 24 h. Resorptive activity in cells switched to pH 7.0 was visible within 8 h, and by 24 h resorption area was comparable to continually acidified osteoclasts. MicroCT analysis of dentine discs revealed 24-fold and 6.4-fold increases in resorption pit number in pH-switched osteoclasts relative to control and acidified cultures, respectively. RNAseq showed changes in extracellular pH differentially regulated gene expression, particularly metabolic and cell cycle-associated genes. Our results reveal previously unknown effects of extracellular pH on osteoclasts. Specifically, they show pH is an important modulator of osteoclast fusion and size that regulates the transcriptome. Furthermore, small changes in pH can induce significant morphological changes in osteoclasts and act as on/off switch between formation and resorption in ≤4 h.