Background: The present study aimed to analyse the expression of osteoclastogenesis-related molecules and the cellular location of the B2 V-ATPase subunit (B2 V-ATPase), which is related to resorption activity, in osteoclast precursors from mouse marrow cells treated with different bisphosphonates.
Methods: Tibia and femur marrow cells obtained from 30 days-old Balb/c mice were cultured in α-MEM supplemented with calcitriol onto the bone slices for 5 days. The bone substracts were previously soaked in α-MEM containing alendronate (AL), etidronate (ET) or zoledronic acid (ZA) or only in medium (V). The bone resorption was analysed by SEM, and the cells were incubated for TRAP, TUNEL or submitted to MTT assay. Additional cells were harvested from the surface of the bone substrate and the expression of osteoclastogenesis-related genes (RANK, RANKL, OPG, CSF-1 and CSF-1R) was analysed by qPCR. For B2 V-ATPase analysis, the cells were harvested from the bone surfaces and spun at 50x103 rpm in an ultracentrifuge. Pellet and supernatant fractions were resolved in SDS-PAGE, blotted into nitrocellulose membrane and immunoblots were detected by electrochemiluminescence.
Results: V and ET specimens presented more bone resorption than AL and ZA. The V group presented more TRAP-positive cells with more than 5 nuclei than other groups. MTT values were higher in ZA group, while the number apoptotic cells was higher in ET. AL reduced the expression of CSF-1 and RANKL, while ZA reduced the expression of RANK and increased the expression of OPG. The B2 V-ATPase was found in the supernatant only in vehicles and remained bound to the pellet in the treated groups.
Conclusion: Therefore, AL and ZA inhibit osteoclastogenesis by different mechanisms, besides the effects on resorption activity. The reduced bone resorption may be related to the inhibited binding of B2 V-ATPase to the cell membrane by bisphosphonates.
Disclosure: The authors declared no competing interests. Supported by FAPESP #2011/24003-3 and # 2013/02240-9.