Bone Abstracts

Introduction: Nitrogen-containing bisphosphonates (N-BPs) can inhibit the differentiation and function of osteoclasts derived from Peripheral Blood Mononuclear cells (PBMCs) in a dose-dependent manner. MUTZ-3 cells are a potentially useful human cell line for studying osteoclast differentiation. The aim of this study was to elucidate the action of N-BPs on MUTZ-3 cells.

Methods: Human PBMCs and MUTZ-3 cells were cultured in α-MEM supplemented with heat inactivated foetal calf serum, 25 ng/ml hMCSF, 100 ng/ml soluble hRANKL and 25 ng/ml hTNF-α. To determine the effects of BPs, cells were cultured in the presence of alendronate, ibandronate, risedronate and zoledronate at concentrations ranging from 10nM to 50 μM. The extent of osteoclast formation (expressed as the number of TRAcP+ multinucleated cells) as well as cell morphometry of newly-generated osteoclasts (osteoclast area and numbers of nuclei/osteoclast) were also determined. The osteoclast markers cathepsin-K, calcitonin receptor (CTR) and osteoclast-associated receptor (OSCAR) were demonstrated by western blotting. Experiments were performed in the presence of inhibitors of cell internalisation and fluorescent FAM-Risedronate to determine the pathway(s) by which these cells internalise BPs.

Results: Multinucleated osteoclast-like cells were evident at day 12 and the osteoclast markers, cathepsin-K, CTR and OSCAR were detectable from day 12 in both cell populations. Analysis of N-BP treatment compared with controls revealed significant dose-dependent effects only in PBMC cultures. Although N-BPs altered osteoclast area and number of nuclei/osteoclast in PBMC cultures, these drugs failed to induce such effects with MUTZ-3 cells. Sodium vanadate, a specific inhibitor of ATPase-dependent endocytosis blocked internalization of N-BPs in PBMCs, whereas caffeine, a specific inhibitor of Ca2+-dependent endocytosis, blocked uptake by MUTZ-3 cells.

Conclusion: Although MUTZ-3 cells can differentiate into TRAP-positive multinucleated osteoclast-like cells which express osteoclast markers, these cells do not respond to BPs in a similar manner to PBMCs, even though BPs can be internalised by both cell types. The reasons why MUTZ-3 cells are unresponsive is so far unexplained.