Purpose: Osteolysis associated with periprosthetic loosening is generally associated with the presence of wear particle-associated macrophages which (i) release inflammatory cytokines (e.g. TNFα and IL-1α) and (ii) are capable of osteoclast differentiation and bone resorption. The recently identified molecule, RANKL (expressed on osteoblastic cells) has been shown to play a central role in the macrophage-osteoclast differentiation observed in aseptic loosening. However, as TNFα and IL-1α are abundant in periprosthetic tissues and have been shown to mediate wear particle (bone cement)-associated osteolysis in animal models, and as we have recently shown that TNFa can induce osteoclastogenesis in a manner independent of RANKL mechanism, the aim of the present study was to determine whether wear particles, in particular bone cement particles, can affect RANKL- and TNFα-induced osteoclast formation and bone resorption in vitro.

Methods: Murine monocytes were cultured on glass coverslips and dentine slices with or without PMMA particles in presence of:- (i) macrophage colony stimulating factor (M-CSF) alone, (ii) M-CSF + soluble RANKL (iii) M-CSF + TNFα or (iv) M-CSF + TNFα + IL-1a. All cultures were maintained for 7–10 days after which the extent of osteoclast differentiation was determined by the expression of specific osteoclast markers including tartrate-resistant acid phosphatase (TRAP) on coverslips and evidence of lacunar resorption on dentine slices.

Results: Extensive osteoclast formation and lacunar resorption was evident in monocyte cultures in the presence of soluble RANKL and M-CSF. Addition of PMMA in these cultures increased the extent of RANKL-induced lacunar resorption by about 2 fold. In the absence of soluble RANKL, but in the presence of TNFα (± IL-1α), murine monocytes were also capable of differentiating into active bone resorbing osteoclasts. Addition of PMMA particles to these cultures resulted in a marked increase in the TNFα-induced osteoclas-togenesis. It is worth noting that monocyte cultures containing M-CSF and PMMA particles only did not differentiate into bone resorbing osteoclasts.

Conclusion: These results indicate that PMMA particles can activate both RANKL- and cytokine-induced osteoclast formation and osteolysis. Although, we had previously shown the existence of these two distinct cellular mechanisms in periprosthetic loosening, this is the first report in which wear particles have directly been shown to stimulate these cellular mechanisms independently. Our findings could provide possible therapeutic approaches to control the wear particle-associated early failure of joint replacements.

Purpose: Bone destruction occurs due to the growth of primary malignant bone tumours (sarcomas) that are often not amendable to surgery. Bone resorption is carried out by osteoclasts which are formed from cells of the mononuclear phagocyte system. Primary malignant bone tumours contain tumour-associated macrophages (TAMs) in addition to neoplastic cells. The aim of the study was to determine the cellular and humoral conditions required for TAM-osteoclast differentiation and to assess the affect of an anti-osteolytic agent on osteoclastic bone resorption.

Methods: TAMs were isolated form bone and soft tissue sarcoma by collagenase digestion and cultured in the presence of RANKL and M-CSF on coverslips and dentine slices for up to 21 days. The extent of osteoclast formation and resorption was determined by expression of osteoclast markers (TRAP, VNR, cathepsin K) in cell cultures on coverslips and the extent of lacunar resorption in cell cultures on dentine slices.

Results: Osteoclast formation occurred only when RANKL and M-CSF were added to the TAM cultures. This resulted in the formation of numerous mononuclear multinucleated cells which were strongly TRAP, VNR and cathepsin K positive. In cell cultures on dentine slices, it was noted that these cells were capable of extensive lacunar resorption with formation of multiple large lacunar resorption pits. The addition of the bisphosphonate zoledronate to the cell cultures resulted in inhibition of osteoclast formation and complete absence of lacunar resorption.

Conclusion: These findings indicate that sarcoma-associated macrophages are capable of differentiating into osteoclasts and that both RANKL and M-CSF are required for this to occur. This process is likely to contribute to tumour osteolysis associated with the growth of sarcomas in bone. Further assessment of the use of inhibitors of osteoclast formation/resorption, is also indicated by our results.

In rheumatoid arthritis (RA) and other arthritic disorders e.g. gout, there is destruction of articular cartilage and juxta-articular bone. Osteoclasts are specialised multinucleated cells (MNCs) that carry out bone resorption. It has previously been shown that circulating monocytes and synovial macrophages in RA can be stimulated to differentiate into functional osteoclasts in the presence of RANKL and M-CSF. The aim of this study was to determine whether the mononuclear cells present in synovial fluid of RA patients are capable of differentiating into functional osteoclasts in the presence of osteogenic factors.

Mononuclear cells were isolated from the synovial fluid obtained from patients with Ra, osteoarthritis (OA) gout and joint trauma. The cells were seeded onto dentine slices and coverslips and cultured for up to 21 days in the presence/absence of RANKL (30ng/ml) and M-CSF (25ng/ml). Cells cultured on coverslips for 24h, 14 and 21 days were assessed for the expression of the monocyte-macrophage antigen CD14 that is known to be expressed by osteoclasts, and the osteoclast associated markers; tartrate-resistant acid phosphatase (TRAP) and vitronectin receptor (VNR). After 21 days, dentine slices were assessed for evidence of osteoclastic lacunar resorption.

After 24 h culture on coverslips mononuclear cells isolated from the synovial fluid of all the above joint conditions were largely CD14+, and entirely negative for TRAP and VNR. After 14 days culture, in the presence of RANKL and M-CSF these synovial fluid macrophages were stimulated to form multinucleated osteoclasts which were TRAP+ and VNR+ and capable of forming resorption pits on dentine slices. In the absence of either RANKL or M-CSF osteoclast formation did not occur.

The osteogenic factors RANKL and M-CSF have been shown to be present in the synovial fluid of patients with RA, OA, gout and joint trauma. Results from this study demonstrate that CD14+ mononuclear cells (macrophages) in the synovial fluid of patients with the above conditions have the capacity to differentiate into functional multinucleated osteoclasts in the presence of RANKL and M-CSF. These findings show that one cellular mechanisms whereby bone erosions many occur in arthritic disorders is through increased osteoclast formation of synovial fluid macrophages; this process requires RANKL and m-CSF, both of which are produced by inflammatory cells e.g. T Cells found in the synovial fluid and the arthritic synovial membrane.