Metal-on-metal (MoM) bearing technology, made of cobalt-chromium (Co-Cr) alloys, is being used in anticipation of extending the durability of hip replacements. Increasingly, concern has been expressed that long term exposure to Co2+ and Cr3+ could cause DNA damage and immune dysfunction; specifically a reduction in the circulating number of CD8+ cytotoxic cells. More recently, we reported that Co2+ and Cr3+ affected the differentiation of osteoclast precursors into bone-resorbing osteoclasts. Despite these observations the effects of metal ions on osteoblast activity have been poorly investigated. The aim of the current study was to elucidate the effects of various metal ions on osteoblast activity in vitro.

Cells of the human osteosarcoma cell line SaOS-2 were cultured in the presence of 0, 1, 10 and 100 μM Co2+ and Cr3+. The morphology, viability, cytokine release (TNFalpha, IL-1beta, IL-6, LIGHT, MIP-1alpha and VEGF) and alkaline phosphatase activity were investigated after 24h and 48h in contact with metal ions. Finally the capacity of SaOS-2 to produce and mineralize a new bone matrix was assessed by the Alizarin red method. All experiments were repeated at least 5 times and the differences between each were determined using non-parametric Mann-Whitney test.

Compared to untreated cultures, although the morphology looked normal after 48h, the viability indicated that Co2+ and Cr3+ ions at high concentrations induced some significant and irreversible damages to the osteoblast cells. Interestingly, any of the cytokines investigated were released in contact with metal ions after 24h or 48h. The alkaline phosphatase activity was significantly increased by low concentrations of Co2+ and decreased by high concentrations of Cr3+ after 24h and 48h. Moreover, the degree of mineralization of a new bone matrix in vitro was significantly reduced when the SaOS-2 cells were exposed to high concentrations of Cr3+, but significantly increased when they were exposed to Co2+.

Our results indicated that irreversible damages are caused to the cells as soon as 24h with high concentrations of metal ions. For osteoblasts cells, Co2+ appeared to be less toxic than Cr3+ at high concentrations.

This study was supported by Furlong Research Charitable Foundation

Introduction: Osteoclast-like multinucleated giant cells (MNGCs) are found in a number of soft tissue sarcomas including malignant fibrous histiocytoma and leiomyosarcoma. The nature of these MNGCs is poorly understood and the cellular mechanisms underlying their accumulation in sarcomas is not known.

Methods: We analysed by immunohistochemistry the expression of osteoclast, macrophage and smooth muscle markers by mononuclear and multinucleated cells in two cases of giant cell-rich leiomyosarcoma. We also characterised the role of mononuclear stromal cells and tumour-associated macrophages in the formation of MNGCs by RT-PCR, cell culture studies and immunohistochemistry/histochemistry for macrophage, osteoclast and smooth muscle markers

Results: MNCGs in giant cell-rich leiomyosarcoma expressed an osteoclast-like phenotype, being negative for smooth muscle actin and CD14 but positive for tartrate-resistant acid phophatase (TRAP), CD45, CD68 and vitronectin receptor (VNR). Scattered mononuclear cells expressing an osteoclast-like antigenic phenotype were also noted. An analysis of 25 conventional (non-giant cell-containing) leiomyosarcomas found isolated CD68+ MNGCs in three cases (approximately 12%); all of these cases were Grade-II/III leiomyosarcomas in which there was a prominent tumour-associated macrophage (TAM) infiltrate. Leiomyosarcoma TAMs isolated from two cases of conventional leiomyosarcoma and cultured in the presence of the osteoclastogenic factors RANKL and M-CSF differentiated into TRAP+/VNR+ MNGCs that were capable of lacunar resorption. RT-PCR studies showed that cultured leiomyosarcoma mononuclear stromal cells expressed RANKL, OPG and TRAIL.

Discussion: These findings show that the MNGCs which are found in leiomyosarcomas are osteoclast-like in nature and that these MNGCs are formed from TAMs by a RANKL dependent mechanism which involves an interaction with RANKL-expressing mononuclear stromal cells. A similar mechanism is likely to account for MNGC accumulation in other soft tissue sarcomas.

Introduction: A major concern with cemented hip resurfacing arthroplasty (HRA) femoral components is the thermal damage to femoral head during cement curing; this maybe linked to fracture (reported incidence ~2%) and early failure. We investigated the effect of a modifid surgical technique using pulse lavage, lesser trochanter suction and early reduction on the maximum temperature recorded in the femoral head during HRA, compared to manual lavage and reduction after cement curing.

Methods: Patients undergoing total hip replacement (THR) were given a dummy HRA procedure, during which a temperature probe was inserted into the femoral head and the measuring tip placed close to the reamed surface; the position of the probe was confirmed by inter-operative xray. Four subjects received a dummy HRA femoral component using manual lavage and Simplex cement. The implanted femur was kept dislocated until the cement cured. The implanted heads were then removed and sectioned to locate the temperature probes, the THR surgery was then performed. Five patients receiving a definitive HRA were also measured; for these subjects suction on the lesser trochanter was used, pulse lavage given for 30 seconds prior to cementing with Simplex, and pulse lavage of the femoral head for 2 minutes, applied 1 minute after cementing the femoral component. The implanted joint was then immediately reduced and a further two minutes of pulse lavage applied to the reduced joint. Temperatures were recorded until the cement finally cured. In every case the cement was hand mixed for 1 minute and the component implanted at 2 minutes 30 seconds after mixing began.

Results: Sectioning showed that probe tips were < 0.5mm from cement mantle. The maximum temperature recorded in the femoral head was significantly (p=0.014) greater for the manual technique, median value of 47.2°C (37.0 to 67.9°C), than for the pulse lavage technique, median value of 32.7°C (31.7 to 35.6°C).

Discussion: The results show that excessive bone temperatures can occur during hip resurfacing. Temperatures above 45°C kill bone cells, the manual technique may lead to substantial thermal necrosis. Technique modification, with the use of suction on the lesser trochanter, generous use of pulse-lavage and joint reduction prior to cement curing, significantly reduced the temperatures recorded. With the modified technique, the maximum temperatures were well below the threshold of thermal damage. This modified technique is recommended as the potential for thermal bone necrosis is significantly reduced.

Osteomyelitis commonly causes bone destruction and is most frequently due to infection by Staphylococcus aureus. S. aureus is known to secrete a number of surface-associated proteins which are extremely potent stimulators of bone resorption in the mouse calvarial assay system. The precise cellular and humoral mechanisms whereby this stimulatory effect is mediated, in particular whether osteoclast formation or activity is directly promoted by these factors, have not been determined by this study. Surface-associated material (SAM)(0.001ug/ml)obtained from 24 hour cultures of S. aureus was added to cultures of mouse and human osteoclast precursors (RAW 264.7 cells and human peripheral blood mononuclear cells respectively). These cultures were incubated in the presence and absence of receptor activator of nuclear factor kappa B ligand (RANKL) and macrophage colony stimulating factor (M-CSF). It was found that independent of RANKL, SAM was capable of inducing osteoclast formation in cultures of RAW cells and human monocytes. This was evidenced by the generation of tartrate-resistant acid phosphatase-positive multinucleated cells, which formed lacunar resorption pits when these cells were cultured on dentine slices. In cultures where M-CSF, RANKL and SAM were added, osteoclast formation was increased, but did not exceed the osteoclast formation in cultures with M-CSF and RANKL. These findings indicate that S. aureus produces a soluble factor which can promote osteoclast formation. Identification of this factor may help to develop therapeutic strategies for treating bone destruction due to Staphylococcal osteomyelitis.

Introduction: Osseous metastases from melanoma are relatively common (7% of cases), and occur most often in the axial skeleton. Bone destruction in skeletal metastases of solid tumours is due to stimulation of osteoclast formation and bone resorption. Osteoclasts are formed by the fusion of marrow-derived mononuclear phagocyte precursors which express RANK (receptor activator of nuclear factor κB) which interacts with RANKL expressed by osteoblasts/bone stromal cells in the presence of macrophage colony-stimulating factor (M-CSF). Osteoclast formation by a RANKL-independent, tumour necrosis factor α (TNFα)-induced mechanism has also been reported. Tumour-associated macrophages (TAMs) are present in both primary and secondary tumours and TAMs are known to be capable of osteoclast differentiation. Our aim in this study was to determine the role of TAMs and the humoral mechanisms of osteolysis associated with melanoma metastases.

Materials and Method: In this study we isolated TAMs from extraskeletal primary melanomas and lymph node metastases. TAMs were cultured for up to 21 days in the presence and absence of M-CSF and RANKL or TNF. In a separate experiment, conditioned medium was extracted from the melanoma cell line, SK-Mel-29, and cultured with human peripheral blood mononuclear cells in the presence of M-CSF.

Results: TAM-osteoclast differentiation, as evidenced by the expression of tartrate-resistant acid phosphatase, vitronectin receptor and lacunar resorption pit formation, occurred by both RANKL-dependent and RANKL-independent mechanisms. Osteoclast formation induced by RANKL-independent mechanism was not abolished by the addition of osteoprotegerin or RANK:Fc, decoy receptors for RANK. Conditioned medium from SK-Mel-29 could support osteoclast differentiation in the absence of RANKL. This effect was not abolished by antibodies to RANKL, TNFα, TGFβ, IL-8 or gp130.

Discussion: These results indicate that melanoma TAMs are capable of differentiation into osteoclasts and that both RANKL-dependent and RANKL-independent (TNFα) mechanisms are involved. Melanoma tumour cells also secrete a soluble factor that supports osteoclastogenesis.

Conclusion: Inhibitors of osteoclast formation targeting TAM-osteoclast differentiation and osteoclast activity and identification of the osteoclastogenic factor produced by melanoma cells may have a therapeutic potential in controlling tumour osteolysis.

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.

Aseptic loosening is generally associated with the presence of wear particle-associated macrophages in the pseudomembrane commonly formed around failed prosthetic implants. The extent of the macrophage response evoked by the wear particles has been shown to correlate with the amount of periprosthetic osteolysis. Numerous studies have shown that wear particle-associated macrophages contribute to osteolysis by (i) releasing inflammatory cytokines and/or (ii) differentiating into bone resorbing osteoclasts. Although macrophages and macrophage polykaryons are the main inflammatory cells found in periprosthetic tissues, numerous fibroblasts are also present in the connective tissue pseudomembrane. The recently identified molecule, RANKL has been shown to play a central role in the osteoclast formation and bone resorption observed in aseptic loosening. We have shown that arthroplasty macrophages, which express RANK, the receptor for RANKL, are capable of osteoclast differentiation; this process is inhibited by osteoprotegerin (OPG), the soluble decoy receptor for RANKL. As fibroblasts are known to express RANKL, the aim of the present study was to determine whether fibroblasts isolated from periprosthetic tissues could induce the generation of bone resorbing osteoclasts that would contribute to the osteolysis commonly seen in the periprosthetic loosening.

Fibroblast-like cells were isolated from pseudomembrane from patients (n=5) undergoing hip revision due to aseptic loosening, by routine collagenase enzyme digestion. The isolated cells were seeded in flasks for 2–4 weeks before being passaged for a further 3–4 times. Generated fibroblast-like cells (10⁴) were then co-cultured with 5x10⁵ normal human peripheral blood monocytes (n=5) on glass coverslips and dentine slices in the presence of (i) no added factors, (ii) macrophage colony stimulating factor (M-CSF) and (iii) M-CSF plus OPG. All cultures were maintained for 1,17 and 21 days. The extent of osteoclast differentiation was then determined by the expression of specific osteoclast markers including tartrate-resistant acid phosphatase (TRAP) and vitronectin receptor (VNR) and evidence of lacunar resorption.

In the absence M-CSF, no osteoclast formation was noted in 24 hours, 17 or 21 days in fibroblast/monocyte cultures. However, in the presence of M-CSF alone, large numbers of TRAP⁺ and VNR⁺ multinucleated cells capable of lacunar resorption were noted in these co-cultures. The addition of OPG, which is known to inhibit RANKL-mediated osteoclast formation, significantly reduced the extent of osteoclast formation and lacunar resorption in these co-cultures.

These results indicate that one means whereby peri-prosthetic osteolysis may occur is by fibroblasts in the arthroplasty pseudomembrane inducing macrophage-osteoclast differentiation. Fibroblasts express RANKL and interact with arthroplasty macrophages, which express RANK and function as osteoclast precursors. These findings indicate that suppression of osteoclast formation by OPG may be a possible form of therapy for reducing prosthetic loosening.

Mast cells (MC), the tissue-based effector cells in allergic diseases, have many functions. Within bone tissue, they have been linked with new blood vessel formation and marrow fibrosis and it has been proposed that they are capable of promoting osteoclastic bone resorption. MC numbers are known to increase in a number of osteolytic conditions e.g. osteoporosis, hyperparathyroidism and periodontitis. In fracture callus, too, large numbers of MC are present, especially during the onset of remodelling where it is believed they may be responsible for osteoclast recruitment and/or differentiation. The aim of this study was to look for further evidence of mast cell (MC) involvement in pathological bone resorption. MC activity was assessed in tissue sections of osteolytic conditions including Paget’s disease of bone, rheumatoid arthritis and fibrous dysplasia together with several benign and malignant bone tumours. MCs were identified by toluidine blue staining and by immunostaining with a commercial antibody against MC tryptase.

Extensive infiltration of mast cells was observed in fibrous dysplasia, rheumatoid arthritis and Paget’s disease of bone and mast cell accumulation was seen at the bone resorbing margin of a number of enlarging bone tumours including osteosarcoma, giant cell tumour of bone, osteoma and osteoid osteoma.

MCs, along with other inflammatory cells, are known to accumulate at the margins of soft tissue tumours where they are thought to promote tumour growth. The current findings are consistent with a similar role for mast cells in the primary bone tumours examined. In each of the conditions studied, an additional role for MC may be that of promoting bone lysis. MC are known to contain numerous factors including TNF-alpha and IL-1, which are potent stimulators of osteoclast formation and activity.

It is concluded that MCs may contribute to the fibrosis, angiogenesis and increased bone resorption seen in certain metabolic bone diseases. MC activity may also be an important factor contributing to the lysis that occurs in numerous other pathological situations including at the margins of aggressive primary bone tumours and skeletal metastases, leading to the expansion of these lesions.

The cellular mechanisms which account for the formation of osteoclasts and bone resorption associated with enlarging benign and malignant mesenchymal tumours of bone are uncertain. Osteoclasts are marrow-derived, multinucleated, bone-resorbing cells which express a macrophage phenotype. We have determined whether tumour-associated macrophages (TAMs) isolated from benign and malignant mesenchymal tumours are capable of differentiating into osteoclasts. Macrophages were cultured on both coverslips and dentine slices for up to 21 days with UMR 106 osteoblastic cells in the presence of 1,25 dihydroxyvitamin D₃ (1,25(OH)₂D₃) and human macrophage colony-stimulating factor (M-CSF) or, in the absence of UMR 106 cells, with M-CSF and RANK ligand.

In all tumours, the formation of osteoclasts from CD14-positive macrophages was shown by the formation of tartrate-resistant-acid-phosphatase and vitronectin-receptor-positive multinucleated cells which were capable of carrying out lacunar resorption. These results indicate that the tumour osteolysis associated with the growth of mesenchymal tumours in bone is likely to be due in part to the differentiation of mononuclear phagocyte osteoclast precursors which are present in the TAM population of these lesions.

Mononuclear osteoclast precursors are present in the wear-particle-associated macrophage infiltrate found in the membrane surrounding loose implants. These cells are capable of differentiating into osteoclastic bone-resorbing cells when co-cultured with the rat osteoblast-like cell line, UMR 106, in the presence of 1,25(OH)₂ vitamin D₃. In order to develop an in vitro model of osteoclast differentiation which more closely parallels the cellular microenvironment at the bone-implant interface in situ, we determined whether osteoblast-like human bone-derived cells were capable of supporting the differentiation of osteoclasts from arthroplasty-derived cells and analysed the humoral conditions required for this to occur.

Long-term co-culture of arthroplasty-derived cells and human trabecular-bone-derived cells (HBDCs) resulted in the formation of numerous tartrate-resistant-acid-phosphatase (TRAP) and vitronectin-receptor (VNR)-positive multinucleated cells capable of extensive resorption of lacunar bone. The addition of 1,25(OH)₂ vitamin D₃ was not required for the formation of osteoclasts and bone resorption. During the formation there was release of substantial levels of M-CSF and PGE₂. Exogenous PGE₂ (10⁻⁸ to 10⁻⁶M) was found to stimulate strongly the resorption of osteoclastic bone. Our study has shown that HBDCs are capable of supporting the formation of osteoclasts from mononuclear phagocyte precursors present in the periprosthetic tissues surrounding a loose implant. The release of M-CSF and PGE₂ by activated cells at the bone-implant interface may be important for the formation of osteoclasts at sites of pathological bone resorption associated with aseptic loosening.

We present a case in which the growth of an intraosseous cyst arising from the proximal tibiofibular joint appeared to have been increased by polyethylene wear particles from a medial unicompartmental knee replacement. Histological examination of the cyst wall showed a histiocytic response associated with numerous polyethylene wear particles. This case demonstrates that there is a direct communication between the joint cavity and the cyst. Such communication is probably through openings in the articular cartilage large enough to allow the passage of these particles.

A heavy infiltrate of foreign-body macrophages is commonly seen in the fibrous membrane which surrounds an aseptically loose cemented implant. This is in response to particles of polymethylmethacrylate (PMMA) bone cement and other biomaterials. We have previously shown that monocytes and macrophages responding to particles of bone cement are capable of differentiating into osteoclastic cells which resorb bone.

To determine whether the radio-opaque additives barium sulphate (BaSO₄) and zirconium dioxide (ZrO₂) influence this process, particles of PMMA with and without these agents were added to mouse monocytes and cocultured with osteoblast-like cells on bone slices. Osteoclast differentiation, as shown by the presence of the osteoclast-associated enzyme tartrate-resistant acid phosphatase (TRAP) and lacunar bone resorption, was observed in all cocultures.

The addition of PMMA alone to these cocultures caused no increase in TRAP expression or bone resorption relative to control cocultures. Adding PMMA particles containing BaSO₄ or ZrO₂, however, caused an increase in TRAP expression and a highly significant increase in bone resorption. Particles containing BaSO₄ were associated with 50% more bone resorption than those containing ZrO₂.

Our results suggest that radio-opaque agents in bone cement may contribute to the bone resorption of aseptic loosening by enhancing macrophage-osteoclast differentiation, and that PMMA containing is BaSO₄ likely to be associated with more osteolysis than that containing ZrO₂.