Adherent endotoxin mediates biological responses of titanium particles without stimulating their phagocytosis.

Aseptic loosening of orthopaedic implants is thought to be primarily due to stimulation of cytokine production by wear particles from the implants. The cytokines increase osteoclast differentiation, leading to osteolysis and implant loosening. Accumulating evidence indicates that adherent endotoxin mediates the biological responses induced by the wear particles. One mechanism by which adherent endotoxin may act is by increasing phagocytosis of the wear particles. To test this hypothesis, the effect of adherent endotoxin on phagocytosis of titanium particles was determined. First, we developed reliable confocal and fluorescence microscopy methods to examine both the attachment and internalization steps of phagocytosis. Use of these methods showed that adherent endotoxin does not detectably alter the rate or the extent of phagocytosis of titanium particles by RAW 264.7 cells. Despite this lack of an effect on phagocytosis, adherent endotoxin dramatically increases the ability of RAW 264.7 cells to produce TNF-alpha and induce osteoclast differentiation. Thus, adherent endotoxin mediates these biological responses by a mechanism that does not rely on increased phagocytosis. These results also demonstrate that phagocytosis is not sufficient to induce cytokine production and osteoclast differentiation but do not rule out the possibility that phagocytosis is required for induction of these responses by titanium particles with adherent endotoxin.     

Inhibitory effect of icariin on Ti-induced inflammatory osteoclastogenesis

Background

Wear particle-induced periprosthetic osteolysis that results in aseptic loosening is the most common cause of long-term failure after total joint replacement.

Materials and methods

Icariin (ICA), a flavonoid isolated from Epimedium pubescens, inhibits osteoclast formation, but its effects on wear particle-induced inflammatory osteoclastogenesis remains unclear. We investigated the role of ICA in the regulation of osteoclast differentiation in a murine macrophage cell line (RAW264.7), which is stimulated by titanium (Ti) particles and the receptor activator of NF-κB ligand.

Results

ICA effectively inhibited osteoclast formation and bone resorption in the differentiation medium. ICA (10⁻⁷ mol/L) significantly reduced the number of tartrate-resistant acid phosphatase-positive cells compared with the control, and significantly reduced the percentage of the surface covered by resorption lacunae. Quantitative real-time polymerase chain reaction analysis showed that ICA inhibited messenger RNA expression for the receptor activator of nuclear factor-κB, cathepsin K, tartrate-resistant acid phosphatase-positive, and matrix metalloproteinase-9 in RAW264.7 cells stimulated by Ti particles and receptor activator of NF-κB ligand. ICA also reduced pro-inflammatory cytokine expression of interleukin-1β and tumor necrosis factor-α in RAW264.7 cells cultured with Ti particles. In addition, incubation with cholecystokinin-8 showed that ICA had no toxic effects on RAW264.7 cells.

Conclusions

ICA possibly elicited inhibitory effects on inflammatory osteoclastogenesis induced by Ti particles, indicating that ICA may be useful for the prevention and treatment of wear particle-induced osteolysis.     

钛颗粒诱导破骨细胞分化过程中活化T细胞核因子c1的表达

目的探讨钙调磷酸酶（CN）／活化T细胞核因子（NFAT）途经在磨损颗粒诱导破骨细胞分化调节中的作用。方法MTT法检测11R—VIVIT肽和钛（Ti）颗粒对小鼠骨髓单核／巨噬细胞系细胞（BMMs）活力的影响;RT—PCR法分析Ti颗粒诱导破骨细胞分化过程中NFATcl mRNA表达,并观察1R—VIVIT肽对Ti颗粒诱导NFATcl表达的影响;TRAP染色进行破骨细胞分化鉴定。结果11R—VIVIT肽和Ti颗粒均不影响体外培养中的BMMs活力;Ti颗粒显著刺激破骨细胞分化（P〈0.01）和NFATcl mRNA表达;应用11R—VIVIT肽阻断CN／NFAT途径可显著抑制Ti颗粒诱导的NFATcl表达。结论Ti颗粒刺激BMMs向破骨细胞分化;其作用机制可能与激活CN／NFAT途径有关。     

Biological responses of human mesenchymal stem cells to titanium wear debris particles

Wear debris-induced osteolysis is a major cause of orthopedic implant aseptic loosening, and various cell types, including macrophages, monocytes, osteoblasts, and osteoclasts, are involved. We recently showed that mesenchymal stem/osteoprogenitor cells (MSCs) are another target, and that endocytosis of titanium (Ti) particles causes reduced MSC proliferation and osteogenic differentiation. Here we investigated the mechanistic aspects of the endocytosis-mediated responses of MSCs to Ti particulates. Dose-dependent effects were observed on cell viability, with doses >300 Ti particles/cell resulting in drastic cell death. To maintain cell viability and analyze particle-induced effects, doses <300 particles/cell were used. Increased production of interleukin-8 (IL-8), but not IL-6, was observed in treated MSCs, while levels of TGF-β, IL-1β, and TNF-α were undetectable in treated or control cells, suggesting MSCs as a likely major producer of IL-8 in the periprosthetic zone. Disruptions in cytoskeletal and adherens junction organization were also observed in Ti particles-treated MSCs. However, neither IL-8 and IL-6 treatment nor conditioned medium from Ti particle-treated MSCs failed to affect MSC osteogenic differentiation. Among other Ti particle-induced cytokines, only GM-CSF appeared to mimic the effects of reduced cell viability and osteogenesis. Taken together, these results strongly suggest that MSCs play both responder and initiator roles in mediating the osteolytic effects of the presence of wear debris particles in periprosthetic zones.     

The balance between endotoxin accumulation and clearance during particle-induced osteolysis in murine calvaria.

Bacterial endotoxin may contribute to aseptic loosening of orthopedic implants even in the absence of clinical or microbiological evidence of infection. One potential source of endotoxin during aseptic loosening is systemically circulating endotoxin, derived from intestinal flora, minor infections, or dental procedures, that may bind to wear particles. The current study demonstrates that systemically derived endotoxin accumulates when 'endotoxin-free' titanium and polyethylene particles are implanted on murine calvaria. Time-course experiments and experiments using germ-free mice rule out the possibility that the observed endotoxin accumulation may be due to bacterial contamination. In contrast, endotoxin is cleared from titanium particles that originally carry high amounts of adherent endotoxin. The mechanism of endotoxin clearance is not dependent on induction of a respiratory burst. Taken together, these results indicate that a balance between endotoxin accumulation and endotoxin clearance controls the steady-state level of endotoxin surrounding orthopedic wear particles implanted on murine calvaria. This balance may regulate the rate of osteolysis in the murine calvaria model as well as in patients with aseptic loosening.     

Comparison of the roles of IL-1, IL-6, and TNFalpha in cell culture and murine models of aseptic loosening

Pro-inflammatory cytokines, such as IL-1, IL-6, and TNF, are considered to be major mediators of osteolysis and ultimately aseptic loosening. This study demonstrated that synergistic interactions among these cytokines are required for the in vitro stimulation of osteoclast differentiation by titanium particles. In contrast, genetic knock out of these cytokines or their receptors does not protect murine calvaria from osteolysis induced by titanium particles. Thus, the extent of osteolysis was not substantially altered in single knock out mice lacking either the IL-1 receptor or IL-6. Osteolysis also was not substantially altered in double knock out mice lacking both the IL-1 receptor and IL-6 or in double knock out mice lacking both TNF receptor-1 and TNF receptor-2. The differences between the in vivo and the cell culture results make it difficult to conclude whether the pro-inflammatory cytokines contribute to aseptic loosening. One alternative is that in vivo experiments are more physiological and that therefore the current results do not support a role for the pro-inflammatory cytokines in aseptic loosening. We however favor the alternative that, in this case, the cell culture experiments can be more informative. We favor this alternative because the role of the pro-inflammatory cytokines may be obscured in vivo by compensation by other cytokines or by the low signal to noise ratio found in measurements of particle-induced osteolysis.     

Efficacy of etanercept for wear debris-induced osteolysis.

A major limitation of total joint arthroplasty is that up to 20% of patients require revision surgery to correct prosthetic loosening. Aseptic loosening is believed to result from the phagocytosis of wear debris particles by macrophages, which secrete proinflammatory cytokines that stimulate osteolysis. Tumor necrosis factor alpha (TNF-alpha) has been shown to be one of the prominent cytokines in this cascade and to be involved critically in the generation of particle-induced osteolysis. Etanercept is a soluble inhibitor of TNF-alpha, which is widely used for the treatment of rheumatoid arthritis. Here, we show this agent's ability to prevent wear debris-induced osteolysis. In vitro we show that Etanercept can inhibit directly osteoclastic bone resorption in a bone wafer pit assay, as well as cytokine production from titanium (Ti)-stimulated macrophages. Using a quantitative in vivo model of wear debris-induced osteolysis, we show that Etanercept prevents bone resorption and osteoclastogenesis. In mice treated with Etanercept at the time of osteolysis induction, bone resorption and osteoclast numbers were reduced to background levels in both normal and human TNF-alpha (hTNF-alpha) transgenic mice. In an effort to evaluate its effect on established osteolysis, Etanercept was administered 5 days after Ti implantation, and we observed that further osteolysis was prevented. These data support the concept that TNF-alpha is involved critically in osteoclastogenesis and bone resorption during periprosthetic osteolysis and suggest that soluble TNF-alpha inhibitors may be useful as therapeutic agents for the treatment of prosthetic loosening in humans.     

Measurement and removal of adherent endotoxin from titanium particles and implant surfaces.

Aseptic loosening is thought to be due primarily to osteolysis induced by cytokines and prostaglandins that are produced in response to implant-derived wear particles. Because endotoxin has many of the same effects as have been reported for wear particles, we hypothesized that adherent endotoxin may be responsible for the biological responses induced by wear particles. We demonstrated the presence of significant levels of adherent endotoxin on commonly used preparations of titanium particles as well as on titanium and titanium-alloy implant surfaces. In contrast, supernatants obtained by centrifugation of particle suspensions contained approximately 1% as much endotoxin as did the particles. Therefore, it is erroneous to assume that particles do not contain endotoxin on the basis of data that it cannot be detected in their supernatants or filtrates. These results emphasize the importance of considering the potential role of adherent endotoxin when examining the in vitro effects of wear particles and the in vivo performance of orthopaedic implants. We also developed a protocol that removed more than 99.94% of the adherent endotoxin from the titanium particles without detectably affecting their size or shape. The removal of adherent endotoxin will allow comparison of the biological responses induced by particles with or without adherent endotoxin.     

The effectiveness of polyethylene versus titanium particles in inducing osteolysis in vivo.

Bearing surface wear and periprosthetic osteolysis due to wear particles are among the most common reasons for joint replacement failure. A murine calvarial model of wear particle-induced osteolysis has been used to identify different biologic factors associated with this problem and to test nonsurgical methods of modulating the host response to particulate debris. This model has utilized titanium particles, however, in clinical practice the most common source of particulate debris is polyethylene particles from bearing surface wear. We now report a calvarial model of wear particle-induced osteolysis based on commercially available polyethylene particles. We found that compared to sham surgery osteoclast recruitment and bone resorption can be induced by introduction of the titanium particles or polyethylene particles. However, bone resorption was significantly higher with polyethylene particles compared to titanium particles (p=0.02). We consider the polyethylene based murine calvarial model of wear particle-induced osteolysis a reliable and clinically relevant tool to understand the host factors and potential pharmacologic interventions that can influence wear debris generated osteolysis. This model might serve as an extension of the well-established titanium based bone resorption model.     

The osteoclastogenic molecules RANKL and RANK are associated with periprosthetic osteolysis

Extensive osteolysis adjacent to implants is often associated with wear particles of prosthetic material. We have investigated if RANKL, also known as osteoprotegerin ligand, osteoclast differentiation factor or TRANCE, and its natural inhibitor, osteoprotegerin (OPG), may be important in controlling this bone loss. Cells isolated from periprosthetic tissues containing wear particles expressed mRNA encoding for the pro-osteoclastogenic molecules, RANKL, its receptor RANK, monocyte colony-stimulating factor (M-CSF), interleukin (IL)-1beta, tumour necrosis factor (TNF)alpha, IL-6, and soluble IL-6 receptor, as well as OPG. Osteoclasts formed from cells isolated from periprosthetic tissues in the presence and absence of human osteoblastic cells. When osteoclasts formed in the absence of osteoblastic cells, markedly higher levels of RANKL mRNA relative to OPG mRNA were expressed. Particles of prosthetic materials also stimulated human monocytes to express osteoclastogenic molecules in vitro. Our results suggest that ingestion of prosthetic wear particles by macrophages results in expression of osteoclast-differentiating molecules and the stimulation of macrophage differentiation into osteoclasts.     

The role of osteoclast differentiation in aseptic loosening.

The major cause of orthopaedic implant loosening is thought to be accelerated osteoclastic bone resorption due to the action of cytokines produced in response to phagocytosis of implant-derived wear particles. This accelerated osteoclastic bone resorption could be due to increases in any of the following processes: recruitment of osteoclast precursors to the local microenvironment, differentiation of precursors into mature multinucleated osteoclasts. activation of mature osteoclasts, and/or survival of osteoclasts. Our studies have focused on differentiation and survival to complement work by others who have focused on recruitment of precursors and activation. Taken together, our studies and those of other investigators provide strong evidence that increased recruitment of osteoclast precursors and their subsequent differentiation play major roles in wear particle-induced osteolysis. In contrast, increased osteoclast activation and survival appear to play minor roles. These studies suggest that development of therapeutic interventions that reduce either recruitment or differentiation of osteoclast precursors would improve the performance of orthopaedic implants.     

RANKL is an essential cytokine mediator of polymethylmethacrylate particle-induced osteoclastogenesis.

RANKL is a TNF superfamily member and an essential cytokine mediator of developmental osteoclastogenesis. We examined the role of RANKL in PMMA particle-induced osteoclastogenesis in vitro. In murine whole bone marrow cultures, PMMA particles stimulate a 2.5 fold increase in secreted RANKL, a 5-8 fold increase in osteoclast number and induce the formation of giant multinuclear osteoclasts. RANKL and TNF, potential cytokine mediators of PMMA, had similar osteoclastogenic effects. The RANKL inhibitor OPG was utilized to define the role of RANKL in mediating the PMMA response and was found to inhibit basal and PMMA particle-induced osteoclastogenesis. Additionally, particles stimulate osteoclast formation in RANKL-primed osteoclast precursor cells (devoid of supporting stromal cells) while RANKL untreated osteoclast precursors demonstrate no osteoclastogenic response to particles. Since TNF can potentiate RANKL action and is thought to mediate implant osteolysis we analyzed TNF(-/-) whole bone marrow cultures to elucidate the role of this cytokine. In TNF(-/-) cultures basal osteoclastogenesis remains intact, yet the PMMA effect is blunted. Finally, we show that PMMA, RANKL and TNF all activate the NF-kB and c-jun/AP-1 signaling pathways which are both fundamental to osteoclast formation and are potential sites of signal convergence in RANKL-mediated particle osteoclastogenesis.     

Polyethylene and titanium particles induce osteolysis by similar, lymphocyte-independent, mechanisms.

Periprosthetic osteolysis is a major clinical problem that limits the long-term survival of total joint arthroplasties. Osteolysis is induced by implant-derived wear particles, primarily from the polyethylene bearing surfaces. This study examined two hypotheses. First, that similar mechanisms are responsible for osteolysis induced by polyethylene and titanium particles. Second, that lymphocytes do not play a major role in particle-induced osteolysis. To test these hypotheses, we used the murine calvarial model that we have previously used to examine titanium-induced osteolysis. Polyethylene particles rapidly induced osteolysis in the murine calvaria 5-7 days after implantation. The polyethylene-induced osteolysis was associated with large numbers of osteoclasts as well as the formation of a thick periosteal fibrous tissue layer with numerous macrophages containing phagocytosed polyethylene particles. Polyethylene-induced osteolysis was rapidly repaired and was undetectable by day 21 after implantation. Lymphocytes were noted in the fibrous layer of wild-type mice. However, the amount of osteolysis and cytokine production induced by polyethylene particles was not substantially affected by the lack of lymphocytes in Pfp/Rag2 double knock out mice. All of these findings are similar to our observations of osteolysis induced by titanium particles. These results provide strong support for both of our hypotheses: that similar mechanisms are responsible for osteolysis induced by polyethylene and titanium particles and that lymphocytes do not play a major role in particle-induced osteolysis.     

Efficacy of ex vivo OPG gene therapy in preventing wear debris induced osteolysis.

Aseptic loosening of prosthetic implants remains a serious orthopaedic problem and the greatest limitation to total joint arthroplasty. Central to the etiology of aseptic loosening is periprosthetic osteolysis at the bone-implant interface, which is caused by wear debris-induced inflammation. This inflammation produces the critical osteoclast differentiation factor RANKL, which directly stimulates osteoclastogenesis and osteoclastic bone resorption. A dominant factor known to counteract this process is the natural RANKL receptor antagonist protein OPG. Here we explore the potential of ex vivo OPG gene therapy for aseptic loosening by evaluating the eflicacy of stably transfected fibroblast-like synoviocytes (FLS) expressing OPG in preventing wear debris-induced osteoclastogenesis, in a mouse calvaria model. Although the stably transfected fibroblasts produced small amounts of OPG (0.3 ng/ml/72 h/10(6) cells), this protein was very effective in preventing osteoclastic resorption as determined in a bone wafer assay. More importantly. implantation of 10(7) FLS-OPG, together with 30 mg of Ti wear debris, onto the calvaria of mice, completely inhibited osteoclastogenesis 3 days after surgery. Animals given FLS-LacZ control cells, which persisted for 3 days as determined by X-gal staining, together with the Ti particles, had a 6-fold increase in osteoclastogenesis compared to controls without Ti. This increased osteoclastogenesis was completely inhibited by the FLS-OPG, as osteoclast numbers in the calvaria of these animals were similar to that seen in the SHAM controls.     

Inhibition of the PI3K-Akt signaling pathway reduces tumor necrosis factor-alpha production in response to titanium particles in vitro

BACKGROUND: Wear debris contributes to implant loosening after total joint arthroplasty, and few advances have been made in our ability to inhibit the biological response to wear particles. Bacterial endotoxins augment the effects of wear particles in vitro and in vivo. The cytokine, tumor necrosis factor-alpha (TNF-alpha), is produced by macrophages in response to bacterial endotoxins and wear particles, and it increases osteoclast activity resulting in bone resorption and implant loosening. The phosphoinositol-3-kinase (PI3K)-Akt intracellular signal transduction pathway contributes to cytokine production in response to soluble endotoxin. We investigated the role of the PI3K-Akt pathway in the production of TNF-alpha in response to wear particles with adherent endotoxin and so-called endotoxin-free wear particles. METHODS: Cultured RAW264.7 murine macrophages were incubated with titanium particles with adherent endotoxin or with endotoxin-free titanium particles in the presence and absence of specific inhibitors of PI3K (LY294002) or Akt (SH-5). Akt activation was assessed with use of Western blot. TNF-alpha production was measured with use of enzyme-linked immunosorbent assay. Cytotoxicity was determined by measuring lactic dehydrogenase release. RESULTS: Titanium particles with adherent endotoxin increased Akt activation, whereas endotoxin-free titanium particles did not. The PI3K inhibitor reduced TNF-alpha production by 70% in response to titanium with adherent endotoxin without increasing cytotoxicity. Similarly, the Akt inhibitor reduced TNF-alpha production by 83% in response to titanium particles with adherent endotoxin without increasing cytotoxicity. High concentrations of endotoxin-free titanium particles resulted in a small delayed increase in TNF-alpha production that was completely blocked by the PI3K inhibitor. CONCLUSIONS: Inhibition of the PI3K-Akt pathway reduces macrophage TNF-alpha production in response to titanium particles with adherent endotoxin and endotoxin-free particles in vitro.     

Cytokine receptor profile of arthroplasty macrophages, foreign body giant cells and mature osteoclasts

In the arthroplasty pseudomembrane surrounding a loose prosthesis there is a marked macrophage and foreign body giant cell (FBGC) response to implant-derived wear particles. These cells contribute to the osteolysis of loosening by releasing cytokines and growth factors which influence the formation and activity of osteoclasts. Using a panel of monoclonal antibodies directed against known cytokine/growth factor receptors, we have determined by immunohistochemistry whether arthroplasty macrophages, FB-GCs and osteoclasts express receptors for cytokines and growth factors that are known to modulate osteolysis. All these cell types reacted with antibodies directed against the following cytokine/growth factor receptors: gp130, IL-1R type 1, IL-2R, IL-4R, IL-6R, TNFR, M-CSFR, GM-CSFR and SCFR but not with antibodies directed against IL-3R and IL-8R. Arthroplasty macrophages, FBGCs and osteoclasts thus show a similar pattern of cytokine/growth factor receptor expression. This reflects the fact that arthroplasty macrophages are capable of osteoclast differentiation and that these cell types form part of the mononuclear phagocyte system. As regards the osteolysis of aseptic loosening, it also indicates that these cells are targets for numerous cytokines and growth factors which influence osteoclast formation and bone resorption.     

Cytokine receptor profile of arthroplasty macrophages, foreign body giant cells and mature osteoclasts

In the arthroplasty pseudomembrane surrounding a loose prosthesis there is a marked macrophage and foreign body giant cell (FBGC) response to implant-derived wear particles. These cells contribute to the osteolysis of loosening by releasing cytokines and growth factors which influence the formation and activity of osteoclasts. Using a panel of monoclonal antibodies directed against known cytokine/growth factor receptors, we have determined by immunohis-tochemistry whether arthroplasty macrophages, FB-GCs and osteoclasts express receptors for cytokines and growth factors that are known to modulate osteolysis.

All these cell types reacted with antibodies directed against the following cytokine/growth factor receptors: gp130, IL-1R type 1, IL-2R, IL-4R, IL-6R, TNFR, M-CSFR, GM-CSFR and SCFR but not with antibodies directed against IL-3R and IL-8R. Arthroplasty macrophages, FBGCs and osteoclasts thus show a similar pattern of cytokine/growth factor receptor expression. This reflects the fact that arthroplasty macrophages are capable of osteoclast differentiation and that these cell types form part of the mononuclear phagocyte system. As regards the osteolysis of aseptic loosening, it also indicates that these cells are targets for numerous cytokines and growth factors which influence osteoclast formation and bone resorption.     

Blockade of JNK and NFAT pathways attenuates orthopedic particle‐stimulated osteoclastogenesis of human osteoclast precursors and murine calvarial osteolysis

Particles released from orthopedic implants attract immune host defense cells to the bone‐implant interface and contribute to development of inflammation. The inflammatory microenvironment supports recruitment and differentiation of osteoclasts, the primary culprit of osteolysis. Therefore, understanding the complex signals that contribute to osteoclastogenesis and osteolysis is a sensible approach to design strategies to inhibit bone loss. The signaling cascades that coordinate osteoclastogenesis have been widely investigated. These include MAP kinases, Akt/PI3K pathway, NF‐κB signal transduction pathway, and NFAT pathway. We have recently reported that polymethylmethacrylate (PMMA) particles activate the NFAT pathway in murine osteoclast precursors and that NFAT inhibitors dose‐dependently block PMMA‐induced osteoclastogenesis. In the current study, we examined the role of JNK and NFATc1 in mice in response to PMMA particles using murine calvaria model. We show that locally administered MAPK/JNK inhibitor SP600125 and calcineurin/NFAT inhibitor cyclosporine‐A effectively blocked PMMA‐induced osteolysis in murine calvaria. To buttress the clinical relevance of JNK/NFATc1‐based regulation of PMMA‐induced osteoclastogenesis, we evaluated the effect of PMMA using human macrophages. We demonstrate that SP600125 and cyclosporine‐A abolished particle‐induced osteoclastogenesis in human osteoclast progenitors retrieved from patients undergoing total hip replacement. Thus JNK and NFATc1 appear to act as significant mediators of orthopedic particle‐induced osteolysis in humans. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31:67–72, 2012     

Titanium particles stimulate bone resorption by inducing differentiation of murine osteoclasts

BACKGROUND: Loosening of orthopaedic implants is mediated by cytokines that elicit bone resorption and are produced in response to phagocytosis of implant-derived wear particles. This accelerated bone resorption could be due to increased osteoclastic activity, survival, or differentiation. Although a number of in vitro studies have shown that wear particles increase osteoclastic activity, the increase was less than twofold in all cases. The objective of the current study was to test the hypothesis that wear particles stimulate bone resorption by inducing osteoclast differentiation. METHODS: Conditioned media were prepared from murine marrow cells or human peripheral blood monocytes incubated in the presence or absence of titanium particles. The effects of conditioned media on osteoclast differentiation were examined with use of a recently developed assay in which osteoclast precursors are co-cultured with mesenchymal support cells. RESULTS: The present study showed that titanium particles induced both murine marrow cells and human peripheral blood monocytes to produce factors that stimulated osteoclast differentiation. The mean increase in osteoclast differentiation was 29.3+/-9.4-fold. The stimulation of osteoclast differentiation led to a parallel increase in bone resorption. The amount of stimulation was regulated in a dose-dependent manner by the concentration of both titanium particles and conditioned media. The stimulation of osteoclast differentiation required interactions between the cells and the particles themselves and, therefore, was not due to metal ions, soluble contaminants released from the particles, or submicrometer particles. In contrast, conditioned media from control cells incubated in the absence of titanium particles had no detectable effect on any of the examined parameters. CONCLUSIONS: The present study showed that titanium particles stimulate in vitro bone resorption primarily by inducing osteoclast differentiation. In contrast, the titanium particles had only small effects on osteoclast activity or survival.