• RANKL;
  • synovial fibroblast;
  • osteolysis;
  • cyclooxygenase-2;
  • osteoclast


  1. Top of page
  2. Abstract
  7. Acknowledgements

Synovial fibroblasts are possible mediators of osteolysis. Fibroblasts respond directly to titanium particles and increase RANKL expression through a COX-2/PGE2/EP4/PKA signaling pathway. Fibroblasts pretreated with titanium or PGE2 stimulated osteoclast formation, showing the functional importance of RANKL induction. Synovial fibroblasts and their activation pathways are potential targets to prevent osteolysis.

Introduction: Bone loss adjacent to the implant is a major cause of joint arthroplasty failure. Although the cellular and molecular response to microscopic wear debris particles is recognized as causative, little is known concerning role of synovial fibroblasts in these events.

Materials and Methods: Murine embryonic fibroblasts and knee synovial fibroblasts in culture stimulated with titanium particles were examined by FACS, real time RT-PCR, Northern blot, and Western blot for expressions of vascular cell adhesion molecule (VCAM)1, RANKL, cyclooxygenase (COX)-1, and COX-2, and the four prostaglandin E2 (PGE2) receptor isoforms. Experiments were performed in the presence and absence of COX inhibitors, protein kinase A (PKA) and protein kinase C (PKC) inhibitors, and various EP receptor agonists. Osteoclast formation was examined in co-cultures of pretreated glutaraldehyde-fixed fibroblasts and primary murine spleen cells treated with macrophage-colony stimulating factor (M-CSF) for 7-days.

Results: TNF-α stimulated VCAM1 expression, consistent with a synovial fibroblast phenotype. Titanium particles stimulated RANKL gene and protein expressions in fibroblasts in a dose-dependent manner. Gene expression was increased 5-fold by 4 h, and protein levels reached a maximum after 48 h. Within 1 h, titanium particles also induced COX-2 mRNA and protein levels, whereas both indomethacin and celecoxib blocked the stimulation of RANKL, suggesting a COX-2-mediated event. Furthermore, PGE2 induced RANKL gene and protein expression and rescued RANKL expression in titanium-treated cultures containing COX-2 inhibitors. Fibroblast cultures pretreated with either PGE2 or titanium particles enhanced osteoclast formation, indicating the functional importance of RANKL induction. EP4 was the most abundant PGE2 receptor isoform, EP1 and EP2 were expressed at low levels, and EP3 was absent. The EP1 selective agonist iloprost and the EP2 selective agonist butaprost minimally stimulated RANKL. In contrast, the EP2 and EP4 agonist misoprostol induced RANKL to a magnitude similar to PGE2. Finally, PKA antagonism strongly repressed RANKL stimulation by PGE2.

Conclusion: Fibroblasts respond directly to titanium particles and increase RANKL expression through a COX-2/PGE2/EP4/PKA signaling pathway. Thus, the synovial fibroblast is important mediator of osteolysis and target for therapeutic strategies.


  1. Top of page
  2. Abstract
  7. Acknowledgements

TOTAL JOINT ARTHROPLASTY is the optimal treatment for severe arthritis involving major joints, including the hip, knee, shoulder, and elbow. Arthroplasty results in a dramatic reduction in pain and a marked improvement in function and is increasingly being used, with >1.3 million arthroplasties performed around the world each year.(1) However, in up to 20% of cases, bone loss develops adjacent to the implant and results in mechanical instability, renewed pain, and the need for revision surgery.(2,3)

The pathogenesis of prosthetic loosening is still unclear, but both biological and mechanical factors likely contribute.(1) Motion at the bearing surfaces of the prosthetic joint results in the generation of submicron particles composed of polyethylene and metallic debris. These particles are shed into the joint space and become imbedded in the surrounding tissues where >109 particles are found per gram of tissue.(4–6) Prior work has shown a direct relationship between the generation of particles, as measured by volumetric polyethylene wear, and aseptic loosening.(7,8) In response to the particles, an inflammatory membrane composed of a fibrovascular tissue containing macrophages and occasional lymphocytes develops in the joint lining and invades the prosthesis-bone interface.(9) Both in vitro and in vivo studies show that this inflammatory membrane mediates the bone loss that occurs in aseptic loosening.

Macrophages compose ∼15% of the cells in the inflammatory membrane. In situ hybridization experiments with retrieved human membranes showed that interleukin (IL)-1 expression was limited to macrophages and did not occur in other cell types.(9,10) Thus, macrophages are considered the major source of proinflammatory mediators and on stimulation with particulate debris release inflammatory cytokines that directly and indirectly stimulate bone resorption.(9,10) Moreover, recent studies show that membrane derived macrophages can form multinucleated, TRACP+ cells that resorb bone. As such, the macrophage has been extensively studied as a mediator of osteolysis.(1) In contrast, there is much more limited information regarding the role of other cells, especially fibroblasts, which compose ∼70% of the cells in the periprosthetic membrane.(9,10)

Recent information suggests that synovial fibroblasts present in the periprosthetic membrane are important targets of wear debris during osteolysis. The pathogenesis of inflammatory joint diseases, including rheumatoid arthritis, have been shown to involve the synovial fibroblast.(11,12) In inflammatory arthritis, synovial fibroblasts secrete proteases that stimulate matrix degradation and release inflammatory mediators.(13–15) Importantly, synovial fibroblasts participate in the bone resorption that characterizes inflammatory arthritis by secreting osteoclastogenic factors including RANKL.(11,12,15,16) Fibroblasts have also been shown to be directly responsive to particles and release metalloproteinases.(17) Work in our laboratory has shown that titanium particles induce prostaglandin E2 (PGE2) and IL-6 expression in fibroblasts, but this effect is absent in fibroblasts lacking cyclooxgenase (COX)-2 gene expression and in cultures containing COX-2 inhibitors.(18) Immunohistochemical examinations have confirmed COX-2 expression in fibroblasts in human periprosthetic membranes and in fibroblasts adjacent to murine calvaria treated with titanium particles.(18,19) Others have shown that fibroblasts in the periprosthetic membrane express RANKL.(20,21)

RANKL is a member of the TNF/TNF receptor (TNFR) superfamily and is present primarily as a membrane protein and is produced by osteoblasts/stromal cells.(22,23) The receptor RANK is located on macrophage/osteoclast precursors, and on activation by RANKL, stimulates the expression of genes required for osteoclast differentiation.(24) Both gain and loss of function experiments have established RANKL/RANK as essential for osteoclast formation.(25,26) The importance of RANKL for the aseptic loosening is indicated by the complete absence of wear debris-induced osteolysis in a murine model after loss of RANK-RANKL signaling.(27)

Here we perform a series of in vitro experiments that define the fibroblasts as an important target of wear debris particles. In response to micron-sized titanium particles, fibroblasts express RANKL and directly stimulate osteoclast formation. We show that this effect is dependent on the induction of COX-2 and is mediated by prostaglandins. Finally, we show that the induction of RANKL likely involves PGE2 through the EP4 receptor and protein kinase A (PKA) signaling pathway. These findings provide a mechanism for earlier work in our laboratory that showed complete absence of inflammatory osteolysis in COX-2-deficient mice using an in vivo murine calvarial model of particle-induced bone loss.(19)


  1. Top of page
  2. Abstract
  7. Acknowledgements

Cell culture

The fibroblastic cell line was previously established from embryos of mouse CBAXBL6 strain.(28) Confirmatory experiments were also performed using fibroblast-like synoviocytes obtained from the murine knee joint.(29) Fibroblasts and ST-2 cells were both cultured in DMEM medium in the presence of 10% heat-inactivated FBS, 1% penicillin, and 5% CO2 at 37°C. As a control for RANKL expression, ST-2 cells were cultured in the presence or absence of 10−8 M 1,25-dihydroxyvitamin D3 [1,25(OH)2D3; Biomol; Plymouth Meeting, PA, USA] and 10−7 M dexamethasone (Sigma, St Louis, MO, USA) for 48 h. Fibroblasts were stimulated with particles (from 5 × 105/ml to 1 × 107/ml) or PGE2 (1 × 10−6 M; Cayman Chemicals, Ann Arbor, MI, USA), IL-6 (100 ng/ml; R&D, Minneapolis, MN, USA), IL-1 (100 ng/ml; R&D), or TNF-α (10 ng/ml; R&D). In various experiments, the cyclooxygenase inhibitors indomethacin (1 μM; Cayman Chemicals) and celecoxib (10 μM; G.D. Searle, Chicago, IL, USA) or the EP receptor agonists, iloprost (2 μM), butprost (10 μM), or misoprostol (1 μM; Cayman Chemicals) were added to the cultures. The PKA and protein kinase C (PKC) inhibitors H-89 (10 μM) and Go6976 (10 μM; Calbiochem, San Diego, CA, USA) were used in some experiments.

Titanium particles

Titanium particles were obtained from Johnson Matthey Chemicals (Ward Hill, MA, USA) and contain 1- to 3-μm-diameter particles. Particles were suspended in PBS and autoclaved for sterilization. In addition, particles were also prepared by alternately washing three times in nitric acid and sodium hydroxide solutions, and then washing three times in sterile PBS as described by Bi et al.(30) Experiments showed similar induction of RANKL with washed and unwashed particles, and the data shown use unwashed titanium particles. For use in cell culture, the particles were suspended in PBS at 1 × 108/ml. As a final check on particle size and count, the suspension was evaluated by Coulter ZM Channelizer analysis.

FACS analysis

Untreated fibroblasts and fibroblasts treated with mouse TNF-α (R&D) for 24 h were washed and harvested in PBS containing 5 mM EDTA at 4°C. Cells (1 × 106) were stained with biotin conjugated antibody specific for anti-mouse vascular cell adhesion molecule (VCAM)-1 and fluorescein isothiocyanate (FITC)-conjugated streptavidin (Pharmingen, San Diego, CA, USA). Isotype-matched monoclonal antibodies were used as negative controls (Pharmingen). A total of 1 × 104 stained cells was assessed by a FACSCalibur cytometer and the Cell Quest plotting program (Becton Dickinson, Franklin Lakes, NJ, USA).

RT-PCR and quantitative real-time PCR

Total RNA was isolated from cell cultures at various times using the RNeasy kit (Qiagen, Valencia, CA, USA) and was reverse transcribed to cDNAs using Superscript II according to manufacturer's instructions (Invitrogen, Carlsbad, CA, USA). Primers specific for murine RANKL, COX-2, GAPDH, and the EP receptors were used. For quantitative real-time PCR, RANKL was amplified using 5′-CACCATCAGCTGAAGATAGT-3′; 5′-CCAAGATCTCTAACATGACG-3′, COX-2 was amplified using 5′-CACAGCCTACCAAAACAGCCA-3′; 5′-GCTCAGTTGAACGCCTTTTGA-3′, and GAPDH was amplified using 5′-AACGACCCCTTCATTGAC-3′; 5′-TCCACGACATACTCAGCAC-3′) primer sets. Duplicate PCR reactions were carried out in Rotor-Gene 3000 (Corbett Research) using n = 3 for each sample. SYBR green dye was used for detection of the product using the SYBR Green PCR Master Mix assay (Applied Biosystems, Warrington, UK). The standard curve used a series of duplicate dilutions of plasmid for RANKL and GAPDH cDNA. The amplification reaction was performed for 45 cycles with denaturation at 95°C for 15 s, followed by annealing at 58°C for 20 s and extension and detection at 72°C for 10 s. For EP receptor expression, primers were selected to give products of similar size for the various receptors, and annealing temperature was selected for the relative same amplification efficiency. The primers were EP1 (5′-TACATGGGATGCTCGAAACA-3′; 5′-TTTTAAGCCCGTGTGGGTAG-3′), EP2 (5′-ATGCTCCTGCTGCTTATCGT-3′; 5′-TAATGGCCAGGAGAATGAGG-3′), EP3 (5′-GGATCATGTGTGTGCTGTCC-3′; 5′-AACTGGAGACAGCGTTTGCT-3′), and EP4 (5′-CCATCGCCACATACATGAAG-3′; 5′-TGCATAGATGGCGAAGAGTG-3′). PCR amplification was performed in 95°C for 15 s, 50°C for 20 s, and 72°C for 30 s for 30 cycles. The cDNA products were separated on a 1% agarose gel.

Northern blot

Fifteen micrograms of total RNA was separated on a 1.2% agarose gel containing 17.5% formaldehyde, and the RNA was transferred to a Gene Screen Plus membrane (New England Nuclear, Boston, MA, USA). After RNA cross-linking to the membrane by UV light, prehybridization was performed in QuickHyb solution (Stratagene, La Jolla, CA, USA) for 60 minutes at 68°C. A mouse cDNA probe for RANKL (from Dr Lianping Xing, Department of Pathology, University of Rochester) and the murine GAPDH probe were used.(29) The probes were labeled with [32P]deoxycytidine 5′-triphosphate using a random priming kit (Stratagene, La Jolla, CA, USA) and hybridized to the blots overnight at 68°C. The blots were washed under stringent conditions and exposed to X-OMAT AR film (Kodak, Rochester, NY, USA) for autoradiography or a phosphor storage screen for PhosphorImage (Molecular Dynamics, Sunnyvale, CA, USA).

Western blot

Total protein extracts were prepared from cells at various times in the presence of protease inhibitors as previously described.(31) Fifteen micrograms of the protein extract was separated by SDS-PAGE. After transfer to a polyvinylidene fluoride (PVDF) membrane, the blots were probed overnight with antibodies specific for murine COX-1, COX-2 (Cayman Chemicals), or β-actin (Sigma). After washing the blots, horseradish peroxidase (HRP)-conjugated secondary antibodies were used to detect the immune complexes using ECL plus (Amersham Biosciences, Piscataway, NJ, USA). For RANKL, IP-Western was performed as described.(32) Briefly, ∼500 μg of total protein extracts were immunoprecipitated by RANK-Fc recombinant protein (R&D) and protein A sepharose (Amersham Biosciences). The complex was boiled in 2× SDS sample buffer and separated by SDS-PAGE and assayed by Western blotting with anti-RANKL antibody (Calbiochem, San Diego, CA, USA). Control experiments were performed in ST-2 cells as previously described.(32)

PGE2 enzyme-linked immunosorbent assay

Levels of PGE2 in the culture supernatants were measured using commercially available enzyme-linked immunosorbent assay (EIA) kits (Cayman Chemicals) following the manufacturer's instructions, as we have described previously.(18,19) Spectrophotometry was performed at 405 nm using an automated microELISA plate reader (TECAN Sunrise, ResearchTriangle Park, NC, USA), and results were compared with standard concentrations analyzed on the same plate.


A mouse calvarial resorption model was used as previously described.(19,27) Briefly, 8-week-old female mice were anesthetized by peritoneal injection using 80 mg/kg of ketamine and 5-7 mg/kg of xylazine. A 1-cm midline sagittal incision was made, and 30 mg of titanium particles was placed evenly on top of the calvaria. After implantation, the skin was closed with 4-0 Ethilon suture. Sham controls had the surgical incision, but no particles were implanted. At day 3, mice were killed by cervical dislocation, and the calvaria were removed and fixed in 10% neutral buffered formalin. Samples were blocked with a 1:20 dilution of normal goat serum and incubated with a 1:20 dilution of primary antibody (Santa Cruz Biotech, Santa Cruz, CA, USA) overnight at 4°C. The sections were washed with PBS and incubated with a 1:200 dilution of secondary antibody followed by streptavidin-peroxidase complex, and the binding of antibody was visualized with aminoethyl carbazole (AEC; Zymed, San Francisco, CA, USA), according to the supplier instructions.

In vitro osteoclastogenesis assay

Fibroblasts (50,000 cells/well) were plated in 24-well plates overnight and treated with titanium particles (5 × 106/ml) or 1 μM PGE2 (Cayman Chemicals). After 24 h, cells were fixed in 2.5% glutaraldehyde for 1 minute, followed by the addition of 1.5% glycine for 1 minute. The cell layers were subsequently washed three times in PBS and covered with α-MEM containing 10% FBS. Spleen cells were isolated from 4-month-old C57BL/6 female mice. Isolated spleens were washed, minced, and purified by adding 0.83% NH4Cl in 10 mM Tris buffer (pH 7.4).(33) The cells were washed three times with PBS, suspended in α-MEM containing 10% FBS, 10 ng/ml macrophage-colony stimulating factor (M-CSF; R&D), and overlaid on prefixed fibroblasts cell layers. In the positive control group, media also contained soluble 50 ng/ml RANKL protein (R&D). Experiments were also performed in the presence and absence of RANK-Fc (50 ng/ml; R&D) to block RANKL signaling. Media with M-CSF with or without RANKL and RANK-Fc were changed every other day. After a 7-day culture, the osteoclast-like cells were detected by TRACP staining (Sigma). Multinucleated (>3 nuclei per cell) and TRACP+ (purple staining cells) were counted as osteoclasts.

Statistical analysis

Results are given as means ± SE. Comparisons were made by ANOVA and Student's t-test between data from the experimental and control groups. p values < 0.05 were considered statistically significant.


  1. Top of page
  2. Abstract
  7. Acknowledgements

Mouse embryonic fibroblasts stimulated with TNF-α express VCAM-1

We began by assessing the ability of the murine embryonic fibroblast cell line to express a synovial fibroblast phenotype.(28) Because VCAM1 is a marker of synovial fibroblasts,(29) we examined the expression of VCAM1 by flow cytometry in embryonic fibroblast cultures in the presence and absence of TNF-α (Fig. 1). Under basal conditions, embryonic fibroblasts express low levels of VCAM1 (Fig. 1B). However, VCAM1 is markedly induced after treatment with TNF-α (Fig. 1C). Because these findings are similar to those described with synovial fibroblasts,(29) we used the embryonic fibroblast cell line as a model to study the cell response to titanium particles in vitro.

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Figure FIG. 1.. VCAM-1 is expressed in embryonic fibroblastic cells. Immortalized embryonic fibroblasts from E18.5 mouse embryos were examined for VCAM-1 expression in the absence and presence of 10 ng/ml of TNF-α for 24 h. VCAM-1 expression was detected by FACS analysis: (A) isotype-matched antibody as negative control; (B) antibody specific for mouse VCAM-1; and (C) VCAM-1 expression in cells treated with TNF-α. MFI, mean fluorescence intensity.

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Titanium particles stimulate RANKL gene and protein expression in fibroblasts

RANKL expression was examined in fibroblast cultures to determine if these cells might directly participate in the induction of osteoclasts in response to wear debris particles. Fibroblasts were treated with titanium particles (5 × 106 particles/ml) for various times up to 24 h, and RANKL expression was determined by quantitative real-time RT-PCR. RANKL was stimulated at 1 h, and whereas peak levels occurred at 4 h (5-fold), expression remained elevated for 24 h after exposure to titanium particles (Fig. 2A). RANKL stimulation was confirmed by Northern blot after 4 and 8 h of titanium exposure (Fig. 2B). RANKL induction occurred over a broad range of particle concentrations (Fig. 2C). A small level of stimulation was observed at 5 × 105 particles and progressively increased up to a concentration of 1 × 107 particles, where maximal stimulation was observed (Fig. 2C). Because more than a 5-fold stimulation was observed with concentrations of 5 × 106 particles, this concentration was used in subsequent experiments.

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Figure FIG. 2.. RANKL expression is induced by titanium particles in fibroblastic cells. Fibroblasts were placed in subconfluent monolayer cell culture and treated with titanium particles. (A) After treatment with 5 × 106 particles/ml, total RNA was harvested at various times ranging from 15 minute to 24 h, and RANKL expression was examined by real time RT-PCR. (B) Total RNA was separated by gel electrophoresis, and Northern blot was performed to confirm the induction of RANKL by 5 × 106 particles/ml titanium particles at 4 and 8 h after treatment. (C) Total RNA was collected, and RANKL expression was examined by Northern blot after 4 h of treatment with various concentrations of titanium particles. Total cellular protein extracts were obtained from cell cultures treated with either control medium or medium containing 5 × 106 particles/ml for 0, 8, 24 or 48 h. RANKL was immuno-precipitated using recombinant RANK-Fc. The precipitate was separated by SDS-PAGE and immunostain performed with anti-RANKL antibody. (D) ST-2 cells treated with vitamin D3 and dexamethasone were used as a positive control. The arrows point to RANKL.

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To determine whether induction of RANKL gene expression is associated with an increase in protein expression, whole cell lysates were examined by immunoprecipitation-Western (IP-Western). The mouse stromal cell line ST-2 was used as a control for RANKL protein induction. After 48 h of treatment with 10−8 M 1,25(OH)2D3 and 10−6 M dexamethasone, RANKL protein was markedly enhanced in ST-2 cells (Fig. 2D, left), as previously described.(32) Whole cell extracts from fibroblast cultures treated with titanium particles for 0, 8, 24, and 48 h were similarly harvested and subjected to IP-Western for RANKL expression. Similar to ST-2 cells, RANKL protein was undetected under basal conditions but was induced by 8 h with maximal levels occurring after 48 h (Fig. 2D, right). Altogether, the findings show that titanium particles activate fibroblasts to produce RANKL, an important osteoclastogenic factor.

Titanium particles induce COX-2 and stimulate PGE2 secretion in fibroblasts

Prior work in our laboratory established that particles induce PGE2 in fibroblast cultures and suggested a COX-2-mediated effect.(18) For this reason, we examined whether titanium particles induced COX-2 gene and protein expression in fibroblast cultures (Fig. 3). Within 30 minutes after exposure to 5 × 106 titanium particles/ml, COX-2 gene expression was induced 2-fold, and a maximal 12-fold increase was present by 2 h (Fig. 3A). Under basal conditions, COX-2 protein expression was undetectable, but expression was observed within 1 h of exposure to titanium particles (Fig. 3B). Maximal levels were present by 2 h and were similar at 4 h after treatment. In contrast, COX-1 expression was constitutive and was not altered after treatment with titanium particles. β-Actin Western blot was used as a loading control and was similar in all of the extracts (Fig. 3B). Finally, COX-2 induction was associated with a massive accumulation of PGE2 in the conditioned medium (Fig. 3C).

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Figure FIG. 3.. Titanium particles induce COX-2 expression and PGE2 secretion in fibroblasts. Fibroblasts were placed in subconfluent monolayer cell culture and treated with control medium or 5 × 106 titanium particles/ml. (A) Total RNA was harvested at various times ranging from 15 minutes to 24 h, and COX-2 expression was examined by real time RT-PCR. Total cellular protein extracts were obtained from cell cultures after 0, 0.5, 1, 2, and 4 h. (B) The protein extract was separated by SDS-PAGE, and immunostain was performed with anti-COX-1 antibody, anti-COX-2 antibody, or anti-β actin antibody. (C) Conditioned medium was obtained at various times after titanium treatment, and PGE2 levels were determined by enzyme-linked immunosorbent assay.

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RANKL induction in fibroblasts is mediated by PGE2

Fibroblasts were treated with titanium particles in the presence and absence of combinations of indomethacin and PGE2 to determine if PGE2 is involved in the induction of RANKL (Fig. 4A). Indomethacin was selected because it inhibits both COX-1 and COX-2 enzyme function.(34,35) As previously shown, 4 h of treatment with titanium particles induced RANKL expression. However, treatment with indomethacin completely blocked RANKL induction by titanium, whereas addition of 10−6 M PGE2 restored the expression of RANKL in cultures treated with titanium particles and indomethacin. The level of RANKL expression under these conditions was similar to that observed in fibroblast cultures treated with PGE2 alone and slightly more than that which occurred after titanium treatment (Fig. 4A).

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Figure FIG. 4.. RANKL induction in fibroblasts is mediated by PGE2. Fibroblast cultures were pretreated with control medium or with medium containing indomethacin (1 μM) for 1 h. Cultures were treated with or without 5 × 106 particles/ml in the presence and absence of PGE2 (10−6 M). (A) Total RNA was harvested at 4 h, and RANKL expression was examined by Northern blot. Fibroblast cultures were pretreated with or without the COX-2-specific inhibitor celecoxib for 1 h, followed by exposure to 5 × 106 titanium particles/ml for 4 h. (B) Total RNA was harvested, and RANKL expression was assessed by Northern blot. (C) Fibroblast cultures were treated with PGE2 (10−6 M), IL-6 (100 ng/ml), IL-1 (100 ng/ml), TNF-α (10 ng/ml), or control medium for 4 h, and RANKL expression was assessed by Northern blot. Total cellular protein extracts were obtained from fibroblast cell cultures treated with control medium or medium containing PGE2 (10−6 M) for 24 h and ST-2 cells treated with 1,25(OH)2D3 and dexamethasone for 48 h. RANKL was immunoprecipitated using recombinant RANK-Fc. (D) The precipitate was separated by SDS-PAGE, and immunostain was performed with anti-RANKL antibody. The arrow points to RANKL.

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To determine if RANKL stimulation by titanium particles is related to the increase in COX-2 expression, fibroblast cultures were treated with titanium particles for 4 h in the presence and absence of the COX-2-specific inhibitor, celecoxib. Celecoxib markedly reduced the induction of RANKL consistent with a COX-2-mediated effect (Fig. 4B). Because the interfascial membrane adjacent to loose prosthetic joints contains high levels of proinflammatory cytokines, the ability of PGE2 to induce RANKL expression in comparison with the osteoclastogenic factors IL-1, IL-6, and TNF-α was assessed (Fig. 4C).(9,36) IL-1 and IL-6 had no effect, whereas TNF-α resulted in a small increase in RANKL expression in the fibroblast cultures.

Finally, to confirm the role of PGE2 in the induction of RANKL, total protein extracts from control and PGE2-treated fibroblast cultures were examined by IP-Western blot (Fig. 4D). Twenty-four hours of treatment with PGE2 resulted in a marked induction of RANKL protein. The level of induction was comparable with the degree of protein expression that occurred in ST-2 cell cultures treated with 1,25(OH)2D3 and dexamethasone for 48 h. Altogether, the findings show that PGE2 is induced by titanium particles through COX-2 and is a powerful stimulator of RANKL in fibroblasts.

Titanium particles stimulate RANKL induction in cultures of murine knee synovial fibroblasts and in calvarial tissues in vivo

To verify the induction of RANKL expression by titanium in synovial fibroblasts, experiments were repeated in authentic fibroblast-like synoviocytes (FLSs) obtained from the murine knee joint.(29) Titanium and PGE2 both induced RANKL expression in FLS cultures between 9- and 11-fold, and the induction by titanium was inhibited by the addition of indomethacin (Fig. 5A). The real time RT-PCR findings were confirmed by Northern blot (Fig. 5B). Thus, the findings are identical to those observed in the embryonic fibroblasts.

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Figure FIG. 5.. Titanium induction of RANKL in knee joint-derived synovial fibroblasts and in calvarial tissues. Fibroblast-like synoviocytes previously obtained from the murine knee joint and characterized were cultured and pretreated with control medium or with medium containing indomethacin (1 μM) for 1 h. Cultures were treated with or without 5 × 106 particles/ml or with PGE2 (10−6 M). (A) Total RNA was harvested at 4 h, and RANKL expression was examined by real time RT-PCR. (B) Total RNA was isolated after 4 h in a separate experiment with similar treatments, and Northern blot was performed. An incision was made overlying the calvaria and closed (Sham) or implanted (Ti) with 30 mg of titanium particles. (C) After 3 days, mice were killed, the calvaria tissues were fixed and sectioned, and immunohistochemistry was performed to assess the expression of RANKL.

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Additionally, experiments were performed using immunohistochemistry to localize RANKL in the inflammatory tissues adjacent to the calvaria of sham-operated mice and mice 3 days after the implantation of titanium particles onto the calvarial surface. In sham-operated mice, minimal or no RANKL was observed, whereas RANKL staining was readily apparent in the soft tissues overlying the calvaria implanted with titanium particles (Fig. 5C). The staining was present in fibroblasts adjacent to the calvarial surface and was consistent with the abundant bone resorption that occured 5 days after titanium implantation in vivo and its inhibition by the RANKL antagonists, RANK-Fc and OPG.(27,37)

Titanium particle and PGE2 activated fibroblasts directly support osteoclastogenesis in vitro

To determine the functional significance of fibroblast RANKL expression, an in vitro osteoclast formation assay was performed using glutaraldehyde-fixed fibroblasts co-cultured with primary murine splenocytes in the presence of 10 ng/ml M-CSF (Fig. 6). Subconfluent fibroblast cell cultures were treated with control medium or medium containing PGE2 (10−6 M) or titanium particles (5 × 106 particles/ml). After 24 h, the cell layers were fixed with glutaraldehyde and glycine and were overlaid with primary murine splenocytes. Splenocytes cultured alone in the absence of recombinant RANKL failed to form multinucleated TRACP+ cells (Fig. 6A). Co-culture with fibroblasts treated in the presence of control medium similarly resulted in minimal osteoclast formation (Fig. 6B), whereas RANKL addition as a positive control resulted in the abundant formation of large multinucleated TRACP+ cells (Fig. 6C). When fibroblasts were treated with either titanium or PGE2 for 24 h before glutaraldehyde fixation, a significant stimulation of multinucleated TRACP+ cells was observed in the co-cultures (Figs. 6D and 6E). However, whereas the addition of recombinant RANKL resulted in enormous cells, with as many as 50 or 100 nuclei, the multinucleated TRACP+ cells that were formed in the titanium and PGE2 co-cultures were substantially smaller. The mean numbers of multinucleated TRACP+ cells that formed in 7-day splenocyte cultures are summarized in Fig. 6F. The findings show that fibroblasts treated with either titanium or PGE2 support osteoclast formation independent of the addition of recombinant RANKL.

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Figure FIG. 6.. PGE2 and titanium-stimulated fibroblasts induce osteoclast formation in vitro. Fibroblasts were plated in subconfluent conditions and were treated with control medium or with medium containing PGE2 (10−6 M) or titanium particles (5 × 106 particles/ml). After 24 h, the cells were rinsed and fixed with 2.5% glutaraldehyde for 1 minute followed by the addition of 1.5% glycine for 1 minute. Murine splenocytes were cultured on either control plates or on the prefixed fibroblast cell layers in MEM containing 10 ng/ml M-CSF. Osteoclasts were detected by TRACP stain after 7 days of culture in the presence or absence of recombinant RANKL. The various panels represent (A) splenocytes alone; (B) splenocytes + untreated fibroblasts; (C) splenocytes + untreated fibroblasts with RANKL added to the medium; (D) splenocytes + fibroblasts pretreated with titanium particles; and (E) splenocytes + fibroblasts pretreated with PGE2. (F) Multinucleated (>3 nuclei), TRACP+ cells present in each well were counted, and the mean number (n = 6 wells) are depicted. Statistical significance was determined by t-test, and comparisons are shown with regard to the untreated fibroblast and splenocyte culture. *p < 0.05.

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To confirm the role of RANKL in this model of osteoclastogenesis, fibroblasts were treated with PGE2, titanium particles, or control medium and fixed with glutaraldehyde. Splenocytes were added to the fibroblast cultures in the presence or absence of the RANKL antagonist, RANK-Fc. In these experiments, RANKL was added only to the cultures containing fibroblasts treated with control medium (Fig. 7A). Whereas osteoclasts formed in all of the cultures in the absence of RANK-Fc, the addition of this factor markedly inhibited osteoclast formation (Figs. 7A and 7B).

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Figure FIG. 7.. RANK-Fc inhibits the ability of PGE2 and titanium-stimulated fibroblasts to induce osteoclast formation in vitro. Fibroblasts were plated in subconfluent conditions and were treated with control medium or with medium containing PGE2 (10−6 M) or titanium particles (5 × 106 particles/ml). After 24 h, the cells were rinsed and fixed with 2.5% glutaraldehyde for 1 minute followed by the addition of 1.5% glycine for 1 minute. Murine splenocytes were cultured on the pretreated and fixed fibroblast cell layers either under control conditions (MEM containing 10 ng/ml M-CSF) or in the presence of RANK-Fc. RANKL was added to cultures containing fixed fibroblasts that had bee pretreated with control medium. (A) Osteoclasts were detected by TRACP stain after 7 days of culture. (B) Multinucleated (>3 nuclei) TRACP+ cells present in each well were counted, and the mean number (n = 6 wells) are depicted. OCP, splenocytes; SF, synovial fibroblasts; Ti, titanium pretreatment of fibroblast cultures; PGE2, pretreatment of fibroblast cultures; RANK Ligand, addition of RANKL to the co-cultures containing fibroblasts pretreated with control medium; RANK-Fc, addition to co-cultures after the various pretreatments. Statistical significance was determined by t-test and, comparisons are shown with regard to the untreated fibroblast and splenocyte culture (*p < 0.05). Comparisons between cultures receiving similar fibroblast pretreatment with or without the addition of RANK-Fc to the co-cultures are shown (#p < 0.05).

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RANKL is induced by specific EP receptors in fibroblasts

Four different receptors (EP1-4) specific for PGE2 have been described and mediate separate downstream signaling events to influence gene expression.(38–40) RT-PCR was performed to examine which of the EP receptors are expressed on fibroblasts (Fig. 8A). After 30 cycles of amplification, EP3 expression was absent, and low levels of EP1 and EP2 were observed. Substantially higher levels of EP4 expression was observed, suggesting that this is the major receptor subtype found on these cells.

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Figure FIG. 8.. RANKL is induced by specific EP receptors in fibroblasts. Total RNA was isolated from subconfluent fibroblast cultures and specific primers for the prostaglandin receptors EP1, EP2, EP3, and EP4 were used for RT-PCR. (A) The products were analyzed after 30 cycles of PCR amplification by gel electrophoresis. Fibroblast cultures were treated with control medium, PGE2 (10−6 M), iloprost (2 μM), or misoprostol (1 μM) for 4 h. (B) Total RNA was harvested, and RANKL expression was assessed by Northern blot. (C) Fibroblast cultures were treated with PGE2 in the presence or absence of either H-89 or Go6976 for 4 h, total RNA was harvested, and RANKL expression was assessed by Northern blot.

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Using receptor agonists with relative specificity, the functional role of the various receptors in RANKL induction was assessed (Fig. 8B). As previously observed, PGE2, which binds to all of the receptors, results in a large induction of RANKL. Iloprost, which binds to the EP1 receptor, and also has affinity for the EP3 and IP receptors, caused only a small increase in RANKL.(41–44) Similarly, the EP2 receptor agonist, butaprost,(41,45) minimally induced RANKL. However, misoprostol, an activator of both EP2 and EP4 receptors, stimulated RANKL to a magnitude similar to that observed with PGE2.(41) Altogether, the findings show that the EP4 receptor is the most abundant isoform expressed by fibroblasts and that induction of RANKL is mediated by activation of this receptor isoform.

Because the EP4 receptor activates adenylate cyclase and PKA signaling,(46) the effect of inhibition of PKA (H89, 10 μM) and PKC (Go6976, 10 μM) signaling on PGE2 effects on fibroblasts was examined (Fig. 8C). Whereas the induction of RANKL by PGE2 was strongly inhibited by the H89, weak repression was observed with PKC inhibition.


  1. Top of page
  2. Abstract
  7. Acknowledgements

These experiments define the fibroblast as an important mediator osteolysis adjacent to joint replacements. After stimulation with titanium particles, fibroblasts synthesize COX-2, secrete prostaglandins, and express RANKL. The induction of RANKL was functionally important because glutaraldehyde-fixed fibroblasts stimulated with titanium particles directly induced osteoclast formation. The induction of RANKL by particles could be blocked by both the nonselective nonsteroidal anti-inflammatory drug indomethacin and by the COX-2-specific inhibitor celecoxib and could be compensated for by the addition of PGE2 to the cultures. Analysis of EP receptor expression and the use of EP receptor agonists implicate the EP4 receptor as the major mediator of the prostaglandin effect. Inhibition of PKA signaling caused a strong suppression of RANKL in response to PGE2, consistent with an EP4 signaling event. Finally, addition of RANK-Fc to fibroblast-splenocyte co-cultures blocked osteoclast formation. Altogether the findings suggest that fibroblasts in the periprosthetic membrane are important targets in the cell and molecular events that regulate bone loss around total joint arthroplasties. The data also account for the lack of in vivo osteolysis observed in mice genetically deficient for COX-2 or RANK and wildtype mice treated with COX-2 or RANKL inhibitors.(19,27,37)

Multiple methods were used to confirm the induction of RANKL by titanium particles, including real time RT-PCR, Northern blot, and Western blot, and showed that the stimulation of the RANKL gene is linked to an increase in protein expression. Furthermore, fibroblasts stimulated with both titanium particles and PGE2 were able to induce osteoclast formation in splenocyte cultures in the presence of M-CSF. Because RANKL is necessary for the induction of osteoclasts, this is further evidence supporting the increase in expression and activity of this important mediator. Confirmatory evidence for the role of RANKL was provided by experiments showing inhibition of osteoclast formation in RANKL, PGE-2, and titanium treated cultures in the presence of RANK-Fc.

PGE2 is the primary product of arachidonic acid metabolism in many cells.(47) The COX enzymes mediate a critical step in prostaglandin biosynthesis and include two isoforms, COX-1, which is constitutive, and COX-2, which is inducible.(47–49) Nonsteroidal anti-inflammatory drugs, which block both COX-1 and COX-2, have been used successfully to reduce inflammatory responses to a variety of different stimuli.(49) More recently, specific inhibitors for COX-2 have been introduced that have the ability to selectively inhibit the COX-2 isoform and avoid potential complications associated with the blockade of COX-1-mediated events.(48,50)

Prior work in our laboratory showed an induction in PGE2 after treatment with titanium particles and suggested a COX-2-mediated effect.(18) Here we show that both COX-2 gene and protein expressions are induced by titanium particles. The initial increase in gene expression is associated with a subsequent elevation in protein levels and PGE2 secretion within 1 h after titanium exposure. The rapid response suggests a direct effect on gene transcription and is consistent with a role for COX-2 at the apex of a response leading to RANKL induction. Further evidence for the role of COX-2 is provided by the finding that both nonselective COX inhibition and selective COX-2 inhibition blocked RANKL stimulation by particles. Finally, the findings are consistent with prior work in our laboratory showing the requirement of COX-2 for in vivo bone loss in a murine model of titanium-induced osteolysis.(19)

There are four different types of G protein-coupled receptors for PGE2.(38,39) The various EP receptors activate different downstream signaling events and are differentially expressed on various cells.(38–40) EP1 is linked with Gq/p and stimulates phospholipase C activity and results in calcium transients and enhanced PKC signaling.(51) EP2 and EP4 are coupled with Gsα and stimulate adenylate cyclase to trigger the cAMP-PKA signaling pathway.(46) In contrast, the EP3 isoforms (α,β,γ) are primarily coupled to Gi and act to inhibit cAMP-PKA signaling, and thus have effects opposite those of the EP2 and EP4 receptors.(39,40)

Relatively low levels of EP1 and EP2 expression were present, EP3 receptor expression was not observed, and EP4 was the most abundantly expressed PGE2 receptor isoform. The importance of the EP4 receptor is supported by the induction of RANKL by misoprostol (EP2 and EP4 receptor agonist) to a level similar to that observed with PGE2. In contrast, the EP1 receptor agonist, iloprost, and the EP2 specific agonist, butaprost, minimally induced RANKL.(41) Altogether the findings most strongly implicate the EP4 receptor as the major target of COX/PGE2 effects on fibroblasts. However, our data do not exclude the possible contribution of other prostaglandin metabolites in the induction of RANKL downstream of COX-2, along with other potential receptors.

For these studies, we used an embryonic fibroblast cell line derived from 18-day-old embryos.(28) The cells represent a useful model because they respond to the inflammatory mediator, TNF-α, with an induction in VCAM1 expression as measured by FACS analysis. Thus, the cells express a synovial fibroblast phenotype.(52–54) The advantage of a cell line is that it allows analysis of critical intracellular signaling events in a stable population of cells over time. Confirmatory experiments were performed using synovial-like fibroblasts derived from the murine knee joint(29) and also showed a marked induction in RANKL expression by titanium particles and PGE2. Recent work has shown that fibroblasts in human periprosthetic tissues express RANKL, and we obtained similar findings in immunohistochemical stains of murine calvarial tissues 3 days after the implantation of titanium particles. Altogether these studies show a role for COX-2/PGE-2/EP4/PKA-mediated signals in the induction of RANKL.(20,21)

The commercially available titanium particles used in the experiments have previously been established as a model to study the biological response to debris particles both in vitro and in vivo.(18,27,30,55–57) Qualitatively similar cell-mediated inflammatory responses occur with a variety of particles, including native particles isolated from periprosthetic tissues as well as commercially prepared metal, polymethylmethacrylate, and polyethylene particles.(58,59) Whereas macrophages phagocytose particles, controversy remains regarding whether phagocytosis is required to incite the inflammatory response.(57,60) There is considerably less information regarding fibroblasts.(17,58) Whereas this study focused on particle-mediated RANKL induction and showed a mechanistic role for COX-2, PGE2, and subsequent signals, the mechanisms through which particles initiate and activate a cellular response in fibroblasts remains unknown.

Osteoclast formation requires interaction between stromal cells and preosteoclasts that are derived from cells of the macrophage/monocyte lineage.(61) Murine splenocyte cultures provide abundant preosteoclasts, and in the presence of M-CSF and RANKL, these cells readily form large, TRACP+ multinucleated osteoclasts.(33) The number of nuclei and the size of the cells that form with the addition of soluble recombinant RANKL are unique to cell culture and are not found under in vivo conditions. In contrast, the TRACP+ multinucleated osteoclasts that form in fibroblast co-cultures were smaller, but more closely resembled the size of osteoclasts found in vivo. The morphology of these cells was also similar to osteoclasts produced in fibroblast co-cultures by others.(62) The smaller number of osteoclasts in the co-cultures is likely caused by reduced concentrations and/or availability of expressed RANKL on the surface of the fibroblasts.

Various stromal cell populations express RANKL. RANKL is induced in osteoblasts by PTH, vitamin D3, and IL-6(22,61,63,64) and is inhibited by TGF-β.(65) Similar to our findings in fibroblasts, RANKL is induced in murine osteoblast cultures by PGE2 primarily through an EP4 receptor mechanism.(66,67) In human periodontal ligament fibroblasts, RANKL is induced by lipopolysaccharide (LPS) through a mechanism that involves the induction of IL-1 and TNF-α.(68) However, substantially less in known about factors regulating RANKL expression in fibroblast-like synoviocytes. Cultured rheumatoid fibroblast-like synoviocytes expressed RANKL in response to vitamin D3.(16) However, whereas high levels of RANKL are present in rheumatoid synovial tissue, minimal expression occurs in osteoarthritic synovium.(16) Thus, it is likely that synovial cells and tissue are involved in the pathogenesis of inflammatory bone resorption.(11,15) This study is the first directly showing RANKL stimulation in fibroblasts in response to titanium particles and that PGE2 is a stronger stimulus than the proinflammatory cytokines, IL-1, IL-6, and TNF-α, which are more frequently associated with osteolysis.

Despite the fact that proinflammatory cytokines are highly expressed in inflammatory tissues, little is known regarding the role of these factors in RANKL induction in synovial fibroblasts. Sen et al.(69) used human rheumatoid fibroblast-like synoviocytes to study the effects of Wnt 5A on IL-6, IL-15, and RANKL expression. Whereas inhibition of Wnt signaling blocked IL-6, IL-15, and RANKL expression, the effect of IL-6 on RANKL expression was not examined. Quinn et al.(62) showed that 1,25(OH)2D3 induced RANKL in skin fibroblasts and enhanced osteoclast formation in splenocyte and fibroblast co-culture. Although the effects of other factors on RANKL expression were not directly studied, the relative ability of TNF-α, IL-l, PTH-related protein (PTHrP), IL-11, PGE2, and vitamin D to induce osteoclast formation in co-culture was examined. IL-1 and TNF-α minimally increased the number of osteoclasts and were markedly less stimulatory than PGE2. Thus, although the information is indirect, it suggests that the proinflammatory cytokines minimally induce RANKL compared with PGE2 and are consistent with our findings.

Thus, evidence is accumulating that fibroblasts can regulate bone remodeling, particularly in the setting of inflammatory joint disease.(15,53) Synovial fibroblasts are the most abundant cell type found in the inflammatory membrane adjacent to loose implants and have extensive contact with wear debris particles.(9,10) Previously we have shown that wear debris-induced osteolysis is ablated in COX-2−/− mice and in wildtype mice with COX-2 inhibition.(19) Here we further our studies by implicating the synovial fibroblast as a potential target of COX-2 inhibition. The findings define the PGE2/EP4/PKA pathway as a potentially important target to regulate particle mediated osteolysis and have relevance for inflammatory bone loss in general.


  1. Top of page
  2. Abstract
  7. Acknowledgements

The authors would like to acknowledge the help of Christine Clark and Lin Ma. Public Health Service Award AR46545 supported the experiments.


  1. Top of page
  2. Abstract
  7. Acknowledgements
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