RNA-seq analysis of chondrocyte transcriptome reveals genetic heterogeneity in LG/J and SM/J murine strains.

OBJECTIVE: To investigate the transcriptomic differences in chondrocytes obtained from LG/J (large, healer) and SM/J (small, non-healer) murine strains in an attempt to discern the molecular pathways implicated in cartilage regeneration and susceptibility to osteoarthritis (OA). DESIGN: We performed RNA-sequencing on chondrocytes derived from LG/J (n = 16) and SM/J (n = 16) mice. We validated the expression of candidate genes and compared single nucleotide polymorphisms (SNPs) between the two mouse strains. We also examined gene expression of positional candidates for ear pinna regeneration and long bone length quantitative trait loci (QTLs) that display differences in cartilaginous expression. RESULTS: We observed a distinct genetic heterogeneity between cells derived from LG/J and SM/J mouse strains. We found that gene ontologies representing cell development, cartilage condensation, and regulation of cell differentiation were enriched in LG/J chondrocytes. In contrast, gene ontologies enriched in the SM/J chondrocytes were mainly related to inflammation and degeneration. Moreover, SNP analysis revealed that multiple validated genes vary in sequence between LG/J and SM/J in coding and highly conserved noncoding regions. Finally, we showed that most QTLs have 20-30% of their positional candidates displaying differential expression between the two mouse strains. CONCLUSIONS: While the enrichment of pathways related to cell differentiation, cartilage development and cartilage condensation infers superior healing potential of LG/J strain, the enrichment of pathways related to cytokine production, immune cell activation and inflammation entails greater susceptibility of SM/J strain to OA. These data provide novel insights into chondrocyte transcriptome and aid in identification of the quantitative trait genes and molecular differences underlying the phenotypic differences associated with individual QTLs.

Notch signaling in postnatal joint chondrocytes, but not subchondral osteoblasts, is required for articular cartilage and joint maintenance.

OBJECTIVE: Notch signaling has been identified as a critical regulator in cartilage development and joint maintenance, and loss of Notch signaling in all joint tissues results in an early and progressive osteoarthritis (OA)-like pathology. This study investigated the targeted cell population within the knee joint in which Notch signaling is required for normal cartilage and joint integrity. METHODS: Two loss-of-function mouse models were generated with tissue-specific knockout of the core Notch signaling component, RBPjκ. The AcanCre(ERT2) transgene specifically removed Rbpjκ floxed alleles in postnatal joint chondrocytes, while the Col1Cre(2.3kb) transgene deleted Rbpjκ in osteoblast populations, including subchondral osteoblasts. Mutant and control mice were analyzed via histology, immunohistochemistry (IHC), real-time quantitative polymerase chain reaction (qPCR), X-ray, and microCT imaging at multiple time-points. RESULTS: Loss of Notch signaling in postnatal joint chondrocytes results in a progressive OA-like pathology, and triggered the recruitment of non-targeted fibrotic cells into the articular cartilage potentially due to mis-regulated chemokine expression from within the cartilage. Upon recruitment, these fibrotic cells produced degenerative enzymes that may lead to the observed cartilage degradation and contribute to a significant portion of the age-related OA-like pathology. On the contrary, loss of Notch signaling in subchondral osteoblasts did not affect normal cartilage development or joint maintenance. CONCLUSIONS: RBPjκ-dependent Notch signaling in postnatal joint chondrocytes, but not subchondral osteoblasts, is required for articular cartilage and joint maintenance.

BMP-2 induces ATF4 phosphorylation in chondrocytes through a COX-2/PGE2 dependent signaling pathway.

OBJECTIVE: Bone morphogenic protein (BMP)-2 is approved for fracture non-union and spine fusion. We aimed to further dissect its downstream signaling events in chondrocytes with the ultimate goal to develop novel therapeutics that can mimic BMP-2 effect but have less complications. METHODS: BMP-2 effect on cyclooxygenase (COX)-2 expression was examined using Real time quantitative PCR (RT-PCR) and Western blot analysis. Genetic approach was used to identify the signaling pathway mediating the BMP-2 effect. Similarly, the pathway transducing the PGE2 effect on ATF4 was investigated. Immunoprecipitation (IP) was performed to assess the complex formation after PGE2 binding. RESULTS: BMP-2 increased COX-2 expression in primary mouse costosternal chondrocytes (PMCSC). The results from the C9 Tet-off system demonstrated that endogenous BMP-2 also upregulated COX-2 expression. Genetic approaches using PMCSC from ALK2(fx/fx), ALK3(fx/fx), ALK6(-/-), and Smad1(fx/fx) mice established that BMP-2 regulated COX-2 through activation of ALK3-Smad1 signaling. PGE-2 EIA showed that BMP-2 increased PGE2 production in PMCSC. ATF4 is a transcription factor that regulates bone formation. While PGE2 did not have significant effect on ATF4 expression, it induced ATF4 phosphorylation. In addition to stimulating COX-2 expression, BMP-2 also induced phosphorylation of ATF4. Using COX-2 deficient chondrocytes, we demonstrated that the BMP-2 effect on ATF4 was COX-2-dependent. Tibial fracture samples from COX-2(-/-) mice showed reduced phospho-ATF4 immunoreactivity compared to wild type (WT) ones. PGE2 mediated ATF4 phosphorylation involved signaling primarily through the EP2 and EP4 receptors and PGE2 induced an EP4-ERK1/2-RSK2 complex formation. CONCLUSIONS: BMP-2 regulates COX-2 expression through ALK3-Smad1 signaling, and PGE2 induces ATF4 phosphorylation via EP4-ERK1/2-RSK2 axis.

Runx2-mediated activation of the Bax gene increases osteosarcoma cell sensitivity to apoptosis.

The Runx family of transcription factors regulate cell growth and differentiation, and control the expression of target genes involved in cell fate decisions. We examined the role of the bone-related member of this family, Runx2, in regulating apoptosis via modulation of the Bcl2 family of genes in the osteosarcoma cell line Saos2. Our data demonstrate that Runx2 directly binds to two Runx-specific regulatory elements on the human bax promoter thereby inducing Bax expression. Furthermore, bone morphogenetic protein-induced or vector-mediated expression of Runx2 resulted in upregulation of Bax expression, and subsequent increased sensitivity of Saos2 cells to apoptosis. Finally, the observed upregulation of Bax expression and increased apoptosis were Runx2 dependent as Runx2 loss of function abrogated these effects. Our study provides the first evidence for Bax as a direct target of Runx2, suggesting that Runx2 may act as a proapoptotic factor in osteosarcoma cells.

Tamoxifen-inducible Cre-recombination in articular chondrocytes of adult Col2a1-CreER(T2) transgenic mice.

OBJECTIVE: To determine the specificity and efficiency of the tamoxifen (TM)-induced Cre-recombination in articular chondrocytes of adult Col2a1-CreER(T2) transgenic mice. METHODS: Col2a1-CreER(T2) transgenic mice were bred with Rosa26 reporter mice. Two-week-old Col2a1-CreER(T2);R26R mice were administered TM for 5 days and were sacrificed 1 and 6 months after TM induction. X-Gal staining was performed. RESULTS: Efficient Cre-recombination is achieved in adult articular chondrocytes 1 and 6 months after TM induction. CONCLUSION: Our findings demonstrate that the Col2a1-CreER(T2) transgenic mouse model is a valuable tool to target genes specifically expressed in articular chondrocytes in a temporally controlled manner in adult mice.

RANK, RANKL and OPG in inflammatory arthritis and periprosthetic osteolysis.

Elucidation of the receptor activator of nuclear factor kappa B (RANK), its ligand (RANKL) and osteoprotegerin (OPG) as the final effectors of bone resorption has transformed our understanding of metabolic bone diseases and revealed novel therapeutic targets. Activation of the RANK-RANKL signaling pathway is directly responsible for dramatic focal erosions that are observed in inflammatory arthritis and aseptic loosening of orthopaedic implants. While these conditions share many features common to all metabolic bone disorders (e.g., osteoclastic resorption), they exhibit several unique properties, which are highlighted in this review. Most important is the relative inability of bisphosphonate therapy to inhibit osteolysis in joint inflammation and periprosthetic joint loosening and the unexpected effectiveness of anti-cytokine therapy in both rheumatoid and psoriatic arthritis. Herein, we provide a review of the role of RANK, RANKL and OPG in erosive arthritis and periprosthetic osteolysis and discuss the potential of anti-RANKL therapy for these conditions.

In vivo gene delivery to articular chondrocytes mediated by an adeno-associated virus vector.

PURPOSES: (1) To investigate the efficiency of direct in vivo adeno-associated virus (AAV) vector-mediated gene transduction to chondrocytes in relation to normal and injured articular cartilage. (2) To evaluate the effects of ultra-violet light-activated gene transduction (LAGT) in chondrocytes in vivo. (3) To determine dissemination of active rAAV vector after intra-articular administration. METHODS: Rabbit knees with either normal or injured cartilage received an intra-articular injection with 1.5x10(12) infectious rAAV-eGFP particles. The right knees received rAAV-eGFP alone, whereas the left knees were given LAGT-treatment. The transduction efficiencies were determined at 1 and 3 weeks after infection by fluorescence-activated cell scanning. The occurrence of active shedding was monitored in serum and various tissues. RESULTS: After 1 week, 7% of the chondrocytes in normal cartilage were transduced by direct rAAV transduction technique. Chondrocytes in cartilage defects demonstrated higher transduction rates compared to chondrocytes in normal cartilage. LAGT increased the cellular eGFP expression in the internal zones to 12%, but did not have any effect in the external zones in defects. Finally, infectious particles were not detected in either serum or tissue samples. CONCLUSIONS: Direct rAAV-mediated gene transfer in vivo to articular chondrocytes is possible. LAGT improves rAAV transduction of chondrocytes in vivo but appears to have a very limited range of effect induction. Expression of eGFP was not determined in other tissues than synovium and cartilage in the treated joints.

Light-activated gene transduction of recombinant adeno-associated virus in human mesenchymal stem cells.

Deficiencies in skeletal tissue repair and regeneration lead to conditions like osteoarthritis, osteoporosis and degenerative disc disease. While no cure for these conditions is available, the use of human bone marrow derived-mesenchymal stem cells (HuMSCs) has been shown to have potential for cell-based therapy. Furthermore, recombinant adeno-associated viruses (rAAV) could be used together with HuMSCs for in vivo or ex vivo gene therapy. Unfortunately, the poor transduction efficiency of these cells remains a significant obstacle. Here, we describe the properties of ultraviolet (UV) light-activated gene transduction (LAGT) with rAAV in HuMSCs, an advance toward overcoming this limitation. Using direct fluorescent image analysis and real-time quantitative PCR to evaluate enhanced green fluorescent protein (eGFP) gene expression, we found that the optimal effects of LAGT with limited cytotoxicity occurred at a UV dose of 200 J/m(2). Furthermore, this UV irradiation had no effect on either the chondrogenic or osteogenic potential of HuMSCs. Significant effects of LAGT in HuMSCs could be detected as early as 12 h after exposure and persisted over 21 days, in a time and energy-dependent manner. This LAGT effect was maintained for more than 8 h after irradiation and required only a 10-min exposure to rAAV after UV irradiation. Finally, we show that the production of secreted TGFbeta1 protein from rAAV-TGFbeta1-IRES-eGFP infected to HuMSCs is highly inducible by UV irradiation. These results demonstrate that LAGT combined with rAAV is a promising procedure to facilitate gene induction in HuMSCs for human gene therapy.

A rapid multiparameter approach to study factors that regulate osteoclastogenesis: demonstration of the combinatorial dominant effects of TNF-alpha and TGF-ss in RANKL-mediated osteoclastogenesis.

Macrophages differentiate into osteoclasts in response to the critical cytokine RANKL. However, the efficiency of RANKL-mediated osteoclastogenesis can be profoundly influenced by various cytokines. While studies describing the isolated effects of particular cytokines on osteoclastogenesis have been performed, combinatorial effects of cytokines have not been addressed routinely due to the absence of an efficient assay system. To study the effects of cytokine combinations on osteoclast formation, we performed in vitro assays using either the RAW293 cell line or primary murine splenic macrophages as osteoclast precursors. Using a multiparameter cytokine plating method, we analyzed osteoclastogenesis in response to multiple combinations of the following inflammation-related cytokines: RANKL, IFN-gamma, TNF-alpha, IL-1beta, IL-6, IL-10. We further investigated the role of T-cell-related cytokine combinations on osteoclastogenesis by measuring osteoclast area in response to RANKL with IFN-gamma, IL-2, IL-4, IL-6, TGF-ss, and TNF-alpha. Treatments with RANKL, TNF-alpha, and TGF-ss induced maximal osteoclast formation, suggesting a role for these cytokines in the most aggressive forms of inflammatory bone loss. TNF-alpha alone, however, was unable to induce osteoclast formation in the absence of RANKL despite co-administration of other proinflammatory cytokines. IFN-gamma was a potent inhibitor under all conditions, implicating T cells and NK cells in osteoclast inhibition. These studies demonstrate a rapid screening approach for identifying the potential collective effects of multiple factors on osteoclastic bone resorption.

Effect of anti-tumor necrosis factor-alpha gene therapy on wear debris-induced osteolysis.

BACKGROUND: Particle phagocytosis by macrophages induces the secretion of tumor necrosis factor-alpha, which is involved in the development of an osteolytic response. Therefore, we aimed to determine whether gene delivery of a soluble inhibitor of tumor necrosis factor-alpha (sTNFR:Fc) could prevent wear debris-induced osteolysis in a mouse model. sTNFR:Fc is a fusion protein containing the extracellular domain of the human type-I tumor necrosis factor receptor fused to the Fc region of mouse immunoglobulin. It acts by binding to tumor necrosis factor-alpha and preventing signaling through the membrane-bound tumor necrosis factor receptors. METHODS: An adenoviral vector encoding the LacZ gene (Ad.CMV-NlacZ) was propagated and was tested for its ability to transduce calvarial tissue. Ad.CMV-TNFR:Fc (encoding sTNFR:Fc) or Ad.CMV-NlacZ was administered to CBAxB6 mice in the presence or absence of titanium particles implanted onto the calvaria. Serum levels of sTNFR:Fc were measured with enzyme-linked immunosorbent assay, and the mice were killed on the tenth postoperative day for histological analysis. The experiments were repeated in athymic nude mice to avoid complications associated with the adenovirus-specific immune response. RESULTS: Administration of the control virus (Ad.CMV-NlacZ) transduced 10% of the cells in the periosteum. Ad.CMV-NlacZ treatment of sham-treated or titanium-treated animals induced significant bone resorption and osteoclastogenesis above control levels (that is, those in animals not treated with a virus). Treatment with the sTNFR:Fc virus did not reduce bone resorption or osteoclast numbers below control levels in CBAxB6 mice. In the athymic mice, no increase in the midline sagittal suture area or osteoclastogenesis was observed after treatment with the control vector and sTNFR:Fc gene therapy reduced the suture area to background levels. CONCLUSIONS: An immunologic response to Ad.CMV-NlacZ was most likely responsible for the increase in bone resorption and osteoclastogenesis in the animals treated with the control vector alone. In the athymic mice, in the absence of this immune response, sTNFR:Fc gene therapy reduced bone resorption in the midline sagittal suture area but had no effect on osteoclastogenesis.

Oral pentoxifylline inhibits release of tumor necrosis factor-alpha from human peripheral blood monocytes : a potential treatment for aseptic loosening of total joint components.

BACKGROUND: Pentoxifylline (Trental) is a methylxanthine-derivative drug that has been used for more than twenty years in the treatment of peripheral vascular disease. Pentoxifylline is also a potent inhibitor of tumor necrosis factor-alpha (TNF-alpha) secretion, both in vitro and in vivo, and has demonstrated efficacy in the treatment of certain animal and human inflammatory diseases. Pentoxifylline has a potential therapeutic role in the treatment of aseptic loosening of total joint replacement components because it inhibits TNF-alpha secretion by particle-stimulated human peripheral blood monocytes. The purpose of our study was to determine whether the particle-stimulated secretion of TNF-alpha by peripheral blood monocytes was inhibited in volunteers who had received pentoxifylline orally. METHODS: Human peripheral blood monocytes were harvested from eight healthy volunteers and were exposed to three different concentrations of titanium particles or to 500 ng/mL of lipopolysaccharide as a positive control. The same volunteers were then given pentoxifylline (400 mg, five times per day) for seven days. Their peripheral blood monocytes were again isolated and exposed to experimental conditions, and the TNF-alpha levels were measured. RESULTS: The peripheral blood monocytes from all eight volunteers showed a significant reduction in TNF-alpha release following oral treatment with pentoxifylline. This reduction was observed at exposures of 10(7) and 10(6) titanium particles/mL and in the lipopolysaccharide-treated group, but not at 10(5) particles/mL. CONCLUSIONS: To our knowledge, this is the first study to demonstrate the ability of an oral drug to decrease the release of TNF-alpha from human peripheral blood monocytes exposed ex vivo to particle debris. TNF-alpha is involved in the pathogenesis of osteolysis and subsequent loosening of total joint arthroplasty components. The ability to suppress the release of TNF-alpha in patients with a total joint replacement may help to control osteolysis and to reduce the development of aseptic loosening. This effect could increase implant longevity and decrease the need for revision arthroplasty.

PTHrP expression in chick sternal chondrocytes is regulated by TGF-beta through Smad-mediated signaling.

PTHrP regulates the rate of chondrocyte differentiation during endochondral bone formation. The expression of PTHrP and its regulation by TGF-beta, BMP-2, and PTHrP was examined in upper sternal chondrocytes following 1, 3, and 5 days of continuous treatment. While TGF-beta stimulated the expression of PTHrP (5-fold), PTHrP caused a slight inhibition, and BMP-2 markedly inhibited PTHrP mRNA expression. The effect of these factors on PTHrP expression was not simply related to the maturational state of the cells, since BMP-2 increased, while both PTHrP and TGF-beta decreased the expression of type X collagen. TGF-beta isoforms 1, 2, and 3 all stimulated PTHrP expression. Signaling events involved in the induction of PTHrP by TGF-beta were further evaluated in a PTHrP-promoter CAT construct. The effect of TGF-beta, BMP-2, and PTHrP on the PTHrP-promoter paralleled their effects on mRNA expression, with TGF-beta significantly increasing CAT activity, BMP-2 decreasing CAT activity, and PTHrP having a minimal effect. Co-transfection of the TGF-beta signaling molecule, Smad 3, mimicked the effect of TGF-beta (induction of PTHrP promoter), while dominant negative Smad 3 inhibited the induction of the PTHrP promoter by TGF-beta. Furthermore, infection with a Smad 3-expressing retrovirus mimicked the effects of exogenously added TGF-beta, and induced PTHrP mRNA expression in the infected chondrocyte culture. In contrast, a dominant negative Smad 3 completely inhibited PTHrP promoter stimulation by TGF-beta, but only partially blocked the effect of TGF-beta on PTHrP mRNA synthesis. These findings demonstrate that PTHrP is expressed in chondrocytes undergoing endochondral ossification, and show regulation, at least in part, by TGF-beta through Smad mediated signaling events.

BMP signaling stimulates chondrocyte maturation and the expression of Indian hedgehog.

Mutant BMP receptors were transfected into cultured embryonic upper sternal chrondrocytes using retroviral vectors to determine if BMP signaling is required for chondrocyte maturation and the expression of a key regulatory molecule, Indian hedgehog (Ihh). Chondrocytes infected with replication competent avian retroviruses (RCAS) viruses carrying constitutive active (CA) BMPR-IA and BMPR-IB had enhanced expression of type X collagen and Ihh mRNA. Addition of PTHrP, a known inhibitor of chondrocyte maturation, abolished the expression of type X collagen, BMP-6, and Ihh mRNAs in control cells. In contrast, PTHrP treated cultures infected with of CA BMPR-IA or CA BMPR-IB had low levels of BMP-6 and type X collagen, but high levels of Ihh expression. Although dominant negative (DN) BMPR-IA had no effect, DN BMPR-IB inhibited the expression of type X collagen and BMP-6, and decreased alkaline phosphatase activity, even in the presence of exogenously added BMP-2 and BMP-6. DN BMPR-IB also completely blocked Ihh expression. Overall, the effect of DN BMPR-IB mimicked the effects of PTHrP. To determine if there is an autocrine role for the BMPs in chondrocyte maturation, the cultures were treated with noggin and follistatin, molecules that bind BMP-2/-4 and BMP-6/-7, respectively. While noggin and follistatin inhibited the effects of recombinant BMP-2 and BMP-6, respectively, they had only minimal effects on the spontaneous maturation of chondrocytes in culture, suggesting that more than one subgroup of BMPs regulates chondrocyte maturation. The results demonstrate that: (i) BMP signaling stimulates chondrocyte maturation; (ii) BMP signaling increases Ihh expression independent of maturational effects; and (iii) BMP signaling can partially overcome the inhibitory effects of PTHrP on maturation.

The role of autocrine growth factors in radiation damage to the epiphyseal growth plate.

Radiation therapy plays an important role as part of the multimodality treatment for a number of childhood malignancies. Dose-limiting complications of radiotherapy include skeletal abnormalities and disturbances in skeletal development within the irradiated field. The current study was undertaken to investigate the molecular mechanisms involved in radiation-induced arrest of bone growth. Our hypotheses were: (1) Expression of autocrine growth factors that regulate chondrocyte proliferation is inhibited by radiation in a specific pattern; (2) the disparity in radiosensitivity of growth plate chondrocytes and epiphyseal chondrocytes is due to differential modulation of autocrine growth factor expression by radiation. Given the important role these cells play in skeletal growth and development, we examined the comparative effects of radiation on expression of specific mitogenic growth factors in growth plate chondrocytes. The effect of radiation on the expression of autocrine/paracrine growth factors was examined in an established avian model of epiphyseal growth plate maturation. Exposure of growth plate chondrocytes to radiation resulted in a specific pattern of biochemical and morphological alterations that were dependent on dose and were progressive over time. While radiation did not affect the mRNA expression of some of the autocrine and paracrine factors important in endochondral ossification (such as FGF2 and TGFB isoforms), it did lead to a decrease in the mRNA expression of PTHrP, a critically important mitogen in growth plate chondrocytes, and a dose-dependent decrease in the PTH/PTHrP receptor mRNA. Interestingly, PTHrP mRNA levels were not affected in irradiated epiphyseal chondrocytes, the main source of PTHrP. Given evidence indicating a role for intracellular calcium levels in regulating PTHrP expression, basal calcium levels in irradiated growth plate chondrocytes and epiphyseal chondrocytes were examined 24 h after treatment. While cytosolic calcium levels were significantly higher in irradiated growth plate chondrocytes, they were not significantly affected in irradiated epiphyseal chondrocytes. The importance of calcium in mediating radiation damage to growth plate chondrocytes was further demonstrated by the finding that the addition of 4.0 mM EGTA (a calcium chelator) to the cell cultures before irradiation prevented the decrease in PTHrP mRNA levels. Since PTHrP up-regulates BCL2 levels and prevents growth plate chondrocyte maturation and apoptosis, BCL2 mRNA levels were examined in irradiated growth plate chondrocytes, and a dose-dependent decrease was found. An increase in apoptosis was further confirmed by a fivefold increase in caspase 3 levels in irradiated growth plate chondrocytes. The results of the current study suggest that radiation may interfere with proliferation of growth plate chondrocytes in part by causing an increase in cytosolic calcium levels which in turn leads to a decrease in PTHrP mRNA. Growth plate chondrocyte PTHrP receptor mRNA expression is also inhibited by radiation, further decreasing PTHrP signaling. Despite subtle differences between the chick and mammalian growth plates, further studies should provide an enhanced understanding of the mechanism(s) of radiation injury to the growth plate, as well as possibilities for new therapeutic strategies to protect the growing skeleton from the detrimental effects of radiotherapy.

Evidence for a direct role of cyclo-oxygenase 2 in implant wear debris-induced osteolysis.

Aseptic loosening is a major complication of prosthetic joint surgery and is manifested as chronic inflammation, pain, and osteolysis at the bone implant interface. The osteolysis is believed to be driven by a host inflammatory response to wear debris generated from the implant. In our current study, we use a selective inhibitor (celecoxib) of cyclo-oxygenase 2 (COX-2) and mice that lack either COX-1 (COX-1-/-) or COX-2 (COX-2-/-) to show that COX-2, but not COX-1, plays an important role in wear debris-induced osteolysis. Titanium (Ti) wear debris was implanted surgically onto the calvaria of the mice. An intense inflammatory reaction and extensive bone resorption, which closely resembles that observed in patients with aseptic loosening, developed within 10 days of implantation in wild-type and COX-1-/- mice. COX-2 and prostaglandin E2 (PGE2) production increased in the calvaria and inflammatory tissue overlying it after Ti implantation. Celecoxib (25 mg/kg per day) significantly reduced the inflammation, the local PGE2 production, and osteolysis. In comparison with wild-type and COX-1-/- mice, COX-2-/- mice implanted with Ti had a significantly reduced calvarial bone resorption response, independent of the inflammatory response, and significantly fewer osteoclasts were formed from cultures of their bone marrow cells. These results provide direct evidence that COX-2 is an important mediator of wear debris-induced osteolysis and suggests that COX-2 inhibitors are potential therapeutic agents for the prevention of wear debris-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.