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Aseptic implant loosening is a devastating long-term complication of total joint arthroplasty. It is mainly initiated by the interaction of wear debris and macrophages. However, how does the chronic inflammation persist and how to stop it is poorly understood. Sphingosine kinases (SPHKs) are an essential feature of immunosuppressive M2 polarisation in macrophages and a promoter for chronic inflammation. In this study, RAW 264.7 macrophages were exposed to stimulation with titanium particles (0.1 mg/ml), and the subsequent expression of SPHKs and pro-inflammatory cytokines was evaluated. The effect of inhibitors of SPHKs (FTY720, PF543, and ABC294640) on titanium particle-challenged macrophages was analysed. As for results, the amount of sphingosine kinase (SPHK)-1 and SPHK-2 in RAW264.7 macrophages increased in the presence of titanium particles in a time-dependent manner. Two inhibitors of SPHKs (FTY720 and ABC294640) suppressed titanium particle-induced tumour necrosis factor (TNF)-α and interleukin (IL)-6 production in RAW264.7 macrophages. These findings suggest that persistent stimulation with titanium particles may lead to a consistent release of TNF-α and IL-6 via SPHK-2 activity, which may lead to aseptic implant loosening. Appropriate regulation of SPHK-2 may serve as a potential new strategy in the treatment of aseptic implant loosening.

Macrophages play an essential role in inflammation. Protein disulfide isomerase (PDI) is central to the redox system, which is closely linked with the inflammatory function of macrophages. However, the relationship between PDI and inflammation is still unknown. In this study, we tested the effects of PDI on inflammatory responses in RAW 264.7 macrophages stimulated with lipopolysaccharide (LPS). Using CRISPR/Cas9 system, we found that PDI knockout suppressed migration, M1 polarization, and secretion of tumor necrosis factor-α (TNF-α) and interluekin-6 (IL-6). The repression of these inflammatory processes was accompanied by decreased production of reactive oxygen species (ROS). PDI ablation also inactivated the phosphorylation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and activated the phosphorylation of NF-κB inhibitor alpha (IκBα). These findings demonstrate that PDI knockout inhibits the inflammatory function of macrophages by decreasing ROS production and inactivating NF-κB pathway.

Background/aims: Prosthesis loosening is closely associated with chronic inflammatory cytokine secretion by macrophages, which are activated by wear particles or inflammatory stimulants such as lipopolysaccharide (LPS). Reactive oxygen species (ROS) are critical regulators of inflammation, but their enzymatic sources in response to wear particles and their effects on peri-implant LPS-tolerance remain unclear., Methods: Three ROS-related enzymes-nicotinamide adenine dinucleotide phosphate oxidase (NOX)-1 and -2 and catalase-were investigated in interface membrane tissues and in titanium (Ti) particle-stimulated macrophages in vitro. The generation of ROS and downstream inflammatory effects were measured with or without pre-incubation with apocynin, an NOX inhibitor., Results: Pre-exposure to Ti particles attenuated NF-κB activation in LPS-stimulated macrophages, indicating that wear particles suppress immune response, which may lead to chronic inflammation. NOX-1 and -2 were highly expressed in aseptically loosened interface membranes and in macrophages stimulated with Ti particles; the particles induced a moderate amount of ROS generation, NF-κB activation, and TNF-α secretion in macrophages, and these effects were suppressed by apocynin., Conclusion: Wear particles induce ROS generation through the NOX signaling pathway, resulting in persistent inflammation and delayed loosening. Thus, the suppression of NOX activity may be a useful strategy for preventing prosthesis loosening., (© 2015 S. Karger AG, Basel.)

Background: Inflammatory osteolysis induced by wear particles is the major cause of prosthetic loosening after artificial joint replacement, and its prevention and treatment are difficult worldwide. Our previous study confirmed that sphingosine kinases (SPHKs) are important mediators regulating the wear particle-induced macrophage inflammatory response. However, it is unclear whether SPHKs can modulate chronic inflammation and alleviate osteolysis. Zoledronic acid (ZA), an imidazole-containing bisphosphonate, directly affects osteoclasts and prevents bone mineral-related diseases. However, the effects of SPHK inhibitors and ZA used to treat periprosthetic osteolysis are unknown. Methods: We applied tartrate-resistant acid phosphatase (TRAP) staining to evaluate bone destruction in the interface membranes of patients with aseptic loosening and a control group. A murine calvarial osteolysis model was used to examine the preventative effect of SPHK inhibitors and ZA on osteolysis. Micro-CT scanning, immunohistochemistry (IHC), and histomorphometric analysis were conducted to determine the variations in inflammatory osteolysis. The effects of different drug concentrations on cell viability were evaluated using the Cell Counting Kit-8 (CCK-8) assay. Real-time quantitative polymerase chain reaction (RT-qPCR) analysis was performed to confirm the reduced expression of osteoclast-specific genes after drug and titanium treatment. The osteoclast formation and functions of the drugs were analyzed using TRAP staining in vivo and in vitro. The effect of SPHKs/S1P-TRAF2-BECN1 signaling pathways was verified via RT-qPCR and tissue IHC. Results: In this study, we found that SPHK inhibitors (ABC294640 and FTY720) combined with ZA decreased the degree of inflammatory osteolysis in vivo. However, ABC294640 and ZA suppressed osteoclast differentiation and osteoclast-specific genes in vitro. SPHKs regulate the inflammatory osteolysis induced by wear particles by increasing the expression of SPHKs/S1P-TRAF2-BECN1. Conclusion: Our study revealed that wear particles could induce inflammatory osteolysis by upregulating SPHKs/S1P-TRAF2-BECN1 and SPHK inhibitors/ZA inhibit osteoclastogenesis in vitro and prevent inflammatory osteolysis in vivo, suggesting that SPHK inhibitors and ZA can be a new perspective and scientific basis for the prevention and treatment of prosthesis loosening. [ABSTRACT FROM AUTHOR]

Aseptic implant loosening is closely associated with chronic inflammation induced by implant wear debris, and reactive oxygen species (ROS) play an important role in this process. Resveratrol, a plant compound, has been reported to act as an antioxidant in many inflammatory conditions; however, its protective effect and mechanism against wear particle-induced oxidative stress remain unknown. In this study, we evaluated resveratrol's protective effects against wear particle-induced oxidative stress in RAW 264.7 macrophages. At non-toxic concentrations, resveratrol showed dose-dependent inhibition of nitric oxide (NO) production, ROS generation, and lipid peroxidation. It also downregulated the gene expression of oxidative enzymes, including inducible nitric oxide synthase (iNOS) and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX)-1 and NOX-2, and promoted the gene expression and activities of antioxidant enzymes, including catalase (CAT), superoxide dismutase (SOD), glutathione reductase (GR), and glutathione peroxidase (GPx). This protective effect against wear particle-induced oxidative stress was accompanied by a reduction of gene expression and release of tumor necrosis factor-α (TNF-α), and decreased gene expression and phosphorylation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). These findings demonstrate that resveratrol can inhibit wear particle-induced oxidative stress in macrophages, and may exert its antioxidant effect and protect against aseptic implant loosening. [ABSTRACT FROM AUTHOR]