Introduction. Particle-induced oxidative stress in cells is a unifying factor that determines toxicity and carcinogenicity potential in biomaterials. A previous study by Bladen et al. showed the production of significant levels of reactive oxygen species (ROS) following the stimulation of phagocytes by UHMWPE and CoCr wear debris [1]. Latest generation bearing materials such as silicon nitride also need to be tested for potential generation of ROS in phagocytic cells. This study aimed to investigate the production of reactive oxygen species in L929 fibroblasts stimulated with clinically relevant doses of nanoscale and micron-sized silicon nitride (Si. 3. N. 4. ) particles, silica nanoparticles, and CoCr wear debris. Silica nanoparticles were included as a comparison material for situations where the Si. 3. N. 4. particle's surface are oxidised to silicon dioxide [2]. Materials and Methods. Si. 3. N. 4. particles (<50 nm and <1 µm, Sigma), silica nanopowder (<100 nm, Sigma) and clinically relevant CoCr wear particles were heat-treated at 180°C for 4 h to remove endotoxin. Particles were then re-suspended in sterile water by sonication. L929 murine fibroblasts were cultured with low doses (0.5 µm. 3. /cell) and high doses (50 µm. 3. /cell) of Si. 3. N. 4. particles, and high doses (50 µm. 3. /cell) of silica nanoparticles and CoCr wear debris. Cells were incubated for three and six days at 37°C with 5% (v/v) CO. 2. tert-Butyl hydroperoxide (TBHP) was used as a positive control for the production of ROS in the cells. Intracellular ROS was measured using Image-IT LIVE kit (Invitrogen). This assay is based on carboxy-2',7'-dichlorodihydro-fluorescein diacetate (carboxy-H2DCFDA), which forms a non-fluorescent derivative by intracellular esterases and then reacts with intracellular ROS to form green fluoroscence producing derivative carboxy- dichlorodihydro-fluorescein. Images were captured using a confocal microscope and analysed using ImageJ for corrected total cell fluorescence (CTCF). The results were expressed as mean ± 95% confidence limits and the data was analysed using one-way ANOVA and Tukey-Kramer post-hoc tests. Results and Discussion. Si. 3. N. 4. nanoparticles significantly reduced the ROS levels in L929 fibroblasts at low doses (0.5 μm. 3. /cell) and high doses (50 μm. 3. /cell) over a period of six days; whereas no significant change in the levels of ROS was observed in cells treated with micron-sized Si. 3. N. 4. particles [Figure 1]. Only a few cells treated with high doses of CoCr wear particles (50 μm. 3. /cell) survived for up to six days and produced significantly higher levels of ROS [Figure 1, 2]. Interestingly, cells challenged with high doses (50 μm. 3. /cell) of Si. 3. N. 4. and silica nanoparticles produced statistically similar levels of ROS in cells [Figure 1]. This might be due to the potential surface oxidation of Si. 3. N. 4. nanoparticles, which makes their surface chemistry and biological identity similar to silica nanoparticles. Conclusion. Unlike existing implant materials such as UHMWPE and CoCr, silicon nitride has demonstrated the capacity to reduce or maintain normal levels of ROS in macrophages depending on the particle size and dose. Acknowledgements. The research leading to these results has received funding from the European Union's Seventh Framework Programme (FP7/2007-2013) under grant agreement no. GA-310477 LifeLongJoints

Implant-related infection (IRI) is closely related to the local immunity of peri-implant tissues. The generation of reactive oxygen species (ROS) in activated macrophages plays a prominent role in the innate immune response. In previous studies, we indicated that implant wear particles promote endotoxin tolerance by decreasing the release of proinflammatory cytokines. However, it is unclear whether ROS are involved in the damage of the local immunity of peri-implant tissues. In the present study, we assessed the mechanism of local immunosuppression using titanium (Ti) particles and/or lipopolysaccharide (LPS) to stimulate RAW 264.7 cells. The results indicate that the Ti particles induced the generation of a moderate amount of ROS through nicotinamide adenine dinucleotide phosphate oxidase-1 (NOX-1), but not through catalase. Pre-exposure to Ti particles inhibited ROS generation and extracellular regulated protein kinase (ERK) activation in LPS-stimulated macrophages. These findings indicate that chronic stimulation by Ti particles may lead to a state of oxidative stress and persistent inflammation, which may result in the attenuation of the immune response of macrophages to bacterial components such as LPS. Eventually, immunosuppression develops in peri-implant tissues, which may be a risk factor for IRI

The purpose of this study was to evaluate whether AGEs induce annulus fibrosus (AF) cell apoptosis and to further explore the mechanism by which this process occurs. AF cells were treated with various concentrations of AGEs for 3 days. Cell proliferation was measured by the Cell Counting Kit-8 (CCK-8) and EdU incorporation assays. Cell apoptosis was examined by the Annexin V/PI apoptosis detection kit and Hoechst 33342. The expression of apoptosis-related proteins, including Bax, Bcl-2, cytochrome c, caspase-3 and caspase-9, was detected by western blotting. In addition, Bax and Bcl-2 mRNA expression levels were detected by RT-PCR. Mitochondrial membrane potential (MMP) and intracellular reactive oxygen species (ROS) production of AF cell were examined by JC-1 staining and DCFH-DA fluorescent probes, respectively. Our results indicated that AGEs had inhibitory effects on AF cell proliferation and induced AF cell apoptosis. The molecular data showed that AGEs significantly up-regulated Bax expression and inhibited Bcl-2 expression. In addition, AGEs increased the release of cytochrome c into the cytosol and enhanced caspase-9 and caspase-3 activation. Moreover, treatment with AGEs resulted in a decrease in MMP and the accumulation of intracellular ROS in AF cells. The antioxidant N-acetyl-L-cysteine significantly reversed AGE-induced MMP decrease and AF cell apoptosis. These results suggest that AGEs induce rabbit AF cell apoptosis and mitochondrial pathways may be involved in AGE-mediated cell apoptosis, which may provide a theoretical basis for diabetic IVD degeneration

Aim

The purpose of this study is to analyze the demographic and microbiological variables of acute ankle infections posterior to ankle osteosynthesis and to determine the different characteristics of patients withE. cloacae infection.

Introduction. Periprosthetic joint infection (PJI) and particle-induced osteolysis are closely related to peri-implant local immunity and macrophage function. We previously demonstrated that titanium particles attenuate the immune response of macrophages caused by chronic inflammation [1]. In a separate study, we have determined that UHMWPE wear particles containing vitamin E (VE) induce less osteolysis compared to HXL UHMWPE wear particles in a murine calvarium model [2]. For this study we hypothesized that macrophages exposed to HXL UHMWPE particles containing VE would better maintain their ability to respond to S. aureus compared to HXL UHMWPE without VE. Methods. A gamma-sterilized, HXL UHMWPE tibial bearing containing VE (E1, Biomet, “VE-PE”) and 100kGy irradiated and melted UHMWPE (“CISM 100”) were cryomilled to particles by Bioengineering Solutions (Oak Park, IL). In the first in vitro study, RAW 264.7 mouse macrophages were exposed (inverted co-culture) to either VE-PE particles or CISM100 particles and lipopolysaccharide (LPS) for 1–7 days. Macrophage viability was measured using a cell counting kit (CCK-8). Control group with no particles and a LPS group were also included. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining was performed to determine macrophage apoptosis rate in response to particle exposure over time. In the second study, macrophages were exposed to VE-PE or CISM100 particles for 48h, then exposed to LPS for 30 min. Subsequently, reactive oxygen species (ROS) generation and extracellular regulated protein kinase (ERK) phosphorylation were measured. In a third study, after exposure to particles for 48h, fatigued macrophages were co-cultured with bioluminescent S. aureus strain Xen29 for 3h and 6h. Bioluminescence signal was determined to measure the total amount of bacteria. Bacterial live/dead staining and optical density at 600 nm (OD 600) were also performed to determine S. aureus viability. Statistical analysis was performed using one-way or two-way ANOVA with a post hoc examination. *indicates p<0.05. Results. CISM100 particles significantly decreased macrophage viability at day 5 and day 7 (p<0.05, Fig. 1A), while the viability of macrophages exposed to VE-PE particles was similar to controls (macrophages not exposed to particles). After 48h, macrophages exposed to VE-PE particles showed a lower TUNEL-positive rate (less apoptosis) compared to CISM100 particles (Fig. 1B, C). 48h-exposure to VE-PE particles increased ROS generation and ERK phosphorylation in 30 min-LPS-activated macrophages when compared to CISM100 particles (Fig. 2). This immune response caused by VE-PE particles resembles that of macrophages without particles. Furthermore, 48h exposure to E1 particles showed less S. aureus at 6h (Fig. 3). Conclusions. These results suggest that VE-PE particles cause reduced macrophage apoptosis and protect the macrophages' immune response. VE-PE particles also preserved the innate immunity of macrophages, unlike CISM100, as evidenced by the S. aureus co-culture study. Thus, patients with vitamin-E containing implants may be less likely to develop PJI

Implant-associated infection is a major source of morbidity in orthopaedic surgery. There has been extensive research into the development of materials that prevent biofilm formation, and hence, reduce the risk of infection. Silver nanoparticle technology is receiving much interest in the field of orthopaedics for its antimicrobial properties, and the results of studies to date are encouraging. Antimicrobial effects have been seen when silver nanoparticles are used in trauma implants, tumour prostheses, bone cement, and also when combined with hydroxyapatite coatings. Although there are promising results with in vitro and in vivo studies, the number of clinical studies remains small. Future studies will be required to explore further the possible side effects associated with silver nanoparticles, to ensure their use in an effective and biocompatible manner. Here we present a review of the current literature relating to the production of nanosilver for medical use, and its orthopaedic applications.

Cite this article: Bone Joint J 2015; 97-B:582–9.