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

Aim. Osteomyelitis is a difficult-to-treat disease with high chronification rates. The surgical amputation of the afflicted limb sometimes remains as the patients’ last resort. Several studies suggest an increase in mitochondrial fission as a possible contributor to the accumulation of intracellular reactive oxygen species and thereby to cell death of infectious bone cells. The aim of this study is to analyze the ultrastructural impact of bacterial infection and its accompanying microenvironmental tissue hypoxia on osteocytic and osteoblastic mitochondria. Method. 19 Human bone tissue samples from patients with osteomyelitis were visualized via light microscopy and transmission electron microscopy. Osteoblasts, osteocytes and their respective mitochondria were histomorphometrically analyzed. The results were compared to the control group of 5 non-infectious human bone tissue samples. Results. The results depicted swollen hydropic mitochondria including depleted cristae and a decrease in matrix density in the infectious samples as a common finding in both cell types. Furthermore, perinuclear clustering of mitochondria could also be observed regularly. Additionally, increases in relative mitochondrial area and number could be found as a sign for increased mitochondrial fission. Conclusions. The results show that mitochondrial morphology is altered during osteomyelitis in a comparable way to mitochondria from hypoxic tissues. This suggests that manipulation of mitochondrial dynamics in a way of inhibiting mitochondrial fission may improve bone cell survival and exploit bone cells regenerative potential to aid in the treatment of osteomyelitis

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

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

Currently, different techniques to evaluate the biocompatibility of orthopaedic materials, including two-dimensional (2D) cell culture for metal/ceramic wear debris and floating 2D surfaces or three-dimensional (3D) agarose gels for UHMWPE wear debris, are used. Moreover, cell culture systems evaluate the biological responses of cells to a biomaterial as the combined effect of both particles and ions. We have developed a novel cell culture system suitable for testing the all three type of particles and ions, separately. The method was tested by evaluating the biological responses of human peripheral blood mononuclear cells (PBMNCs) to UHMWPE, cobalt-chromium alloy (CoCr), and Ti64 alloy wear particles. Methods. Clinically relevant sterile UHMWPE, CoCr, and Ti64 wear particles were generated in a pin-on-plate wear simulator. Whole peripheral blood was collected from healthy human donors (ethics approval BIOSCI 10–108, University of Leeds). The PBMNCs were isolated using Lymphoprep (Stemcell, UK) and seeded into the wells of 96-well and 384-well cell culture plates. The plates were then incubated for 24 h in 5% (v/v) CO. 2. at 37°C to allow the attachment of mononuclear phagocytes. Adherent phagocytes were incubated with UHMWPE and CoCr wear debris at volumetric concentrations of 0.5 to 100 µm. 3. particles per cell for 24 h in 5% (v/v) CO. 2. at 37°C. During the incubation of cells with particles, for each assay, two identical plates were set up in two configurations (one upright and one inverted). After incubation, cell viability was measured using the ATPlite assay (Perkin Elmer, UK). Intracellular oxidative stress was measured using the DCFDA-based reactive oxygen species detection assay (Abcam, UK). TNF-α cytokine was measured using sandwich ELISA. DNA damage was measured by alkaline comet assay. The results were expressed as mean ± 95% confidence limits and the data was analysed using one-way ANOVA and Tukey-Kramer post-hoc analysis. Results and Discussion. Cellular uptake of UHMWPE, CoCr and Ti64 particles was confirmed by optical microscopy. PBMNCs incubated with UHMWPE particles did not show any adverse responses except the release of significant levels of TNF-α cytokine at 100 µm. 3. particles per cell, when in contact with particles. PBMNCs incubated with CoCr wear particles showed adverse responses at high particle doses (100 µm. 3. particles per cell) for all the assays. Moreover, cytotoxicity was observed to be a combined effect of both particles and ions, whereas oxidative stress and DNA damage were mostly caused by ions. Ti64 wear particles did not show any adverse responses except cytotoxicity at high particle doses (100 µm. 3. particles per cell). Moreover, this cytotoxicity was mostly found to be a particle effect. In conclusion, the novel cell culture system is suitable for evaluating the biological impact of orthopaedic wear particles and ions, separately

Introduction. Wear debris and metal ions originating from metal on metal hip replacements have been widely shown to recruit and activate macrophages. These cells secrete chemokines and pro-inflammatory cytokines that lead to an adverse local tissue reaction (ALTR), frequently requiring early revision. The mechanism for this response is still poorly understood. It is well documented that cobalt gives rise to apoptosis, necrosis and reactive oxygen species generation. Additionally, cobalt stimulates T cell migration, although the effect on macrophage motility remains unknown. This study tests the hypothesis that cobalt ions and nanoparticles affect macrophage migration stimulating an ALTR. Methods. This study used Co. 2+. ions (200µM) and cobalt nanoparticles (CoNPs, 100µM, 2–60nm diameter). PMA differentiation of the U937 cell line was used as macrophage-like cells. The effect of cobalt on macrophage migration was investigated by live cell imaging. After 12 hours of each treatment, timelapse images of 20 cells were collected over a 6 hour period with images captured every 5 min. Migration of individual cells was tracked in 2D using ImageJ software. The transwell migration assay was also applied to study the effect of cobalt on macrophage directional migration. U937 cells in serum free medium were added to the upper chamber of a 8µm pore size Transwell insert in the presence of cobalt, whilst the lower chamber was filled with medium plus 10% FBS. After 6 hours treatment, cells remaining on the membrane were fixed, stained with crystal violet and counted. Cellular F-actin and podosomes were visualized by labeling with TRITCconjugated phalloidin and anti-vinculin antibody after 12 hours of cobalt exposure (Co. 2+. and CoNPs). Results. Cells incubated with cobalt ions and nanoparticles showed a substantial reduction in cell migration compared with control cells. The total migration path length of cells treated with Co. 2+. (362.4±96.6µm) and CoNPs (217.3±128.1µm) were significantly shorter than those for untreated cells (801.1±198.3µm). The ability of macrophages to migrate through the transwell membrane was significantly impaired by pre-treatment with cobalt, with 16±4 and 18± migrated cells/field for Co. 2+. and CoNPs respectively with the control at 42±7 migrated cells/field. In addition, cobalt influenced macrophage morphology and actin cytoskeletal organization with a dramatic increase in the presence of intracellular podosome-type adhesions structure. Discussion. Co. 2+. ions and nanoparticles dramatically inhibited the migration of U937 macrophages in contrast to the enhanced migration reported for T cells. We propose that macrophages recruited into the area of CoCr implants would lose their responsiveness to migration signals and be retained in situ due to cobalt-induced cytoskeleton rearrangement. This enhanced macrophage accumulation and cobalt-induced formation of podosomes may therefore represent a mechanism through which cobalt wear debris and metal ions from joint prostheses exacerbate the ALTR leading to revision surgery

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

Staphylococcus aureus is one of the leading causes of surgical site infection (SSI). Over the past decade there has been an increase in methicillin-resistant S. aureus (MRSA). This is a subpopulation of the bacterium with unique resistance and virulence characteristics. Nasal colonisation with either S. aureus or MRSA has been demonstrated to be an important independent risk factor associated with the increasing incidence and severity of SSI after orthopaedic surgery. Furthermore, there is an economic burden related to SSI following orthopaedic surgery, with MRSA-associated SSI leading to longer hospital stays and increased hospital costs. Although there is some controversy about the effectiveness of screening and eradication programmes, the literature suggests that patients should be screened and MRSA-positive patients treated before surgical admission in order to reduce the risk of SSI.

Cite this article: Bone Joint J 2013;95-B:4–9.