Development of osteoarthritis (OA) correlates with epigenetic alteration in chondrocytes. H3K27me3 demethylase UTX is known to regulate tissue homeostasis, but its role in the homeostasis of articulating joint tissue is poorly understood. Forced UTX expression upregulated H3K27me3 enrichment at the Sox9 promoter region to inhibit key extracellular matrix (ECM) molecules, like e.g. type II collagen, aggrecan, and glycosaminoglycans in articular chondrocytes. Utx loss in vitro altered the H3K27me3-binding epigenomic landscape, which contributes to mitochondrial activity, cellular senescence, and cartilage development. Functional target genes of Utx comprise insulin-like growth factor 2 (Igf2) and polycomb repressive complex 2 (PRC2) core components Eed and Suz12. Specifically, Utx deletion promoted Tfam transcription, mitochondrial respiration, ATP production and Igf2 transcription, but inhibited Eed and Suz12 expression. Igf2 inhibition or forced Eed or Suz12 expression increased H3K27 trimethylation and H3K27me3 enrichment at the Sox9 promoter, compromising Utx loss-induced ECM overproduction. Overexpression of Utx in murine knee joints aggravated OA development, including articular cartilage damage, synovitis, osteophyte formation, and subchondral bone loss. Transgenic mice with a chondrocytespecific Utx knockout develop thicker articular cartilage as compared to wild-type controls and show fewer gonarthrotic symptoms during destabilized medial meniscus- and collagenase-induced joint injury. In summary, UTX represses chondrocytic activity and accelerates cartilage degradation during OA, while Utx loss promotes cartilage integrity through epigenetic stimulation of mitochondrial biogenesis and Igf2 transcription. This highlights a novel noncanonical role of Utx that regulates articular chondrocyte anabolism and OA development.

Introduction and Objective

Senescent bone cell overburden accelerates osteoporosis. Epigenetic alteration, including microRNA signalling and DND methylation, is one of prominent features of cellular senescence. This study aimed to investigate what role microRNA-29a signalling may play in the development of senile osteoporosis.

Purpose: The presence of metal ions in the blood of patients with a metal-on-metal (MM) bearing points to the importance of understanding the long-term effects of these ions. Metal ions have the potential to induce the production of reactive oxygen species (ROS), making them prime suspects for inducing molecular damage in circulating cells. The aim of this study was to analyze the levels of oxidative stress markers in the plasma of patients with hip surface replacement.

Method: Blood was collected up to 3 years after implantation from 66 patients with articular surface replacement (ASRÔ, DePuy Orthopaedics) and 54 patients with 36 mm-head MM THA. Forty (40) pre-operative patients were also assessed as control group. Total anti-oxidant levels were measured by the Oxford Biomedical total antioxidant power assay (Oxford, MI) to obtain an overview of the defense capacity of patient’s oxidative stress. Peroxide concentrations were measured by the Biomedica OxyStat assay (Medicorp, Montreal, QC) to quantify damage to lipids in the systemic circulation. Nitrototyrosine levels were quantified using the BIOXYTECH® Nitrotyrosine-EIA assay (OxisResearch™, Portland OR) to measure damage to proteins. The concentrations of metal ions were analyzed by inductively coupled plasma-mass spectroscopy.

Results: Results showed that there were no statistical differences in the concentrations of total antioxidants, lipid peroxides, and protein nitrotyrosines between the control, the ASR, and the 36 mm-head groups. Furthermore, there was no correlation between the concentrations of these markers and the concentrations of both Co and Cr ions (r2 £ 0.006).

Conclusion: The single most significant obstacle preventing a broader application of metal-on-metal hip arthroplasties and resurfacings continues to be the concerns regarding elevated metal ion levels in the blood and urine of patients. The present results showed that there were no changes in the levels of oxidative stress markers in patients with MM bearings compared to the control group. Given the possible latency periods related to metal ion exposure, longer follow-ups are required to conclusively determine the effects of elevated circulating ions on oxidative stress in the blood of patients with MM bearings.

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 (Si₃N₄) particles, silica nanoparticles, and CoCr wear debris. Silica nanoparticles were included as a comparison material for situations where the Si₃N₄ particle's surface are oxidised to silicon dioxide [2].

Purpose: Several studies have shown elevated levels of metal ions in the blood of patients with metal-on-metal (MM) total hip arthroplasty (THA). Even though there is no conclusive evidence that the elevated levels of ions have any detrimental effects on the patients, the presence of these ions is still a cause of concerns. The potential of metal ions released from MM implants for oxidative stress is unknown. In the present study, we measured the concentrations of oxidative stress markers in the plasma of patients with MM THAs.

Method: Blood from patients having MM THAs was collected up to 10 years post-operatively into Sarstedt Li-Heparin tubes. Plasma was prepared by centrifugation at 500 × g for 10 min. Plasma was chosen as opposed to whole blood because it is known that the assays for oxidative stress are not recommended for blood and can lead to erroneous data. Total antioxidant levels were measured by the Oxford Biomedical total antioxidant power assay to obtain an overview of the defense capacity of patients against oxidative stress. The activity of catalase and glutathione peroxidase, two antioxidant enzymes acting on specific reactive oxygen species, was measured by enzymatic assays. Peroxide concentrations were measured by the Biomedica Oxy-Stat assay to quantify damage to lipids in the systemic circulation. Nitrototyrosine levels were quantified using the BIOXYTECH® Nitrotyrosine-EIA assay to measure damage to proteins. Levels in patients without prostheses were used as control.

Results: There were no statistical differences in the concentrations of total antioxidants, lipid peroxides, and nitrotyrosines throughout the period of study. The activity of catalase and glutathione peroxidase was also stable over time. Moreover, there was no correlation between the concentrations of these markers and the concentrations of both cobalt and chromium ions.

Conclusion: Metal ions have the potential to induce the production of reactive oxygen species (free radicals) and cause oxidative stress in the plasma of patients with MM THAs. The present study showed that there were no changes in the levels of oxidative stress markers or antioxidant enzymes in these patients up to 10 years post-operatively. Taken together, the data strongly suggest that metal ions may not cause a significant oxidative stress in patients with MM THAs.

Introduction: Staphylococcal bacteria, especially the coagulase negative Staphylococci, are responsible for the majority of orthopaedic device related infection. These infections are sub acute, and may not present for months or years following surgery. The virulence of these bacteria is related to their ability to form biofilm, a protective slime which allows them to survive the effects of the host immune system and antimicrobial therapy. Treatment of biofilm based infection almost always necessitates removal of the implant.

Recent work has identified environmental stimuli which induce biofilm formation in Staphylococci. These include stressors such as high temperature, high osmolarity, anaerobiosis, nutrient depletion, salt, ethanol and subinhibitory concentrations of certain antimicrobial drugs. Given the ability of these bacteria to survive the “respiratory burst” from the cells of the mononuclear-macrophage system, we hypothesised that oxidative stress may be one such promoter of biofilm formation by Staphylococci.

Methods and Materials: Staphylococcus epidermidis CSF41498 and Staphylococcus aureus RN422O were selected for study as these are known biofilm forming organisms. Hydrogen peroxide (H₂O₂) was used as an oxidizing agent.

Bacteria were incubated for 24 hours at 37°C in Brain-Heart Infusion (BHI, Oxoid) containing progressively weaker concentrations of H₂O₂ to determine a Minimal Inhibitory Concentration (M.I.C.) for the representative strains. Bacterial viability was assessed by measuring the optical density of the incubated culture using a cell density meter (Ultraspec 10, Amersham Biosciences).

The bacteria were then grown as a biofilm on a 96 well microtitre plate (Nunc) in the presence of subinhibitory concentrations of H₂O₂, using pure BHI as a control. Semiquantative determination of biofilm formation was performed by washing the plates, staining the adherent cells with crystal violet, and measuring the light absorbance of the adherent stained cells at 492 nm using a Multiskan plate reader (Flow Laboratories).

Results: The M.I.C. of H₂O₂ was 18 mM for both Staphylococcus epidermidis CSF41498 and Staphylococcus aureus RN422O. Concentrations of H₂O₂ of 16 mM and below had no normal bacterial growth and replication.

There was no difference in biofilm formation by Staphylococcus epidermidis csf41498 in the presence of 15 mM H₂O₂ when compared to that of the control. However, H₂O₂ had a significant inhibitory effect on biofilm formation by Staphylococcus aureus RN422O, even at a concentration well below the M.I.C.

Conclusion: We conclude that oxidative stress may have an antibiofilm action on certain Staphylococcal species, which is independent from its bactericidal effect, and which is manifest at a concentration below the M.I.C. for that species.

Introduction: Despite significant progress at the molecular level the etiology of aseptic loosening is still unclear. Fibrosis of the new capsule is an invariable finding at revision hip arthroplasty. Tissue fibrosis has been demonstrated in varies pathologic conditions due to elevated oxidative stress. The present retrospective study was designed to proof the hypothesis that peri-prosthetic fibrosis in aseptic loosening may be caused by elevated oxidative stress and represent an initial step in the pathomechanism of aseptic loosening.

Material and methods: Levels of malondialdehyde (MDA), oxidized (GSSG) and reduced (GSH) gluthatione were assayed as markers of oxidative stress in retrieved capsules of 28 loose hips (Group I) and 12 hips revised for high rate of wear (Group II). Collagen in the periprosthetic tissues was measured as hydroxiproline content and semiquantitatively by electrophoresis. In four representative cases electron microscopy was performed.

Results: MDA level as well as GSH/GSSG and GSH/ GSSG² ratios showed elevated oxidative stress in group I compared to group II and controls. SDS-PAGE electrophoresis showed higher molecular bands in 20 patients compared to controls. Hydroxiproline level in group II is significantly higher than in group I (p< 0.05). MDA, GSH and GSSG correlate significantly with hydroxiproline. A negative correlation between collagen content and osteolysis was established.

Discussion and conclusion: Higher oxidative stress plays role in aseptic loosening of hip arthroplasty. The present data support the hypothesis that the process is initiated by excessive fibrosis which consequently might lead to increase of intraarticular pressure and to extension of the joint space.

Hip surface replacement is an alternative for young patients considered for hip replacement. The in vivo release of ions from these surfaces has yet to be well evaluated. In the present study, we compared the concentrations of metal ions in blood of patients with hip surface replacement and metal-on-metal (MM) total hip arthroplasty (THA).

Blood was collected six months and one year after implantation time into Sarstedt Monovette® tubes for trace metal analysis from patients having Articular Surface Replacement (ASR®, DePuy Orthopaedics; n=61), 28 mm-head MM THA (n=18), and 36 mm-head MM THA (n=25). The concentrations of cobalt (Co), chromium (Cr), and molybdenum (Mo) were analyzed by inductively coupled plasma-mass spectroscopy (ICP-MS). Since metal ions are potent inducers of oxidative stress, total antioxidant, peroxide, and nitrotyrosine levels (oxidative stress markers) were also measured in plasma of the patients.

The median Co and Cr levels progressively and significantly increased in the three groups during the first year post-operation (compared to patients without hip bearings (n=25)). After six months, the levels of Co and Cr were significantly higher in patients with ASR and 28 mm MM THA than in patients with 36 mm MM THA. There was no difference after one year. The level of activity, as measured by the UCLA activity score, was higher in ASR patients than in 28 and 36 mm MM THA after one year. No differences were observed for Mo levels in these patients when compared to our control group. There was no increase of oxidative stress marker levels in patients with ASR and 36 mm MM THA and no correlation between the concentrations of Co and Cr ions and the levels of oxidative stress markers.

Our results show that, at one year post-operation, the concentration of ions in patients with ASRs is similar than those in patients with MM THAs. Moreover, results suggest that metal ions liberated from MM bearings do not induce damage to macromolecules by oxidative stress in plasma of patients. Longer follow-ups are still required to characterise the concentration of ions in ASR and to determine conclusively the effects of elevated circulating ions.

The effects of Hypericum perforatum on nerve regeneration after sciatic nerve injury have not yet been evaluated in all its aspects yet. In this experimental study, the effect of Hypericum perforatum on injured nerve tissue was histologically and biochemically investigated. Motor functional healing was surveyed by gait analysis.

Rats were divided into 3 groups: Group I (n=8) was intact control group and no intervention and treatment was applied to this group. Group II (n=16) was surgical control group and Group III (n=16) was Hypericum perforatum group. After the operation, while any treatment was performed on Group II, 30 mg/kg dose Hypericum perforatum extract was intraperitoneally administered to the Group III per day for 8 weeks from the 1^st day of post-op. Gait analysis was made to all rats for functional evaluation at 2^nd, 3^rd, 4^th, 6^th and 8^th weeks, and sciatic functional index (SFI) was evaluated. At the end of the eighth week, sciatic nerve tissue samples were taken from the sacrificed rats. Tissues were examined biochemically, histologically and immnohistochemically. Malondialdehyde (MDA) as an indicator of oxidative stress and main antioxidant enzyme [superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT)] levels were biochemically measured. The nerve degeneration and regeneration were histologically viewed, and also cell count was immnohistochemically done by having done anti-S100 staining.

It was seen that measurement results of SFI were statistically significantly difference between groups (p<0,001). In the sciatic nerve tissue samples taken from the rats, it was not determined a statistically significant difference between MDA, SOD, GPx and CAT levels detected by ELISA method (p>0,05). In the histological evaluation, it was seen that Hypericum perforatum affected positively the regeneration and immunohistochemically, it was found a statistically significant difference between the anti-S-100 positive cell numbers.

The obtained results in this study show that; Hypericum perforatum, which was intraperitoneally administered on rats subjected to nerve injury, has affected positively the nerve regeneration and it can also provide an insight to future studies.

Introduction: Metal on metal hip implants continue to be successful alternatives to conventional bearings in younger patients with osteoarthritis. Levels of metal ions such as cobalt (Co) and chromium (Cr) increase in patients with metal bearing hip replacements and resurfacings. These particles are cytotoxic, induce bone loss, and lead to malignant tumors in rats. A subset of these patients are considered outliers as they have unusually high levels of Co and Cr ions. Given the increasing prevalence of metal bearings and the potential for cellular toxicity, we attempted to determine whether patient or surgical factors could account for abnormally elevated ion levels. Methods: We analyzed the Co and Cr levels from whole blood in 661 patients with metal on metal hip bearings. Patient outliers were defined as those who had ion levels ≥ three-fold the mean value. Twenty-four patients (3.6%) had abnormally high metal ion levels, which included 15 patients that underwent total hip replacements and 9 patients following hip resurfacings. These patients were followed prospectively with the Harris Hip Score (HHS) and the University of California Los Angeles (UCLA) activity score. Serial radiographs and ion levels were analyzed at regular intervals. Oxidative stress markers (total anti-oxidants, peroxide, and nitro-tyrosine) were also measured from whole blood to determine if these correlated with an increase ion levels in outlier patients. Results: Post-operative HHS and UCLA activity scores improved significantly compared to pre-operative values. There was no statistical correlation between outlier ion levels, patient demographics, HHS and UCLA activity scores. Radiologic parameters such as cup inclination and femoral component neck-shaft angle could not account for higher ion levels in these outliers. Oxidative stress markers were similar to the levels observed in the control patients with normal ion values following with metal on metal hip implants. Conclusion: We could not identify any patient or surgical factors that could explain the abnormally high metal ion levels in the outlier patients. This suggests that the cause of ion level increase is multifactorial. The clinical relevance of such high levels of ions remains unknown given that there was no increase in serum oxidative stress markers. Further studies are necessary to better understand the effect of abnormal elevations in metal ions given the recent concerns of pseudotumours following metal on metal hip implants

Introduction: Oxidative stress occurs when reactive oxygen species (ROS) are produced faster than they can be removed by cellular defence mechanisms contributing to ageing, many chronic diseases, such as atherosclerosis, RA, Parkinson and Alzheimer’s disease and skeletal pathologies. Here we address the impact of ROS on the viability of early osteogenic precursors in the bone marrow and study the influence of estrogen on this interaction. Cells have a number of mechanisms to protect themselves from ROS, which are constantly being formed in the cell through normal metabolic pathways, such as Vitamin E, C and estrogen. Estrogen has been shown to prevent intracellular accumulation of peroxide and to attenuate oxidant-induced death of neuronal and endothelial cells. In addition, it contributes significantly to bone turnover and relieves postmenopausal symptoms. This study has focused on the potential anti-oxidant properties of estrogen against oxidative on bone marrow stromal cells. stress induced by H. 2. O. 2. Methods: Primary bone marrow stromal cells were pre-treated with several different doses between 10. −6. M – 10. −8. M of estrogen prior to H. 2. O. 2. administration at 0.08–0.4 mM 30% (v/v) for 2–24h. The cellular production of ROS was determined by using the free radical indicator DCFH-DA. Apoptosis was determined by morphological criteria. Results: H. 2. O. 2. induced an increase in apoptosis of osteoprogenitor cells (p< 0.05). Determination of apoptosis and cell number by nuclear staining, indicated that pre-treatment of bone marrow stromal cells with 17-beta estradiol reduced the apoptotic response induced by H. 2. O. 2. (p< 0.05) and restored cell number to control levels. In order to test the anti-oxidant activity of estrogen, the dye DCFH-DA was introduced in a cell free system in the presence or absence of 17-beta estradiol and H. 2. O. 2. The same experiment was repeated in the presence of bone marrow stromal cells. H. 2. O. 2. increased both intracellularly and extracellularly oxidant activity and estradiol has the capacity of modifying this activity both inside and outside the cell. Discussion: These data demonstrate the ability of estrogen, used at physiological doses, to block oxidant-induced apoptosis of osteoprogenitor cells. Estrogen appears to reduce the generation of ROS in these cells. These data could have important implications on the maintenance of osteogenic stem cells during fractures, ageing and disease