Previous research has shown an increase in chromosomal aberrations in patients with worn implants. The type of aberration depended on the type of metal alloy in the prosthesis. We have investigated the metal-specific difference in the level of DNA damage (DNA stand breaks and alkali labile sites) induced by culturing human fibroblasts in synovial fluid retrieved at revision arthroplasty. All six samples from revision cobalt-chromium metal-on-metal and four of six samples from cobalt-chromium metal-on-polyethylene prostheses caused DNA damage. By contrast, none of six samples from revision stainless-steel metal-on-polyethylene prostheses caused significant damage. Samples of cobalt-chromium alloy left to corrode in phosphate-buffered saline also caused DNA damage and this depended on a synergistic effect between the cobalt and chromium ions. Our results further emphasise that epidemiological studies of orthopaedic implants should take account of the type of metal alloy used

Intervertebral disc (IVD) degeneration is a major cause of low back pain (LBP). Degenerate discs are associated with accelerated cellular senescence. Cell senescence is associated with a secretory phenotype characterised by increased production of catabolic enzymes and cytokines. However, to date, the mechanism of cell senescence within disc degeneration is unclear. Senescence can be induced by increased replication or induced by stress such as reactive oxygen species or cytokines. This study investigated the association of cellular senescence with markers of DNA damage and presence of cytoplasmic DNA (which in cancer cells has been shown to be a key regulator of the secretory phenotype), to determine mechanisms of senescence in disc degeneration. Immunohistochemistry for the senescence marker: p16INK4A was firstly utilised to screen human intervertebral discs for discs displaying at least 30% immunopostivity. These discs were then subsequently analysed for immunopostivity for DNA damage markers γH2AX and cGAS and the presence of cytoplasmic DNA. The number of immunopositive cells for p16 INK4A positively correlated with the expression of γH2AX and cGAS. Senescent cells were also associated with the presence of cytoplasmic DNA. These new findings elucidated a role of cGAS and γH2AX as a link from genotoxic stress to cytokine expression which is associated with senescent cells. The findings indicate that cellular senescence in vivo is associated with DNA damage and presence of cytoplasmic DNA. Whether this DNA damage is a result of replicative senescence or stress induced is currently being investigated in vitro

Objectives. The cytotoxicity induced by cobalt ions (Co. 2+. ) and cobalt nanoparticles (Co-NPs) which released following the insertion of a total hip prosthesis, has been reported. However, little is known about the underlying mechanisms. In this study, we investigate the toxic effect of Co. 2+. and Co-NPs on liver cells, and explain further the potential mechanisms. Methods. Co-NPs were characterised for size, shape, elemental analysis, and hydrodynamic diameter, and were assessed by Transmission Electron Microscope, Scanning Electron Microscope, Energy Dispersive X-ray Spectroscopy and Dynamic Light Scattering. BRL-3A cells were used in this study. Cytotoxicity was evaluated by MTT and lactate dehydrogenase release assay. In order to clarify the potential mechanisms, reactive oxygen species, Bax/Bcl-2 mRNA expression, IL-8 mRNA expression and DNA damage were assessed on BRL-3A cells after Co. 2+. or Co-NPs treatment. Results. Results showed cytotoxic effects of Co. 2+. and Co-NPs were dependent upon time and dosage, and the cytotoxicity of Co-NPs was greater than that of Co. 2+. In addition, Co-NPs elicited a significant (p < 0.05) reduction in cell viability with a concomitant increase in lactic dehydrogenase release, reactive oxygen species generation, IL-8 mRNA expression, Bax/Bcl-2 mRNA expression and DNA damage after 24 hours of exposure. Conclusion. Co-NPs induced greater cytotoxicity and genotoxicity in BRL-3A cells than Co. 2+. Cell membrane damage, oxidative stress, immune inflammation and DNA damage may play an important role in the effects of Co-NPs on liver cells. Cite this article: Y. K. Liu, X. X. Deng, H.L. Yang. Cytotoxicity and genotoxicity in liver cells induced by cobalt nanoparticles and ions. Bone Joint Res 2016;5:461–469. DOI: 10.1302/2046-3758.510.BJR-2016-0016.R1

Summary Statement. DNA damage induced by systemic drugs or local γ-irradiation drives disc degeneration and DNA repair ability is extremely important to help prevent bad effects of genotoxins (DNA damage inducing agents) on disc. Introduction. DNA damage (genotoxic stress) and deficiency of intracellular DNA repair mechanisms strongly contribute to biological aging. Moreover, aging is a primary risk factor for loss of disc matrix proteoglycan (PG) and intervertebral disc degeneration (IDD). Indeed, our previous evidences in DNA repair deficient Ercc1−/Δ mouse model strongly suggest that systemic aging and IDD correlate with nuclear DNA damage. Thus the aim of the current study was to test whether systemic or local (spine) genotoxic stress can induce disc degeneration and how DNA repair ability could help prevent negative effects of DNA damage on IDD. To test this hypothesis a total of twelve Ercc1−/Δ mice (DNA repair deficient) and twelve wild-type mice (DNA repair competent) were challenged with two separate genotoxins to induce DNA damage, i.e. chemotherapeutic crosslinking agent mechlorethamine (MEC) and whole-body gamma irradiation. Local effects of gamma irradiation were also tested in six wild-type mice. Methods. Ercc1. −/Δ. mice (n=6) and their wild-type littermates were chronically exposed to genotoxic stress beginning at 8 wks of age by subcutaneous administration of a subtoxic dose of MEC (8 μg/kg once per week for 6 weeks). Similarly, six Ercc1. −/Δ. mice and their wild-type littermates were exposed to genotoxic stress by whole-body administration of ∼10% radiotherapeutic dose of ionizing radiation (0.5 Gy 1x per week for 10 weeks). A third set of wild-type mice (n=6) were exposed to one shot local spine irradiation at 0, 6, and 10 Gy at 22 weeks old and sacrificed 10 weeks later. Histological staining for proteoglycan (Safranin O) and collagen (Masson's Trichrome), PG synthesis (. 35. S-sulfate incorporation) and GAG content (DMMB assay), disc ADAMTS4, aggrecan and its fragments terminating in NITEGE-. 373. (immunohistochemistry (IHC)) were analyzed. Cellular senescence markers (p16) and apoptosis (TUNEL assay) were also measured. Results. Histological staining revealed substantial reduction in matrix collagen, proteoglycan, and endplate cellularity in the discs of MEC-exposed and irradiated mice. IHC analysis showed decreased aggrecan and increased levels of ADAMTS4 and NITEGE-. 373. containing aggrecan proteolytic fragments. Disc PG synthesis was reduced 2–3 folds in MEC-treated mice and irradiated mice. Locally irradiated mice showed similar effects on disc matrix. Expression of p16 as well as apoptosis significantly increased in MEC-treated and irradiated mice. The overall effect of the treatments on disc matrix and endplate cartilage was more severe in Ercc1−/Δ mice than wild-type mice. Discussion/Conclusion. MEC and IR treatment resulted in loss of disc matrix proteoglycan and collagen in adult wild-type and Ercc1−/Δ mice. The finding that loss of matrix proteoglycan was greater in the DNA repair deficient mice strongly supports the conclusion that DNA damage can drive disc degeneration and DNA repair ability is extremely important to help prevent these effects. Results of this work suggest that patients treated with genotoxic drugs (i.e. long-term cancer survivors) may be at increased risk of IDD

Since 2010, there has been a sharp decline in the use of metal-on-metal joint replacement devices due to adverse responses associated with the release of metal wear particles and ions in patients. Surface engineered coatings offer an innovative solution to this problem by covering metal implant surfaces with biocompatible and wear resistant materials. The present study tests the hypothesis whether surface engineered coatings can reduce the overall biological impact of a device by investigating recently introduced silicon nitride coatings for joint replacements. Biological responses of peripheral blood mononuclear cells (PBMNCs) to Si3N4 model particles, SiNx coating wear particles and CoCr wear particles were evaluated by testing cytotoxicity, inflammatory cytokine release, oxidative stress and genotoxicity. Clinically relevant wear particles were generated from SiNx-on-SiNx and CoCr-on-CoCr bearing combinations using a multidirectional pin-on-plate tribometer. All particles were heat treated at 180°C for 4 h to destroy endotoxin contamination. Whole peripheral blood was collected from healthy donors (ethics approval BIOSCI 10–108, University of Leeds). The PBMNCs were isolated using Lymphoprep (Stemcell) and incubated with particles at various volumetric concentrations (0.5 to 100 µm3 particles/cell) for 24 h in 5% (v/v) CO2 at 37°C. After incubation, cell viability was measured using the ATPlite assay (Perkin Elmer); TNF-alpha release was measured by ELISA (Invitrogen); oxidative stress was measured using H2DCFDA (Abcam); and DNA damage was measured by comet assay (Tevigen). The results were expressed as mean ± 95% confidence limits and the data was analysed using one-way ANOVA and Tukey-Kramer post-hoc analysis. No evidence of cytotoxicity, oxidative stress, TNF-alpha release, or DNA damage was observed for the silicon nitride particles at any of the doses. However, CoCr wear particles caused cytotoxicity, oxidative stress, TNF-alpha release and DNA damage in PBMNCs at high doses (50 µm3 particles per cell). This study has demonstrated the in-vitro biocompatibility of SiNx coatings with primary human monocytic cells. Therefore, surface engineered coatings have potential to significantly reduce the biological impact of metal components in future orthopaedic devices

Chondrocytic activity is downregulated by compromised autophagy and mitochondrial dysfunction to accelerate the development of osteoarthritis (OA). Irisin is a cleaved form of fibronectin type III domain containing 5 (FNDC5) and known to regulate bone turnover and muscle homeostasis. However, little is known about the role of irisin in chondrocytes and the development of OA. This talk will shed light on FNDC5 expression by human articular chondrocytes and compare normal and osteoarthritic cells with respect to autophagosome marker LC3-II and oxidative DNA damage marker 8-hydroxydeoxyguanosine (8-OHdG). In chondrocytes in vitro, irisin improves IL-1β-mediated growth inhibition, loss of specific cartilage markers and glycosaminoglycan production. Irisin further suppressed Sirt3 and UCP- 1 to improve mitochondrial membrane potential, ATP production, and catalase. This attenuated IL-1β-mediated production of reactive oxygen species, mitochondrial fusion, mitophagy, and autophagosome formation. In a surgical murine model of destabilization of the medial meniscus (DMM) intra-articular administration of irisin alleviates symptoms like cartilage erosion and synovitis. Furthermore, gait profiles of the treated limbs improved. In chondrocytes, irisin treatment upregulates autophagy, 8-OHdG and apoptosis in cartilage of DMM limbs. Loss of FNDC5 in chondrocytes correlates with human knee OA and irisin repressed inflammation-mediated oxidative stress and deficient extracellular matrix synthesis through retaining mitochondrial biogenesis and autophagy. The talk sheds new light on the chondroprotective actions of this myokine and highlights the remedial effects of irisin during progression of OA

The use of mesenchymal stromal cells (MSCs) in regenerative medicine and tissue engineering is well established, given their properties of self-renewal and differentiation. However, several studies have shown that these properties diminish with age, and understanding the pathways involved are important to provide regenerative therapies in an ageing population. In this PRISMA systematic review, we investigated the effects of chronological donor ageing on the senescence of MSCs. We identified 3023 studies after searching four databases including PubMed, Web of Science, Cochrane, and Medline. Nine studies met the inclusion and exclusion criteria and were included in the final analyses. These studies showed an increase in the expression of p21, p53, p16, ROS, and NF- B with chronological age. This implies an activated DNA damage response (DDR), as well as increased levels of stress and inflammation in the MSCs of older donors. Additionally, highlighting the effects of an activated DDR in cells from older donors, a decrease in the expression of proliferative markers including Ki67, MAPK pathway elements, and Wnt/ -catenin pathway elements was observed. Furthermore, we found an increase in the levels of SA- -galactosidase, a specific marker of cellular senescence. Together, these findings support an association between chronological age and MSC senescence. The precise threshold for chronological age where the reported changes become significant is yet to be defined and should form the basis for further scientific investigations. The outcomes of this review should direct further investigations into reversing the biological effects of chronological age on the MSC senescence phenotype

Abstract. Objective. The aim of our systematic review was to report the latest evidence on the effects of CoCr particles on local soft tissue with a focus on its clinical relevance. Methods. PubMed, Embase, and Cochrane Library databases were screened to perform an extensive review. Inclusion criteria were studies of any level of evidence published in peer-reviewed journals reporting clinical and preclinical results written in English. Relative data were extracted and critically analyzed. PRISMA guidelines were applied, and the risk of bias was assessed, as was the methodological quality of the included studies. Results. 30 studies were included after applying the inclusion and exclusion criteria. Of these, 24 were preclinical studies (18 in vitro human studies, 6 animal modal studies, including 3 in vitro and 3 in vivo), 5 were clinical studies and 1 was previous review on similar topic. The presence of metal ions causes cell damage by reducing cell viability, inducing DNA damage, and triggering the secretion of cytokines. Mechanisms of apoptosis, autophagy and necrosis are responsible for the inflammatory reaction observed in ALTR. Conclusion. The available literature on the effects of CoCr particles released from MoM implants shows that metal debris can cause damage to skeletal muscle, the capsule, and provoke osteolysis and inflammation. Therefore, the cytotoxic and genotoxic damages, as well as the interaction with the immune system, affect the success of the arthroplasty and lead to a higher rate of revision surgeries. Declaration of Interest. (b) declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported:I declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research project

Histone modifications critically contribute to the epigenetic orchestration of bone development - in part by modifying accessibility of genes to transcription factors. Based on the previous finding that histone H2A deubiquitinase 2A-DUB/Mysm1 interacts with the p53-axis in hematopoiesis and tissue development, we here analyzed the molecular and cellular mechanisms of Mysm1-p53 interplay in bone development. The bone phenotype of 4–5 week-old Mysm1-/- (MKO), Mysm1-/-p53-/- (DKO) and corresponding wildtype (WT) mice was determined using µCT and histology. Primary osteoblasts, mesenchymal stem cells (MSCs) and osteoclasts were isolated from long bones to assess cell proliferation, differentiation, apoptosis and activity. Statistics: one-way ANOVA, p<0.05. MKO mice displayed an osteopenic bone phenotype compared to WT (BV/TV: 5.7±2.9 vs. 12.5±4.2, TbN: 1.3±0.6 vs. 2.7±0.7 1/mm, respectively), and these effects were abolished in DKO mice (BV/TV: 17.8±2.6, TbN: 3.7±0.4 1/mm). MKO mice compared to WT also showed both in vitro and in vivo disturbed osteoclast formation (in vitro: 1.5±1.2 vs. 9.9±1.8 OcN/mm2, in vivo OcN/BPm: 1.4±1.0 vs. 3.0±0.7 cells/mm, respectively) accompanied by increased apoptosis and DNA damage; additional p53 knockout attenuated these effects (7.8±1.8 OcN/mm2 and OcN/BPm: 2.2±1.0 cells/mm). Primary osteoblasts from both MKO and DKO mice showed decreased expression of the transcription factor Runx2 and of the osteogenic markers. ChIP-Seq analysis revealed direct binding of Mysm1 to Runx2 promoter regions in osteoblasts, implying that Mysm1 here regulates osteogenic differentiation. In contrast, MKO-MSCs differentiation did not differ from WT, but DKO-MSCs displayed a significantly increased expression of Alpl, Bglap and Runx2. The different effects of Mysm1-/- in MSCs and osteoblasts presumably resulted from the lower expression level of Mysm1 in MSCs in comparison to mature osteoblasts. Thus, our data demonstrate that H2A deubiquitinase Mysm1 is essential for the epigenetic control of bone development via distinct mechanisms: 1) In osteoclasts, Mysm1 is involved in maturation of osteoclast progenitors and osteoclast survival. 2) In osteoblasts, Mysm1 directly controls Runx2 expression, thereby explaining osteopenic phenotype of MKO mice. 3) In MSCs, Mysm1 may play an inferior role due to low expression level. However, loss of p53 increases Runx2 expression during MSC differentiation, leading to normal bone formation in DKO mice

Disturbed muscular architecture, fatty infiltration and muscular atrophy remain irreversible in chronic rotator cuff tears (RCT) even after repair. Poly-[ADP-ribose]-polymerase 1 (PARP-1), a nuclear factor involved in DNA damage repair, has shown to be a key element in the up-regulation of early muscle inflammation, atrophy and fat deposition. We therefore hypothesized that the absence of PARP-1 would lead to a reduction in muscular architectural damage, early inflammation, atrophy and fatty infiltration subsequent to combined tenotomy and neurectomy in a PARP-1 knock-out mouse model. PARP-1 knock-out (KO group) and standard wild type C57BL/6 (WT group) mice were randomly allocated into three different time points (1, 6 and 12 weeks, total n=72). In all mice the supraspinatus (SSP) and infraspinatus (ISP) tendons of the left shoulder were detached and the SSP muscle was denervated according to a recently established model. Macroscopic muscle weight analysis, retraction documentation using macroscopic suture, magnetic resonance imaging, immunohistochemistry gene expression analysis using real time qPCR (RTqPCR) and histology were used to assess the differences in muscle architecture, early inflammation, fatty infiltration and atrophy between knock out and wild type mice in the supraspinatus muscle. The SSP did retract in both groups, however; the KO muscles and tendons retracted less than the WT muscles (2.1±21mm vs 3.4±0.41mm; p=0.02). Further assessment of muscle architecture demonstrated that the pennation angle was significantly higher in the KO groups at 6 and 12 weeks (28±5 vs 36±5 and 29±4 vs 34±3; p<0.0001). Combined Tenotomy and neurectomy resulted in a significant loss of muscle mass in both groups compared to the contralateral unoperated side (KO group 62±11% and WT group 52±11%, p=0.04) at 6 weeks. But at 12 weeks postoperatively, there was a significant increase in muscle mass to near normal levels in KO group compared to the WT group (14±6% and 42±7% lower muscle mass respectively; p<0.0001) and less fatty infiltration (12.5 ± 1.82% and 19.6 ± 1.96%, p=0.027). Immunohistochemistry revealed a significant decrease in the expression of inflammatory, apoptotic, adipogenic and muscular atrophy genes at both the 1 week and 6 weeks time points, but not at 12 weeks in the KO group compared to the WT group. This was confirmed by histology. Our study is the first to show that knocking out PARP-1 leads to decreased loss of muscle architecture, early inflammation, fatty infiltration and atrophy after combined tenotomy and neurectomy of the rotator cuff muscle. Although the macroscopic muscles reaction to injury is similar in the first 6 weeks, its ability to regenerate is much greater in the PARP-1 group leading to a near normalization of the muscle substance and muscle weight, less retraction, and less fatty infiltration after 12 weeks

Objectives

Recently, high failure rates of metal-on-metal (MOM) hip implants have raised concerns of cobalt toxicity. Adverse reactions occur to cobalt nanoparticles (CoNPs) and cobalt ions (Co²⁺) during wear of MOM hip implants, but the toxic mechanism is not clear.

Objectives

The surface of pure titanium (Ti) shows decreased histocompatibility over time; this phenomenon is known as biological ageing. UV irradiation enables the reversal of biological ageing through photofunctionalisation, a physicochemical alteration of the titanium surface. Ti implants are sterilised by UV irradiation in dental surgery. However, orthopaedic biomaterials are usually composed of the alloy Ti6Al4V, for which the antibacterial effects of UV irradiation are unconfirmed. Here we evaluated the bactericidal and antimicrobial effects of treating Ti and Ti6Al4V with UV irradiation of a lower and briefer dose than previously reported, for applications in implant surgery.

The biological significance of cobalt-chromium wear particles from metal-on-metal hip replacements may be different to the effects of the constituent metal ions in solution. Bacteria may be able to discriminate between particulate and ionic forms of these metals because of a transmembrane nickel/cobalt-permease. It is not known whether wear particles are bacteriocidal.

We compared the doubling time of coagulase negative staphylococcus, Staphylococcus aureus and methicillin resistant S. aureus when cultured in either wear particles from a metal-on-metal hip simulator, wear particles from a metal-on-polyethylene hip simulator, metal ions in solution or a control.

Doubling time halved in metal-on-metal (p = 0.003) and metal-on-polyethylene (p = 0.131) particulate debris compared with the control.

Bacterial nickel/cobalt-transporters allow metal ions but not wear particles to cross bacterial membranes. This may be useful for testing the biological characteristics of different wear debris. This experiment also shows that metal-on-metal hip wear debris is not bacteriocidal.

We carried out a cross-sectional study with analysis of the demographic, clinical and laboratory characteristics of patients with metal-on-metal hip resurfacing, ceramic-on-ceramic and metal-on-polyethylene hip replacements. Our aim was to evaluate the relationship between metal-on-metal replacements, the levels of cobalt and chromium ions in whole blood and the absolute numbers of circulating lymphocytes. We recruited 164 patients (101 men and 63 women) with hip replacements, 106 with metal-on-metal hips and 58 with non-metal-on-metal hips, aged < 65 years, with a pre-operative diagnosis of osteoarthritis and no pre-existing immunological disorders.

Laboratory-defined T-cell lymphopenia was present in13 patients (15%) (CD8⁺ lymphopenia) and 11 patients (13%) (CD3⁺ lymphopenia) with unilateral metal-on-metal hips. There were significant differences in the absolute CD8⁺ lymphocyte subset counts for the metal-on-metal groups compared with each control group (p-values ranging between 0.024 and 0.046). Statistical modelling with analysis of covariance using age, gender, type of hip replacement, smoking and circulating metal ion levels, showed that circulating levels of metal ions, especially cobalt, explained the variation in absolute lymphocyte counts for almost all lymphocyte subsets.