Objective. Osteoarthritis (OA) is polygenic, with more than 90 risk loci currently mapped, including at the singlenucleotide polymorphism rs6516886. Previous analysis of OA cartilage DNA identified 6 CpG dinucleotides with methylation levels that correlated with the rs6516886 genotype, forming methylation quantitative trait loci (mQTLs). We undertook this study to investigate these mQTLs and to map expression quantitative trait loci (eQTLs) across joint tissues in order to identify a particular gene as a target of the rs6516886 association effect. Methods. Nucleic acids were extracted from the cartilage, fat pad, synovium, and peripheral blood from OA patients. Methylation of CpGs and allelic expression imbalance of potential target genes were assessed by pyrosequencing. A chondrocyte cell line expressing deactivated Cas9 (dCas9)-TET1 was used to directly alter CpG methylation levels, with effects on gene expression quantified by polymerase chain reaction. Results. Multiple mQTLs were detected, with effects strongest in joint tissues and methylation at CpG cg20220242 correlating most significantly with the rs6516886 genotype. CpG cg20220242 is located upstream of RWDD2B. Significant rs6516886 eQTLs were observed for this gene, with the OA risk-conferring allele of rs6516886 correlating with reduced expression. CpG methylation also correlated with allelic expression of RWDD2B, forming methylation-expression QTLs (meQTLs). Deactivated Cas9-TET1 reduction in the methylation of cg20220242 increased expression of RWDD2B. Conclusion. The rs6516886 association signal is a multi-tissue meQTL involving cg20220242 and acting on RWDD2B. Modulating CpG methylation reverses the impact of the risk allele. RWDD2B codes for a protein about which little is currently known. Further analysis of RWDD2B as a target of OA genetic risk will provide novel insight into this complex disease.

Metal-on-metal (MoM) hip replacements, often manufactured from a cobalt-chrome alloy, are associated with adverse reactions including soft tissue necrosis and osteolysis. Histopathological analysis of MoM peri-implant tissues reveals an inflammatory cell infiltrate that includes macrophages, monocytes and neutrophils.Toll-like receptor 4 (TLR4) is an innate immune receptor activated by bacterial lipopolysaccharide. Recent studies have demonstrated that cobalt ions from metal-on-metal joints also activate human TLR4, increasing cellular secretion of inflammatory chemokines including interleukin-8 (IL-8, CXCL8) and CCL2. Chemokines recruit immune cells to the site of inflammation, and their overall effect depends on the chemokine profile produced.This study investigated the effect of cobalt on the secretion of inflammatory cytokines CCL20 and IL-6. The chemotactic potential of conditioned media from a cobalt-stimulated human monocyte cell line on primary monocytes and neutrophils was investigated using an in vitro transwell migration assay. The role of TLR4 in observed effects was studied using a small molecule TLR4-specific antagonist.Cobalt ions significantly increased release of CCL2 and IL-6 by MonoMac 6 cells (P<0.001). Conditioned media from cobalt-stimulated cells significantly increased monocyte and neutrophil chemotaxis in vitro (P<0.001). These effects were abrogated by the TLR4 antagonist (P<0.001) suggesting that they occur through cobalt activation of TLR4.This study demonstrates the role of TLR4 in cobalt-mediated immune cell chemotaxis and provides a potential mechanism by which cobalt ions may contribute to the immune cell infiltrate surrounding failed metal hip replacements. It also highlights the TLR4 signalling pathway as a potential therapeutic target in preventing cobalt-mediated inflammation.

Cobalt-containing metal-on-metal hip replacements are associated with adverse reactions to metal debris (ARMD), including inflammatory pseudotumours, osteolysis, and aseptic implant loosening. The exact cellular and molecular mechanisms leading to these responses are unknown. Cobaltions (Co2+) activate human Toll-like receptor 4 (TLR4), an innate immune receptor responsible for inflammatory responses to Gram negative bacterial lipopolysaccharide (LPS).We investigated the effect of Co2+-mediated TLR4 activation on human microvascular endothelial cells (HMEC-1), focusing on the secretion of key inflammatory cytokines and expression of adhesion molecules. We also studied the role of TLR4 in Co2+-mediated adhesion molecule expression in MonoMac 6 macrophages.We show that Co2+ increases secretion of inflammatory cytokines, including IL-6 and IL-8, in HMEC-1. The effects are TLR4-dependent as they can be prevented with a small molecule TLR4 antagonist. Increased TLR4-dependent expression of intercellular adhesion molecule 1 (ICAM1) was also observed in endothelial cells and macrophages. Furthermore, we demonstrate for the first time that Co2+ activation of TLR4 upregulates secretion of a soluble adhesion molecule, sICAM-1, in both endothelial cells and macrophages. Although sICAM-1 can be generated through activity of matrix metalloproteinase-9 (MMP-9), we did not find any changes in MMP9 expression following Co2+ stimulation.In summary we show that Co2+ can induce endothelial inflammation via activation of TLR4. We also identify a role for TLR4 in Co2+-mediated changes in adhesion molecule expression. Finally, sICAM-1 is a novel target for further investigation in ARMD studies.

Osteoarthritis is a painful, disabling condition which is increasing in prevalence as a result of an ageing population. With no recognized disease-limiting therapeutics, arthroplasty of the hip and knee is the most common and effective treatment for lower limb osteoarthritis, however lower limb arthroplasty has a finite life-span and a proportion of patients will require revision arthroplasty. With increasing life expectancy and an increasing proportion of younger (<65 years) patients undergoing arthroplasty, the demand for revision arthroplasty after implant failure is also set to increase. Statins are cholesterol-modulating drugs widely used for cardiovascular risk reduction which have been noted to have pleiotropic effects including potentially influencing arthroplasty survival. In vitro studies have demonstrated pleiotropic effects in human bone cells, including enhancement of osteoblastogenesis following simvastatin exposure, and in vivo studies have demonstrated that intraperitoneal simvastatin can increase peri-implant bone growth in rats following titanium tibial implant insertion. There is evidence that statins may also influence osseointegration, enhancing bone growth at the bone–implant interface, subsequently improving the functional survival of implants. Data from the Danish Hip Arthroplasty Registry and the Clinical Practice Research Datalink in the UK suggest a reduction in the risk of lower limb revision arthroplasty in statin ever-users versus never-users, and a time-dependent effect of statins in reducing the risk of revision. In this article we review the clinical and experimental evidence linking statins and risk of revision arthroplasty.

regulation of key ECM components in OA synovial fibroblasts. Next, we employed "Watson Discovery tool" to identify direct targets of miR-27b-3p to further elucidate its signalling pathway. This tool identified a panel of 11 genes including peroxisome proliferator-activated receptor gamma (PPARg) as direct targets for miR-27b-3p. Indeed, we confirmed that miR-27b-3p mimic treatment results in a marked suppression of PPARg expression, while inhibition of miR-27b-3p upregulates PPARg, suggesting that miR-27b-3p may impart its ECM regulatory effect through modulation of PPARg. Furthermore, ISH of DMM-induced OA mice knee joints further show that miR-27b-3p expression is prominent in the synovial lining and fibrocartilage compared to sham control mice in vivo. Conclusions: Our results thus far suggest that, in OA synovial fibroblasts, miR-27b-3p is involved in the regulation of key ECM components. Our data also suggest that miR-27b-3p may signal via negative regulation of PPARg. Our ongoing studies are confirming if miR-27b-3p signalling via PPARg controls ECM regulation in OA synovial fibroblasts. Our in vivo studies show that miR-27b-3p expression is elevated in the synovium during OA. To comprehensively investigate the endogenous role of miR-27b-3p in synovial pathology, we have intra-articularly injected mouse knee joints with the in-vivo grade mimic of miR-27b-3p to determine any changes in the synovium upon miR-27b-3p treatment in vivo.

Osteoarthritis (OA) is a metabolically active condition characterised by loss of protective hyaline cartilage on joint surfaces. The subsequent contact of articulating bone surfaces within joints leads to subchondral sclerosis, cyst formation as well as osteophytes. This manifests clinically as joint pain, stiffness, swelling and loss of function for 10% of men and 18% of women aged over 60 years [1]. There are no licenced medications to limit OA progression and when symptoms are refractory to conservative treatments such as analgesia and physiotherapy, total joint arthroplasty (TJA) is indicated. Total joint replacementTJA is an effective treatment in end-stage OA using biomaterials to replace diseased joint surfaces and in 2019 there were 95,677 primary total hip replacements performed in England, Wales and Northern Ireland with osteoarthritis being indicated in 91.5% of cases[2]. This number is set to increase with our ageing population and rising obesity. As implants last on average for 78.9% at 20 years there is an urgent need to understand mechanisms driving implant failure, as revision surgery is associated with increased morbidity, mortality, venous thromboembolism rates and poorer subsequent function [3].The most common combination of hip replacement bearing surfaces are a metal alloy femoral head component articulating with a polyethylene acetabular cup, which has been in use since the development of the primary total hip replacement by Sir John Charnley in the 1960s [4]. However, metal-onpolyethylene (MoP) implants have been implicated in adverse reactions to debris as a result of phagocytosis of particulate debris by peri-implant macrophages, leading to osteolysis [5]. Ceramics have become increasingly popular as an alternative bearing surface in femoral head components as compared to MoP, as they are considered to be more biologically inert and have high wear resistance rates, in addition to reduced metal ion release from taper corrosion, which is observed in metal implants complicated by aseptic, lymphocyte-dominated vasculitis lesion (ALVAL)(6, 7). Given the increasing proportion of use of ceramic-on-polyethylene (CoP)

Osteoarthritis is a painful, disabling condition which is increasing in prevalence as a result of an ageing population. With no recognised disease limiting therapeutics, arthroplasty of the hip and knee is the most common and effective treatment for lower limb osteoarthritis, however lower limb arthroplasty has a finite life-span and a proportion of patients will require revision arthroplasty. With increasing life expectancy and an increasing proportion of younger (&lt;65 years) patients undergoing arthroplasty, the demand for revision arthroplasty after implant failure is also set to increase. Statins are cholesterol modulating drugs widely used for cardiovascular risk reduction which have been noted to have pleiotropic effects including potentially influencing arthroplasty survival. In vitro studies have demonstrated pleiotropic effects in human bone cells, including enhancement of osteoblastogenesis following simvastatin exposure, and in vivo studies have demonstrated that intraperitoneal simvastatin can increase peri-implant bone growth in rats following titanium tibial implant insertion. There is evidence also that statins may also influence osseointegration, enhancing bone growth at the bone-implant interface, subsequently improving the functional survival of implants. Data from the Danish Hip Arthroplasty Registry and Clinical Practice Research Datalink in the UK suggest a reduction in the risk of lower limb revision arthroplasty in statin ever-users vs never users, and a time dependent effect of statin administration on reduction in risk of revision. In this article we review the clinical and scientific evidence linking statins and risk of revision arthroplasty.

Toxoplasma gondiihas a relict plastid, the apicoplast; for most proteins that are localized in the apicoplast, N-terminal signal sequences precisely direct this localization. Proteins exclusively found in the apicoplast use a Golgi-independent route for trafficking, while dually targeted proteins found in both the apicoplast and the mitochondrion use a Golgi-dependent route. This report shows that the N-terminus of a dually targeted protein,TgTPx1/2, has all the signals for targeting both to the apicoplast and the mitochondrion yet exhibits flexibility in organellar targeting depending on the reporter protein. When enhanced Green Fluorescent Protein (eGFP) is used as a reporter,TgTPx1/2 targeting signals direct the protein to the mitochondrion, while the same targeting signals directT. gondiiendogenous proteinsTgSOD2 andTgSOD3 to the apicoplast. Chimeric proteins consisting of N-termini and coding sequences from different proteins were tested for both their apicoplast localization and their trafficking route to the apicoplast. This report shows, for the first time, that in addition to the N-terminal signal sequences, targeting and trafficking signals also reside within the coding sequences of apicoplast proteins. Our results hint that during evolution when the apicoplast endosymbiont was transferring its genes to the host nucleus, signal sequences such as those ofTgTPx1/2 enabled sampling of different sub-cellular compartments before the final destination of their protein products was fixed.