Synovitis has been shown to play a role in pathophysiology of OA promoting cartilage destruction and pain. Synovium is mainly composed of synovial fibroblast (SF) and macrophage (SM) that guide synovial inflammation. Adipose stromal cells (ASC) promising candidate for cell therapy in OA are able to counteract inflammation. Two different subsets of macrophages have been described showing a pro-inflammatory (M1) and an anti-inflammatory (M2) phenotype. Macrophage markers: CD68, CD80 (M1-like) and CD206 (M2-like) were evaluated in osteoarthritic synovial tissue. GMP-clinical grade ASC were isolated from subcutaneous adipose tissue and M1-macrophages were differentiated from CD14+ obtained from peripheral blood of healthy donors. ASC were co-cultured in direct and indirect contact with activated (GM-CSF+IFNγ)-M1 macrophages for 48h. At the end of this co-culture we analyzed IL1β, TNFα, IL6, MIP1α/CCL3, S100A8, S100A9, IL10, CD163 and CD206 by qRT-PCR or immunoassay. PGE2 blocking experiments were performed. In moderate grade OA synovium we found similar percentages of CD80 and CD206. M1-activated macrophage factors IL1β, TNFα, IL6, MIP1α/CCL3, S100A8 and S100A9 were down-modulated both co-culture conditions. Moreover, ASC induced the typical M2 macrophage markers IL10, CD163 and CD206. Blocking experiments showed that TNFα, IL6, IL10, CD163 and CD206 were significantly modulated by PGE2. We confirmed the involvement of PGE2/COX2 also in CD14+ OA synovial macrophages. In conclusion we demonstrated that ASC are responsible for the switching of activated-M1-like to a M2-like anti-inflammatory phenotype, mainly through PGE2. This suggested a specific role of ASC as important determinants in therapeutic dampening of synovial inflammation in OA

Staphylococcus aureus is a highly virulent pathogen and implicated in approximately 50% of cases of septic arthritis. Studies investigating other S. aureus-related infections suggest that alpha-(Hla), beta-(Hlb) and gamma-(Hlg) toxins are key virulence factors, with the ‘pore-forming’ alpha-toxin considered the most potent. Here, we have assessed the influence of alpha-toxin alone on in situ chondrocyte viability. Osteochondral explants were harvested from the metacarpophalangeal joints of 3-year-old cows and cultured in Dulbecco's Modified Eagle's Medium. The flasks were then inoculated with isogenic ‘knockout’ strains of S. aureus: DU5946 (Hla+Hlb-Hlg-: alpha-toxin only strain) or DU1090 (Hla-Hlb+Hlg+: beta- and gamma-toxin only strain). Explants were incubated (37°C) and stained after 18, 24 and 40hrs with chloromethylfluorescein-di-acetate and propidium iodide, labelling living chondrocytes green and dead cells red, respectively. Axial sections were imaged by confocal microscopy and the percentage cell death determined. Alpha-toxin-producing S. aureus caused 24.8+/−3.7% chondrocyte death at 18hrs and 44.6+/−7.2% death at 24hrs. At 40hrs, there was significantly more chondrocyte death (87.4+/−3.6%;p<0.001) compared to the alpha-toxin knockout strain, which was negligible (4.1+/−1.7%; means+/−SEM; N=4 independent experiments). In this in vitro bovine cartilage explant model, whereby the effects of defined toxins were determined in isolation of a complex host immune response, in situ chondrocyte viability was dramatically and exclusively reduced by alpha-toxin. This work forms the basis for developing a rational treatment to reduce the extent of cartilage destruction during an episode of septic arthritis. IDMS was supported by Orthopaedic Research UK and The Royal College of Surgeons of Edinburgh

Few studies have investigated the direct effect of bacteria and their products on articular cartilage chondrocytes ex vivo. An ex vivo model that allows the analysis of chondrocytes in situ would therefore be an important and exciting area of future research. It was hypothesised that a bovine cartilage explant model of septic arthritis would be an ideal model for providing fundamental information on the basic cellular mechanisms of cartilage destruction and chondrocyte death induced by bacterial infection uncomplicated by the immune response. A fresh metacarpophalangeal joint from an abattoir slaughtered 3-year-old cow was skinned, rinsed in water and opened under sterile conditions. The cartilage explants were harvested using surgical scalpels and placed into a total of three tissue culture bottles (2 explants per bottle) containing 10ml Dulbecco's Modified Eagle Medium (DMEM). 50ml of a knee aspirate from a patient with septic arthritis, containing Group B streptococci (GBS), was added to bottle 1, 50ml of a negative knee aspirate was added to bottle 2 and 50ml DMEM to bottle 3. The explants were incubated at 37°C for 24 hours. They were then stained with the fluorescent probes Chloromethylfluorescein Di-acetate (CMFDA) and Propidium Iodide and analysed using a Confocal Scanning Laser Microscope. Cell counts to assess percentage cell death were performed using Velocity 4 software. There was strikingly more cell death observed at 24 hours in the cartilage explant exposed to bacteria in comparison to the non-infected controls. The percentage chondrocyte death was 43% in the presence of GBS, 0.8% in the presence of the negative aspirate and 0.2% in the presence of the DMEM control. Although this is a very preliminary pilot study, it demonstrates an extremely rapid effect on the cartilage. Future bovine explant studies of septic arthritis will therefore be feasible and achievable

Summary Statement. Dickkopf-3 is upregulated in OA cartilage and synovial tissue. In vitro studies show Dkk3 can prevent cartilage degradation and antagonise Wnt signaling. We hypothesis that Dkk3 can protect against OA-related cartilage destruction. Introduction. Our group has previously shown that Dkk3, a member of the Dkk family of Wnt antagonists, is upregulated in OA cartilage and synovium. Levels of Dkk3 in synovial fluid are also increased in individuals with tricompartmental OA and after arthroscopy. The role of Dkk3 in cartilage or the factors regulating its expression are not currently understood. Correct regulation of cell signalling pathways is integral to cartilage homeostasis and thus the prevention of OA pathogenesis. Dkk3 is a member of the Dkk family of Wnt antagonists and therefore may impact on chondrocyte biology through interaction with the Wnt pathway. Dkk3 has also been found to influence TGFβ signalling in other cell systems. Methods. Expression of Dkk3 was assessed in primary human articular chondrocytes (HAC) following treatment with interleukin-1,-6 (IL1, IL6), TNFα, FGF2 and oncostatin-M (OSM). Dkk3 expression was assessed following ex vivo injury of murine cartilage explants. The effect of Dkk3 on IL1/OSM-induced proteoglycan and collagen release from explants of bovine nasal (BNC)- and primary human-cartilage was assessed. SW1353 chondrosarcoma cells were treated with Dkk3+/−Wnt3a, TGFβ and Activin and TOPFlash and CAGA luciferase reporters used to measure Wnt and Smad signalling. RNA was extracted from primary HAC treated with Dkk3+/−TGFβ or Wnt3a. ADAM12 and TIMP3 expression were measured to assess TGFβ signalling and AXIN2 to assess Wnt signalling. Micromass HAC were treated with Wnt3a +/− Dkk3 and proteoglycan output assessed using alcian blue staining. β-catenin was silenced in primary HAC prior to TGFβ and Activin treatment. Dkk3 was silenced in primary HAC for microarray analysis. Results. Dkk3 expression was decreased in primary HAC following IL1/OSM treatment but increased by TNFα. Dkk3 expression was decreased immediately following injury to murine explants. In BNC explants, IL1/OSM-induced proteoglycan release was inhibited by Dkk3. Dkk3 antagonised chondrocyte Wnt signalling and Wnt3a-induced reductions in proteoglycan production in micromass cultures. Interestingly, Dkk3 enhanced TGFβ signalling, increasing TGFβ-induced TIMP3 and ADAM12 expression and TGFβ-induced luciferase from the CAGA-luc reporter. In contrast Dkk3 antagonised Activin-induced CAGA-luc activity, TIMP3 and ADAM12 expression. β-catenin knockdown did not significantly alter TGFβ- or Activin-induced expression of TIMP3 or ADAM12, suggesting that Dkk3-effects on these pathways is not mediated solely by inhibition of Wnt signalling. Conclusions. Dkk3 expression is increased in OA and can be regulated injury and inflammatory cytokines. This suggests a balance of Dkk3 effects depending upon the biological stimuli within the cartilage. Dkk3 may act in a protective role in the presence of inflammatory cytokines as exemplified by its ability to inhibit matrix loss. Dkk3 knockdown decreases DICER expression and thus changes in Dkk3 expression in OA may alter chondrocyte phenotype through alterations in miRNA activity. The ability of Dkk3 to antagonise Wnt, enhance TGFβ and antagonise Activin signalling would have multiple effects on chondrocyte activity. These results imply that Dkk3 could influence multiple OA-relevant processes, protect cartilage from degradation and be important in cartilage development and homeostasis

Objectives

The objectives of this study were: 1) to examine osteophyte formation, subchondral bone advance, and bone marrow lesions (BMLs) in osteoarthritis (OA)-prone Hartley guinea pigs; and 2) to assess the disease-modifying activity of an orally administered phosphocitrate ‘analogue’, Carolinas Molecule-01 (CM-01).

Objectives

This study looked to analyse the expression levels of microRNA-140-3p and microRNA-140-5p in synovial fluid, and their correlations to the severity of disease regarding knee osteoarthritis (OA).

Objectives

Interleukin 18 (IL-18) is a regulatory cytokine that degrades the disc matrix. Bone morphogenetic protein-2 (BMP-2) stimulates synthesis of the disc extracellular matrix. However, the combined effects of BMP-2 and IL-18 on human intervertebral disc degeneration have not previously been reported. The aim of this study was to investigate the effects of the anabolic cytokine BMP-2 and the catabolic cytokine IL-18 on human nucleus pulposus (NP) and annulus fibrosus (AF) cells and, therefore, to identify potential therapeutic and clinical benefits of recombinant human (rh)BMP-2 in intervertebral disc degeneration.

Objectives

Osteoarthritis (OA) is characterised by articular cartilage degradation. MicroRNAs (miRNAs) have been identified in the development of OA. The purpose of our study was to explore the functional role and underlying mechanism of miR-138-5p in interleukin-1 beta (IL-1β)-induced extracellular matrix (ECM) degradation of OA cartilage.