Introduction. Kashin-Beck disease (KBD) is an endemic degenerative osteoarthropathy affecting approximately 3 million people in China (Stone R, 2009). The precise aetiology of KBD is not clear, but the lack of selenium and the pollution of mycotoxins in food are a suspected cause of KBD. In this pilot study, we use a rat model to investigate the effect of low selenium and T-2 toxin on articular cartilage metabolism. Methods. 140 male Sprague-Dawley rats were fed with selenium-deficient or normal diet for 4 weeks to produce a low selenium or normal nutrition status. The rats were then fed for a further 4 weeks with low selenium or normal diets with or without T-2 toxin (100ng per gram body weight per day). The rat knee joints were fixed and paraffin embedded and histological and immunohistochemical staining was performed to analyse the metabolism of articular cartilage. Results. There was increased cell cluster formation in the middle and/or deep zones in rats fed with both diets. However, an apparent cell loss was observed in the low selenium + T-2 toxin group with an apparent increase in caspase-3 staining, indicating the increased cell apoptosis. Moreover, toluidine blue staining was reduced in the low selenium + T-2 toxin group, suggesting a loss of sulphated glycosaminoglycans. Similarly, there was reduced 2B6 and 6C3 staining in the territorial matrix of chondrocytes, indicating a reduced synthesis in 4-sulhated and native CS motifs. In contrast, increased 1B5 staining was observed in the articular cartilage from the low selenium + T-2 toxin group, suggesting a lack of CS sulphatransferase activity. Interestingly, there was increased 7D4 staining in the superficial zone of articular cartilage from low selenium + T-2 toxin group, suggesting an initiation of an osteoarthritis-like lesion. Discussion. These results indicated that low selenium nutrition and T-2 toxin could promote cell apoptosis and disrupt CS-GAG metabolism in ECM of rat articular cartilage in this animal model, which is similar to that observed in KBD patients. Collectively, our results support the hypothesis that low selenium and T-2 toxin are the cause of KBD

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

We have demonstrated that toxins produced by Staphylococcus aureus, a common infective agent in septic arthritis (SA), cause rapid in situ chondrocyte death. Here, we have compared the sensitivity of chondrocytes within the superficial and deep zones (SZ, DZ) of cartilage to the same toxins. Culture medium containing the toxins produced by S. aureus strain 8325-4, which include alpha-, beta-, and gamma-toxin, was prepared. Cartilage explants free of subchondral bone were taken from the metacarpophalangeal joints of 3-year-old cows, and incubated (37°C) with the toxins. Explants were stained after 6hrs with chloromethylfluorescein-di-acetate and propidium iodide, labelling living chondrocytes green and dead cells red, respectively. Full-thickness coronal sections were imaged by confocal microscopy and the percentage cell death within the SZ (100μm from articular surface) and DZ (100μm from subchondral bone interface) determined. Both zones were incubated with the same toxin culture medium for the same time period. At 0hrs, chondrocytes within all zones were >98% viable. However, after incubation with toxin-containing culture medium for 6hrs, 71.9+/−11.2% of the SZ cells were dead compared to only 47.4+/−6.7% in the DZ (p=0.03;data are means+/−SEM;N=4). These results suggest that SZ chondrocytes are considerably more sensitive to S. aureus toxins than those within deeper zones. As SZ chondrocytes are close to the synovial fluid harbouring bacterial toxins, these data emphasise the need to remove bacteria and their products aggressively as part of the treatment of SA. IDMS was supported by Orthopaedic Research UK and The Royal College of Surgeons of Edinburgh

Chondrocytes are essential to the maintenance of articular cartilage and it is thought that chondrocyte death occurs early in septic arthritis. Understanding the causes of chondrocyte death will allow the development of chondroprotective strategies to improve long-term outcomes following septic arthritis. We utilised a murine model of septic arthritis using intra-articular injection of 10µL of a 107 concentration of S. aureus suspended in PBS. Seventy-five adult male C57/Bl6 mice were randomised to receive injection of either S. aurues 8325-4 (a wild-type of S. aurues capable of alpha toxin production), DU1090 (an isogenic mutant of 8325-4 that is identical to 8325-4 other than being incapable of producing alpha toxin) or a PBS control. Establishment of septic arthritis was confirmed through gait changes (5 mice/group), limb swelling and histological changes (10 mice/group). 10 animals from each group were sacrificed at 48 hours and the injected knee joints were dissected before being stained with CFMDA (labelling live chondrocytes green) and PI (labelling dead chondrocytes red). The samples were imaged using a confocal laser scanning microscope and the percentage of chondrocyte death was calculated. Mice injected with S. aureus 8325-4 or DU1090 developed septic arthritis with evidence of weight loss, limb swelling and gait changes whereas these were absent in the control group. There was a significantly higher level of chondrocyte death in the group infected with 8325-4 (2.7% chondrocyte viability) when compared to DU1090 (73.9% chondrocyte viability) and PBS injected mice (95% chondrocyte viability). One-Way ANOVA revealed that the difference between each group was statistically different (p < 0.05). Alpha toxin is the major damaging toxin in S. aurues septic arthritis. Any adverse effect of the immune system is negligible in comparison. Development of treatments counteracting the effect of alpha toxin is required

Depletion of Scleraxis-lineage (ScxLin) cells in adult tendon recapitulates age-related decrements in cell density, ECM organization and composition. However, depletion of ScxLin cells improves tendon healing, relative to age-matched wildtype mice, while aging impairs healing. Therefore, we examined whether ScxLin depletion and aging result in comparable shifts in the tendon cell environment and defined the intrinsic programmatic shifts that occur with natural aging, to define the key regulators of age-related healing deficits. ScxLin cells were depleted in 3M-old Scx-Cre+; Rosa-DTRF/+ mice via diphtheria toxin injections into the hindpaw. Rosa-DTRF/+ mice were used as wildtype (WT) controls. Tendons were harvested from 6M-old ScxLin depleted and WT mice, and 21-month-old (21M) C57Bl/6 mice (aged). FDL tendons (n=6) were harvested for single-cell RNAseq, pooled, collagenase digested, and sorted for single cell capture. Data was processed using Cell Ranger and then aligned to the annotated mouse genome (mm10). Filtering, unsupervised cell clustering, and differential gene expression (DEG) analysis were performed using Seurat. Following integration and sub-clustering of the tenocyte populations, five distinct subpopulations were observed. In both ScxLin depletion and aging, ‘ECM synthesizers’ and ‘ECM organizers’ populations were lost, consistent with disruptions in tissue homeostasis and altered ECM composition. However, in ScxLin depleted mice retention of a ‘specialized ECM remodeler’ population was observed, while aging tendon cells demonstrated inflammatory skewing with retention of a ‘pro-inflammatory tenocyte population’. In addition, enrichment of genes associated with protein misfolding clearance were observed in aged tenocytes. Finally, a similar inflammatory skewing was observed in aged tendon-resident macrophages, with this skewing not observed in ScxLin depleted tendons. These data suggest that loss of ‘ECM synthesizer’ populations underpins disruptions in tendon homeostasis. However, retention of ‘specialized remodelers’ promotes enhanced healing (ScxLin depletion), while inflammatory skewing may drive the impaired healing response in aged tendons

Staphylococcus aureus is the main cause of osteomyelitis and forms biofilm and staphylococcal abscess communities (SACs) in humans. While S. aureus has several toxins with specificity for human targets and working with human host cells would be preferred, for SACs no in vitro models, two-dimensional (2D) or three-dimensional (3D), have been described in literature to date. Advanced 3D in vitro cell culture models enable the incorporation of human cells and resemble in vivo tissue more closely than conventional 2D cell culture. Therefore, the aim of this study was to develop an in vitro model of SACs by using a 3D system. The model should allow for studies into antibiotic tolerance and S. aureus - human host cells interactions. With a clinical isolate (S. aureus JAR) or a lab strain (S. aureus ATCC 49230-GFP), SACs were grown in a collagen gel (1.78 mg/ml, Gibco) supplemented with 200 µl human plasma at 37 °C. Transmission and scanning electron microscopy was used to obtain a detailed overview of SACs, whereas immunofluorescent stainings were done to determine whether the pseudocapsule around SACs consist of fibrin. Antibiotic tolerance of SACs was assessed with 100× the minimal inhibitory concentration (MIC) of gentamicin (Roth). Bacterial clearance of non-establised SACs and established SACs with or without pseudocapsule was determined by exposure to differentiated PLB neutrophil-like cells (differentiation with 1.25% DMSO and 5% FBS for 5 days; dPLB) or primary neutrophils isolated with lymphoprep from fresh heparin blood. Degradation of the pseudocapsule was done with 7.5 µl/ml plasmin (Sigma). Colony forming unit (CFU) counts were performed as quantification method. Statistical analysis was performed with the ANOVA multiple comparison test or, when data was not normally distributed, with a Mann-Whitney U test. We have developed a 3D in vitro model of SACs which after overnight growth were on average 200 micrometers in diameter, consisted of 8 log10 CFUs and were surrounded by an inner and outer fibrin pseudocapsule. The in vitro grown SACs tolerated 100× the MIC of gentamicin for 24h and did not significantly differ from control SACs (p=0.1000). dPLB neutrophil-like cells or primary neutrophils did not clear established in vitro SACs (p=0.1102 and p=0.8767, respectively). When the fibrin pseudocapsule was degraded by the enzyme plasmin, dPLB neutrophil-like cells or primary neutrophils caused for a significant decrease in total CFU compared the SACs that did had a pseudocapsule (p=0.0333 and p=0.0272, respectively). The in vitro SACs model offers a tool for host-pathogen interaction and drug efficacy assessments and is a valuable starting point for future research

Staphylococcus aureus is the most common bacterial isolate in septic arthritis. From studies on isolated cartilage cells, the ‘pore-forming’ alpha and gamma toxins are considered the most virulent factors. However, understanding the response of in situ chondrocytes is important in order to identify new treatments to reduce the extent of cartilage damage during, and following, episodes of septic arthritis. Animal models can give useful information; however the interpretation of data can be complex because of the strong immune response. Thus, to clarify the role of S. aureus toxins on in situ chondrocytes we have developed a bovine cartilage explant model. Metacarpophalangeal joints, from 3-year-old cows, were opened under sterile conditions within 6hrs of slaughter and cartilage explants harvested. Explants were placed into flasks containing Dulbecco's Modified Eagle Medium (DMEM). Aspirates from a patient with septic arthritis of the hip, containing S. aureus, were compared to negative aspirates (no bacterial growth) from a patient with an inflamed knee joint (controls). The explants were incubated at 37 degrees Celsius and stained after 18, 24 and 40hrs with the fluorescent probes chloromethylfluorescein di-acetate and propidium iodide (10 micromolar each) to label living chondrocytes green and dead cells red respectively. Following imaging of cartilage by confocal laser scanning microscopy, the percentage cell death at each time point was obtained using Volocity 4 software. There was no detectable change in chondrocyte viability (<1% cell death) over 40hrs incubation with the negative aspirate. However, for the aspirate from a patient positive for S. aureus, there was a rapid increase in cell death between 18 and 24hrs (0.2 +/− 0.3% to 23 +/− 5% cell death respectively) and almost complete cell death at 40hrs (80 +/− 12%; data are means +/− s.d; n=4). These results show that a strain of S. aureus capable of manifesting clinical disease exerts a potent effect on in situ chondrocytes. In the absence of an immune response, chondrocyte death was purely the result of the bacteria and their products. This bovine cartilage explant model could therefore be useful for studying the effects of S. aureus on chondrocyte behaviour and, ultimately, cartilage integrity

Summary. Staphylococcus aureus isolates from Fracture fixation device related infections contained fewer isolates that form a strong biofilm in comparison with isolates from Prosthetic joint infections. Both orthopaedic implant related infection groups possessed fnbB and sdrE more frequently than the non-implant related infection groups. Introduction. One of the most common pathogen causing musculoskeletal infections is Staphylococcus aureus. The aim was to characterise S. aureus isolated from these infections and to look for differences between the isolates from orthopaedic implant related infections (OIRI) and those in non-implant related infections (NIRI). The OIRI are further differentiated in those associated with fracture fixation (FFI) devices and those found in prosthetic joint infections (PJI). Methods. Three-hundred and five S. aureus isolates were collected from different Swiss and French hospitals (FFI, n=112; PJI, n=105; NIRI, n=88). The cases of NIRI were composed of 27 osteomyelitis (OM), 23 diabetic foot infections (DFI), 27 soft tissue infections (STI) and 11 postoperative spinal infections (SI). Isolates were tested for their ability to form a biofilm. They were typed by agr (accessory gene regulator) group and genes coding for the 13 most relevant MSCRAMMs, Panton-Valentine leukocidin (PVL), PIA (polysaccharide intercellular adhesin), γ-haemolysin, the five most relevant Staphylococcal enterotoxins (SEA-SEE), exfoliative toxins A and B (ETA and ETB) and toxic shock protein (TST) were screened for by PCR. Results. The majority of the S. aureus isolates were methicillin susceptible (MSSA) with 83.4% for the OIRI and 93.2% for the NIRI. All isolates were able to produce a biofilm. A strong biofilm was produced in 13.8% of the OIRI isolates compared to 10.2% of the NIRI isolates. The difference between the isolates of the PJI versus the FFI was statistically significant (20% vs 8%; p=0.011). All four agr types were present in all groups. agrI predominated in the OIRI (42.4%) as well as in the NIRI (44.4%). Comparing OIRI with NIRI, agrII was present in a higher prevalence in OIRI (30.9% vs 14.8%) and agrIII in a lower incidence (21.2% vs 30.7%). Genes cna, clfA and bbp were exhibited predominantly by isolates from the NIRI, while the fnbB and the sdrE gene were more frequently observed among OIRI. Conclusions. Methicillin susceptible S. aureus (MSSA) was more prevalent than methicillin resistant S. aureus (MRSA) in this collection. Possible trends for the orthopaedic device associated infection groups FFI and PJI could be observed whereby isolates from PJI produced stronger biofilm than isolates from the FFI group. The agr type agrII, the fnbB gene and sdrE gene were more prevalent present in the OIRI compared to the NIRI. In contrast, agrIII, and the bbp gene were more prevalent in the NIRI than in the OIRI

Objectives

Implant-related infection is one of the most devastating complications in orthopaedic surgery. Many surface and/or material modifications have been developed in order to minimise this problem; however, most of the in vitro studies did not evaluate bacterial adhesion in the presence of eukaryotic cells, as stated by the ‘race for the surface’ theory. Moreover, the adherence of numerous clinical strains with different initial concentrations has not been studied.

Using a rat model the characteristics of the sensory neurones of the dorsal-root ganglia (DRG) innervating the hip were investigated by retrograde neurotransport and immunohistochemistry.

Fluoro-Gold solution (FG) was injected into the left hip of ten rats. Seven days later the DRG from both sides between T12 and L6 were harvested. The number of FG-labelled calcitonin gene-related peptide-immunoreactive or isolectin B4-binding neurones were counted.

The FG-labelled neurones were distributed throughout the left DRGs between T13 and L5, primarily at L2, L3, and L4. Few FG-labelled isolectin B4-binding neurones were present in the DRGs of either side between T13 and L5, but calcitonin gene-related peptide-immunoreactive neurones made up 30% of all FG-labelled neurones.

Our findings may explain the referral of pain from the hip to the thigh or lower leg corresponding to the L2, L3 and L4 levels. Since most neurones are calcitonin gene-related peptide-immunoreactive peptide-containing neurones, they may have a more significant role in the perception of pain in the hip as peptidergic DRG neurones.