Aims. This study explored the shared genetic traits and molecular interactions between postmenopausal osteoporosis (POMP) and sarcopenia, both of which substantially degrade elderly health and quality of life. We hypothesized that these motor system diseases overlap in pathophysiology and regulatory mechanisms. Methods. We analyzed microarray data from the Gene Expression Omnibus (GEO) database using weighted gene co-expression network analysis (WGCNA), machine learning, and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis to identify common genetic factors between POMP and sarcopenia. Further validation was done via differential gene expression in a new cohort. Single-cell analysis identified high expression cell subsets, with mononuclear macrophages in osteoporosis and muscle stem cells in sarcopenia, among others. A competitive endogenous RNA network suggested regulatory elements for these genes. Results. Signal transducer and activator of transcription 3 (STAT3) was notably expressed in both conditions. Single-cell analysis pinpointed specific cells with high STAT3 expression, and microRNA (miRNA)-125a-5p emerged as a potential regulator. Experiments confirmed the crucial role of STAT3 in osteoclast differentiation and muscle proliferation. Conclusion. STAT3 has emerged as a key gene in both POMP and sarcopenia. This insight positions STAT3 as a potential common therapeutic target, possibly improving management strategies for these age-related diseases. Cite this article: Bone Joint Res 2024;13(8):411–426

Aim. While metagenomic (microbial DNA) sequencing technologies can detect the presence of microbes in a clinical sample, it is unknown whether this signal represents dead or live organisms. Metatranscriptomics (sequencing of RNA) offers the potential to detect transcriptionally “active” organisms within a microbial community, and map expressed genes to functional pathways of interest (e.g. antibiotic resistance). We used this approach to evaluate the utility of metatrancriptomics to diagnose PJI and predict antibiotic resistance. Method. In this prospective study, samples were collected from 20 patients undergoing revision TJA (10 aseptic and 10 infected) and 10 primary TJA. Synovial fluid and peripheral blood samples were obtained at the time of surgery, as well as negative field controls (skin swabs, air swabs, sterile water). All samples were shipped to the laboratory for metatranscriptomic analysis. Following microbial RNA extraction and host analyte subtraction, metatranscriptomic sequencing was performed. Bioinformatic analyses were implemented prior to mapping against curated microbial sequence databases– to generate taxonomic expression profiles. Principle Coordinates Analysis (PCoA) and Partial Least Squares-Discriminant Analysis were utilized to ordinate metatranscriptomic profiles, using the 2018 definition of PJI as the gold-standard. Results. After RNA metatranscriptomic analysis, blinded PCoA modeling revealed accurate and distinct clustering of samples into 3 separate cohorts (infected, aseptic, and primary joints) – based on their active transcriptomic profile, both in synovial fluid and blood (synovial anosim p=0.001; blood anosim p=0.034). Differential metatranscriptomic signatures for infected versus noninfected cohorts enabled us to train machine learning algorithms to 84.9% predictive accuracy for infection. Multiple antibiotic resistance genes were expressed, with high concordance to conventional antibiotic sensitivity data. Conclusions. Our findings highlight the potential of metatranscriptomics for infection diagnosis. To our knowledge, this is the first report of RNA sequencing in the orthopaedic literature. Further work in larger patient cohorts will better inform deep learning approaches to improve accuracy, predictive power, and clinical utility of this technology

Hip and knee arthroplasty (HKA) are two of the most successful orthopaedic procedures. However, one major complication necessitating revision surgery is osteolysis causing aseptic loosening of the prosthesis. JAK-STAT has been demonstrated to influence bone metabolism and can be regulated by microRNA (miRNA). Adult patients with osteolysis or aseptic loosening undergoing revision HKA were recruited. Age and gender matched patients undergoing primary hip or knee arthroplasty were our controls. Samples of bone, tissue and blood were collected and RNA isolation was performed. The best quality samples were used for RNA-sequencing. Data analysis was performed using RStudio and Galaxy to identify differentially expressed genes. Western blotting of IL6 was used to confirm protein expression. Five circulating miRNA were identified which had 10 differentially expressed genes in bone and 11 differentially expressed genes in tissue related to the JAK-STAT pathway. IL6 in bone and EpoR in bone were highly significant and IL6 in tissue, MPL in bone, SOCS3 in tissue, JAK3 in bone and SPRED1 in bone were borderline significant. Western blot results demonstrated up-expression of IL6 in bone tissue of revision patients. Periprosthetic osteolysis and aseptic loosening can be attributed to miRNA regulation of the JAK-STAT pathway in osteoblasts and osteoclasts, leading to increased bone resorption. These findings can be used for further experiments to determine utility in the clinical setting for identifying diagnostic markers or therapeutic targets

Aim. The management of PJIs is slowed down by the presence of bacteria forming biofilms where they may withstand antibiotic therapy. The use of adjuvant strategies, such as hydrolytic enzymes cocktail targeting biofilm matrices and facilitating their dispersion, is a promising option to limit impact of biofilms. Our aim was to evaluate the effect of enzymes cocktail combined with antibiotic dual therapy of rifampicin and vancomycin in a relevant in-vitro model. Method. Mature methicillin-resistant Staphylococcus aureus biofilms were grown on Ti-6Al-4V coupons by adding 1mL of a 8Log10 ATCC 33591 suspension in TGN (TSB + 1% glucose + 2% NaCl) to 24-wells plates containing the coupons and incubating the plates for 24h at 37°C with a continuous 50rpm agitation. The samples were rinsed and placed in 6 wells plates containing 1ml of the enzymatic cocktail (C.D.D.) solution (tris-buffered (pH 7.0) solution of 400 U/ml of aspecific DNA/RNA endonuclease, 50 U/ml of endo-1,4-b-D-glucanase, and 0.06 U/ml of β-N-acetylhexosaminidase). 9ml of TGN or TGN containing antibiotics RIF/VAN (rifampicin 5µg/mL + vancomycin 8µg/mL) at clinically relevant concentrations found locally in bone or joints, was then added and the samples were incubated in identical conditions for 24h. The samples were then recovered and rinsed. CFU counts were obtained by recovering the bacteria with sonication, serial dilutions, and TSA plating. Biomass was determined via crystal violet staining, followed by dye solubilization in acetic acid, and absorbance measurement using a spectrophotometer. Results. Significant reductions in bacterial counts were observed in biofilms exposed to either RIF/VAN or RIF/VAN+CDD, by respectively 2,6 and 3,7Log10 when compared to samples reincubated with TGN alone (p <0.05). Additionally, CFU counts in samples exposed to RIF/VAN+CDD were reduced by 1,1Log10 when compared to those exposed to RIF/VAN (p<0,05). Significant reduction in biomass (-29,8%, p<0.05) was observed for coupons exposed to RIF/VAN+CDD when compared to C.D.D alone (figure 1). Conclusions. The concurrent utilization of enzymes with rifampicin and vancomycin, holds promise as a feasible method to address periprosthetic joint infections (PJIs). For any tables or figures, please contact the authors directly

Osteoarthritis (OA) is the most common form of arthritis and one of the ten most disabling diseases in developed countries. Total joint replacement (TJR) is considered by far as the most effective treatment for end-stage OA patients. The majority of patients achieve symptomatic improvement following TJR. However, about 22% of the TJR patients either do not improve or deteriorate after surgery. Several potential non-genetic predictors for the TJR outcome have been investigated. However, the results were either inconclusive or had very limited predictive power. The aim of this study was to identify genetic variants for the poor outcome of TJR in primary OA patients by a genome-wide association study (GWAS). Study participants were total knee or hip replacement patients due to primary OA who were recruited to the Newfoundland Osteoarthritis Study (NFOAS) before 2017. The Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) was used to assess pain and functional impairment pre- and 3.99±1.38 years post-surgery. Two non-responder classification criteria were used in our study. One was defined by an absolute WOMAC change score. Participants with a change score less than 7/20 points for pain were considered as pain non-responders; and those with less than 22/68 points for function were classified as function non-responders. The second one was the Outcome Measures in Arthritis Clinical Trials and the Osteoarthritis Research Society International (OMERACT-OARSI) criteria. Blood DNA samples were genotyped using the Illumina GWAS microarrays genotyping platform. The quality control (QC) filtering was performed on GWAS data before the association of the genetic variants with non-responders to TJR was tested using the GenABEL package in R with adjustment for the relatedness of the study population and using the commonly accepted GWAS significance threshold p < 5*10. −8. to control multiple testing. In total, 316 knee and 122 hip OA patients (mean age 65.45±7.62 years, and 58% females) passed the QC check. These study participants included 368 responders and 56 non-responders to pain, and 364 responders and 68 non-responders to function based on the absolute WOMAC point score change classification. While 377 responders and 56 non-responders to pain, and 366 responders and 71 non-responders to function were identified by the OMERACT-OARSI classification criteria. Interestingly, the same results were obtained by both classification methods, and we found that the G allele of rs4797006 was significantly associated with pain non-responders with odds ratio (OR) of 5.12 (p<7.27×10. -10. ). This SNP is in intron one of the melanocortin receptor 5 (MC5R) gene on chr18. This gene plays central roles in immune response, pain sensitivity, and negative regulation of inflammatory response to antigenic stimulus. The A allele of rs200752023 was associated with function non-responders with OR of 4.41 (p<3.29×10. -8. ). The SNP is located in intron three of the RNA Binding Fox-1 Homolog 3 (RBFOX3) gene on chr17 which has been associated with numerous neurological disorders. Our data suggested that two chromosomal regions are associated with TJR poor outcomes and could be the novel targets for developing strategies to improve the outcome of the TJR

The poor prognosis of patients with soft-tissue sarcoma as not changed in the past several decades, highlighting the necessity for new therapeutic approaches. T-cell based immunotherapies are a promising alternative to traditional cancer treatments due to their ability to target only malignant cells, leaving benign cells unharmed. The development of successful immunotherapy requires the identification and characterization of targetable immunogenic tumor antigens. Cancer-testis antigens (CTA) are a group of highly immunogenic tumor-associated proteins that have emerged as potential targets for CD8+ T-cell recognition. In addition to identifying a targetable antigen, it is crucial to understand the tumor immune microenvironment. The level of immune infiltration and mechanisms of immune suppression within the tumor play important roles in the outcome of immunotherapy. The goal of this study is to identify targetable immunogenic antigens for T-cell based immunotherapy and to characterize the tumor immune microenvironment in human dedifferentiated liposarcoma (DDLS) by Nanostring and IHC. To assess the complexity of the human DDLS tumor immune microenvironment and to identify target antigens we used the nCounter NanoString platform to generate a gene expression profile for hundreds of genes from RNA obtained from 29 DDLS and 10 control fat FFPE samples. To classify inflammatory status of DDLS tumors, we performed hierarchical clustering based on expression levels of selected tumor inflammatory signature genes (CCL5, CD27, CD274, CD276, CD8A, CMKLR1, CXCL9, CXCR6, HLA-DQA1, HLA-E, IDO1, LAG3, PDCDILG2, PSMB10, STAT1, TIGIT). To confirm protein expression and distribution of identified antigens, we performed immunohistochemistry on human tissue micro-arrays encompassing DDLPS tumor tissues and matched normal control tissue from 63 patients. IHC for the cancer testis antigens PBK, SPA17, MAGE-A3, NY-ESO-1 and SSX2 was performed, and the staining results were scored by two authors based on maximal staining intensity on a scale of zero to three (absent=0, weak=1, moderate=2, or strong=3) and the percentage of tumor cells that stained. Hierarchical clustering of DDLS tumors based on expression of tumor inflammation signature genes revealed two distinct groups, consisting of 15 inflamed tumor and 14 non-inflamed tumors, demonstrating tumor heterogeneity within the DDLS sarcoma subtype. All antigens were found to be expressed in DDLS at an mRNA level. SPA17 was expressed at the highest levels in DDLS, however, this antigen was expressed at high levels in normal fat. Notably, antigens PBK and TTK had the largest fold change increase in expression in DDLS compared to normal fat controls. Immunohistochemical analysis of selected antigens revealed that PBK was found to be expressed in 96% (52/54) of DDLS samples at high levels. Other antigens were absent or expressed at low levels in DDLS; MAGEA3 in 15.87% (10/63) NY-ESO-1 in 6.35% (4/62) and SSX2 in 12.7% (8/63) and SPA17 in 5.5% (3/54). This data shows considerable inter-tumoral heterogeneity of inflammation, which should be taken into consideration when designing an immunotherapy for DDLS. To date, these results show promising expression of PBK antigen in DDLS, which may be used as a target in the future development of an immunotherapy for sarcoma

Aims. To describe the incidence of adverse clinical outcomes related to COVID-19 infection following corticosteroid injections (CSI) during the COVID-19 pandemic. To describe the incidence of positive SARS-CoV-2 reverse transcriptase polymerase chain reaction (RT-PCR) testing, positive SARS-COV2 IgG antibody testing or positive imaging findings following CSI at our institution during the COVID-19 pandemic. Methods. A retrospective observational study was undertaken of consecutive patients who had CSI in our local hospitals between 1 February and 30June 2020. Electronic patient medical records (EPR) and radiology information system (RIS) database were reviewed. SARS-CoV-2 RT-PCR testing, SARS-COV2 IgG antibody testing, radiological investigations, patient management, and clinical outcomes were recorded. Lung findings were categorized according to the British Society of Thoracic Imaging (BSTI) guidelines. Reference was made to the incidence of lab-confirmed COVID-19 cases in our region. Results. Overall, 1,656 lab-confirmed COVID-19 cases were identified in our upper tier local authority (UTLA), a rate of 306.6 per 100,000, as of 30June 2020. A total of 504 CSI injections were performed on 443 patients between 1 February and 30June 2020. A total of 11 RT-PCR tests were performed on nine patients (2% of those who had CSI), all of which were negative for SARS-CoV-2 RNA, and five patients (1.1%) received an SARS-CoV-2 IgG antibody test, of which 2 (0.5%) were positive consistent with prior COVID-19 infection, however both patients were asymptomatic. Seven patients (1.6%) had radiological investigations for respiratory symptoms. One patient with indeterminate ground glass change was identified. Conclusion. The incidence of positive COVID-19 infection following corticosteroid injections was very low in our cohort and no adverse clinical outcomes related to COVID-19 infection following CSI were identified. Our findings are consistent with CSI likely being low risk during the COVID-19 pandemic. The results of this small observational study are supportive of the current multi-society guidelines regarding the judicious use of CSI. Cite this article: Bone Joint Open 2020;1-9:605–611

Osteoarthritis (OA) is a debilitating disease and the most common joint disorder worldwide. Although the development of OA is considered multifactorial, the mechanisms underlying its initiation and progression remain unclear. A prominent feature in OA is cartilage degradation typified by the progressive loss of extracellular matrix components - aggrecan and type II collagen (Col II). Cartilage homeostasis is maintained by the anabolic and catabolic activities of chondrocytes. Prolonged exposure to stressors such as mechanical loading and inflammatory cytokines can alter the phonotype of chondrocytes favoring cartilage catabolism, and occurs through decreased matrix protein synthesis and upregulation of catabolic enzymes such as aggrecanases (ADAMTS-) 4 and 5 and matrix metalloproteinases (MMPs). More recently, the endoplasmic reticulum (ER) stress response has been implicated in OA. The ER-stress response protects the cell from misfolded proteins however, excessive activation of this system can lead to chondrocyte apoptosis. Acute exposure of chondrocytes to IL-1β has been demonstrated to upregulate ER-stress markers (GADD153 and GRP78), however, it is unclear whether the ER-stress response plays a role on chronic IL-1β exposure. The purpose of this study was to determine whether modulating the ER stress response with tauroursodeoxycholic acid (TUDCA) in human OA chondrocytes during prolonged IL-1β exposure can alter its catabolic effects. Articular cartilage was isolated from donors undergoing total hip or knee replacement. Chondrocytes were recovered from the cartilage of each femoral head or knee by sequential digestion with Pronase followed by Collagenase, and expanded in DMEM-low glucose supplemented with 10% FBS. Chondrocytes were expanded in flasks for one passage before being prepared for micropellet culture. Chondrocyte pellets were cultured in regular growth medium (Control), medium supplemented with IL-1β [10 ng/mL], TUDCA [100 uM] or IL-1β + TUDCA for 12 days. Medium was replaced every three days. Cartilage explants were prepared from the donors undergoing knee replacement, and included cartilage with the cortical bone approximately 1 cm2 in dimension. Explants were cultured in the above mentioned media, however, the incubation period was extended to 21 days. RNA was extracted using Geneaid RNA Mini Kit for Tissue followed by cDNA synthesis. QPCR was performed using Cyber Green mastermix and primers for the following genes: ACAN (aggreacan), COL1A1, COL2A1, COL10A1, ADAMTS-4, ADAMTS-5, MMP-3, and MMP-13, on an ABI 7500 fast qPCR system. Although IL-1β did not significantly decrease the expression of matrix proteins, it did increase the expression of ADAMTS-4, −5, and MMP3 and −13 when compared to controls (Kruskal-Wallis, p < 0 .05, n=3). TUDCA treatment alone did not significantly increase the expression of catabolic enzymes but it did increase the expression of collagen type II. When IL-1β was coincubated with TUDCA, the expression of ADAMTS-4, ADAMTS-5, and MMP-13 significantly decreased by ∼40-fold, ∼10-fold, and ∼3-fold, respectfully. We provide evidence that the catabolic activities of IL-1β on human cartilage can be abrogated through modulation of the ER stress response

Intervertebral discs (IVDs) degeneration is one of the major causes of back pain. Upon degeneration, the IVDs tissue become inflamed, and this inflammatory microenvironment may cause discogenic pain. Cellular senescence is a state of stable cell cycle arrest in response to a variety of cellular stresses including oxidative stress and adverse load. The accumulation of senescent IVDs cells in the tissue suggest a crucial role in the initiation and development of painful IVD degeneration. Senescent cells secrete an array of cytokines, chemokines, growth factors, and proteases known as the senescence-associated secretory phenotype (SASP). The SASP promote matrix catabolism and inflammation in IVDs thereby accelerating the process of degeneration. In this study, we quantified the level of senescence in degenerate and non-degenerate IVDs and we evaluated the potential of two natural compounds to remove senescent cells and promote overall matrix production of the remaining cells. Human IVDs were obtained from organ donors. Pellet or monolayer cultures were prepared from freshly isolated cells and cultured in the presence or absence of two natural compounds: Curcumin and its metabolite vanillin. Monolayer cultures were analyzed after four days and pellets after 21 days for the effect of senolysis. A cytotoxicity study was performed using Alamar blue assay. Following treatment, RNA was extracted, and gene expression of senescence and inflammatory markers was evaluated by real-time q-PCR using the comparative ΔΔCt method. Also, protein expression of p16, Ki-67 and Caspase-3 were evaluated in fixed pellets or monolayer cultures and total number of cells was counted on consecutive sections using DAPI and Hematoxylin. Proteoglycan content was evaluated using SafraninO staining or DMMB assay to measure sulfated glycosaminoglycan (sGAG) and antibodies were used to stain for collagen type II expression. We observed 40% higher level of senescent cells in degenerate compare to the non-degenerate discs form unrelated individuals and a 10% increase when we compare degenerate compare to the non-degenerate discs of the same individual. Using the optimal effective and safe doses, curcumin and vanillin cleared 15% of the senescent cells in monolayer and up to 80% in pellet cultures. Also, they increased the number of proliferating and apoptotic cells in both monolayer and pellets cultures. The increase in apoptotic cell number and caspase-3/7 activity was specific to degenerate cells. Following treatment, mRNA expression levels of SASP factors were decreased by four to 32-fold compared to the untreated groups. Senescent cell clearance decreased, protein expression of MMP-3 and −13 by 15 and 50% and proinflammatory cytokines levels of IL-1, IL-6 and IL-8 by 42, 63 and 58 %. Overall matrix content was increased following treatment as validated by an increase in proteoglycan content in pellet cultures and surrounding culture media. This work identifies novel senolytic drugs for the treatment of IVD degeneration. Senolytic drugs could provide therapeutic interventions that ultimately, decrease pain and provide a better quality of life of patients living with IVDs degeneration and low back pain

Aim. Diagnosing Orthopaedic infection is limited by the sensitivity of culture methods. Next generation sequencing (NGS) offers an alternative approach for detection of microorganisms from clinical specimens. However, the low ratio of pathogen DNA to human DNA often inhibits detection of microorganisms from specimens. Depletion of human DNA may enhance the detection of microbial DNA. 1. Our aim was to compare four DNA extraction methods for the recovery of microbial DNA from orthopaedic samples for NGS. Method. Simulated samples; pooled culture negative sample matrix was spiked with known concentrations of microorganisms, each panel consisting of 7 samples. Broth culture was performed on simulated samples for comparison with NGS. *. . DNA Extraction; total nucleic acid extraction was performed on an automated extraction platform. **. using the viral NA assay. Modifications included: (1) mechanical lysis (glass beads), (2) lysis of human cells (saponin 0.025%), turbo DNase treatment and (3) mechanical lysis and addition of MspJI enzyme post-extraction for methylated DNA digestion. Detection of human and microbial DNA; human endogenous (HE) gene rtPCR. ***. was utilised following manufacturer's recommendations. Microbial DNA was detected using SYBR green 16s ribosomal RNA rtPCR with high resolution melt-curve analysis. ****. . Results. Broth culture recovered 64% (9/14) of the microorganisms from simulated samples. A significant increase (p<0.01) in the cycle threshold (C. T. ) (median C. T. 25.9 IQR 25.5, 26.1) of the HE gene rtPCR was observed using extraction method b, indicating a significant reduction in human DNA. No significant change (p=0.38) in the C. T. of the HE gene rtPCR was observed between the baseline method (median C. T. 19.2 IQR 18.5, 19.7) and modifications a (median C. T. 18.4 IQR 18.2, 19.4) and c (median C. T. 19.3 IQR 18.6, 19.4). Detection of microbial DNA was successful using the base line extraction method and modification a. Microbial DNA was not detected using the 16s ribosomal RNA rtPCR for modifications b and c. Conclusions. This study has demonstrated that modification of DNA extraction methods using selective enzymatic digestion of human DNA negatively impacts on the recovery of microbial DNA from simulated specimens. Total DNA extraction allows the successful recovery of microbial DNA alongside a significant amount of human DNA. The effect of the presence of human DNA will be subsequently assessed through NGS CosmosID analysis to establish if NGS is more sensitive than broth based culture

Staphylococcus aureus gene locus sdr is encoding proteins responsible for adhesion to bone tissue and therefore for the invasiveness of this bacteria. The aim of the study was to determine possible mechanism of activation of this gene and its role in bone infections. S. aureus strain 838/05 was isolated from the bone adjacent to infected joint prostheses. The strain contains all sdrC,D, and E genes. It was grown in liquid TSB medium (Bio Merieux) with gentle aeration or on Columbia agar plates containing 5% sheep blood (Bio Merieux). The presence of putative promoters and transcriptional organization of the sdr region was detected using BPROM and FGENESB algorithms (. www.softberry.com. ) based on region 611262bp-623152bp (GeneBank number CP000730.1) of the Staphylococcus aureus subsp. aureus USA300_TCH1516 complete genome sequence. In order to determine interaction of S. aureus with human blood, 50 ml of bacteria grown in TSB medium to ML growth phase were collected and washed twice with sterile PBS to remove the medium and re-suspended in 50ml of sterile PBS. 10 ml of cells were mixed with ∼100ml of mixed fresh human blood collected from healthy volunteers. Immediately after mixing blood with bacteria, 30 ml of the sample (Time 0) were mixed with two volumes of RNA Protect reagent (Qiagen), centrifuged to sediment cells, and frozen at −800C. Additional samples were collected after 30 and 90 minutes of incubation of bacteria with blood. Samples collected after 30 and 90 minutes were treated the same as samples collected at the beginning of the experiment. RNA was isolated, and cDNA was generated. The expression level of the sdr genes was calibrated to the gyrA level and then normalized to the expression level at time 0 using the ΔΔCT method. Results and conclusions. The study confirms separation of the Sdr region into three transcriptional units and suggests its dissimilar functions, based on differential reaction of the sdrD transcript to environmental conditions and blood. SdrE has been previously proposed to play role in bone infections; our results indicate that sdrD can play a role in the interactions between the pathogen and human immune system

Aim. The aim of this study was to gain insight into the in vivo expression of virulence and metabolic genes of Staphylococcus aureus in a prosthetic joint infection in a human subject. Method. Deep RNA sequencing (RNA-seq) was used for transcriptome profile of joint fluid obtained from a patient undergoing surgery due to acute S. aureus prosthetic joint infection. The S. aureus gene expression in the infection was compared with exponential culture of a S. aureus isolate obtained from the same sample using EdgeR. In addition, the genome of the isolate was sequenced on Miseq, assembled in CLC genomics workbench and annotated by MaGe. Moreover, using nuclear magnetic resonance (NMR) spectroscopy we analysed the metabolites in the joint fluid and in the culture supernatants to determine the biochemical composition of the environments. Results. Antibiotic susceptibility testing by disk diffusion (EUCAST) demonstrated that the strain was susceptible to β-lactams (penicillin and cefoxitin) and macrolides (erythromycin and roxitromycin). This was indirectly confirmed by the annotated genome, because of absence of known resistant genes. The patient showed no signs of improvement during 2-days treatment with antibiotics (different β-lactams and gentamicin) prior to the surgery. The RNA-seq data indicated that the strategy employed by S. aureus to survive and proliferate in the host during antibiotic treatment involved overexpression of various enzymes related to cell-wall synthesis and multidrug efflux pumps. Interestingly, these efflux pumps are only known to be related to fluoroquinolone resistance. Many of the genes encoding virulence factors were upregulated, including toxins and superantigen-like proteins, hemolysins, and immune evasion proteins. A number of chaperones and stress related genes were overexpressed indicating a stress response. Furthermore, the RNA-seq data provided clues of the potential major nutrient sources for the pathogen in vivo. Several amino acid degradation pathways were highly upregulated, e.g. arginine, histidine. Additional carbon sources included N-acetylneuraminate and purine/pyrimidine deoxyribonucleosides as indicated by the upregulation of the genes involved in the degradation pathways of these compounds and higher concentration of these substances in the joint fluid compared to culture supernatants. Conclusions. Our results show that the gene expression pattern of S. aureusin vivo is vastly different from that of an in vitro grown exponential culture, indicating that the pathogen adapts to host environmental conditions by altering gene expression. Finally our study emphasizes the importance of in vivo study in elucidating pathogenesis of S. aureus in prosthetic joint infections

Aims

The aim of this study was to analyze the prevalence of culture-negative periprosthetic joint infections (PJIs) when adequate methods of culture are used, and to evaluate the outcome in patients who were treated with antibiotics for a culture-negative PJI compared with those in whom antibiotics were withheld.

Aims

The new COVID-19 variant was reported by the authorities of the UK to the World Health Organization (WHO) on 14 December 2020. We aim to describe the clinical characteristics and nosocomial infection rates in major trauma and orthopaedic patients comparing the first and second wave of COVID-19 infection.

Introduction. Macrophages phagocytes implant wear debris and produce various cytokines to evoke inflammation and periprosthetic osteolysis of aseptic loosening. It had been reported that expression of Toll-like receptor (TLR) 2 and other TLRs increased in periprosthetic tissues of aseptic loosening. Pathogen-associated molecular patterns (PAMPs) and damaged-associated molecular patterns (DAMPs) have been known as ligands of TLRs and considered to be involved in the osteolytic reactions via TLRs. Another type of immune sensors, nucleotide-binding and oligomerization domain (NOD)-like receptors (NLR) with a pyrin domain 3 (NLRP3) can also recognize PAMPs and DAMPs as their lignds, which has been presumed to participate in the local host response of macrophage cascade via phagocytosis of implant wear particles. However, the contribution of NLRP3 in periprosthetic tissues of aseptic loosening and the correlation between TLR2 and NLRP3 are still unclear. Materials and methods. TLR1, TLR2, TLR6, NLRP3, TNF-α and IL-1β of macrophages in aseptic loose periprosthetic tissues were immnohistorically evaluated and compared to osteoarthritic synovium. RAW264.7 cells, macrophagic cell line, were stimulated by titanium particles (Ti) and lipoteichoic acid (LTA)-coated Ti. The celluar reaction associated with TLR2 and NLRP3 and the correlation of them were analyzed at mRNA expression levels with small-interfering RNA of Irak2, one of adaptor molecules in TLR2 cascades. Results. Macrophages, which expressed abundant TLR2, NLRP3, TNF-α and IL-1β, were observed dominantly in foreign body granuloma of aseptic periprosthetic tissues. The features of abundant expression were quite different from osteoarthritic synovium. In vitro experiment of RAW264.7, mRNA levels of NLRP3 and TNF-α increased after stimulation of Ti. mRNA levels of TLR2, NLRP3, TNF-α and IL-1β were enhanced by LTA-coated Ti. mRNA expression level of NLRP3 were suppressed by silencing Irak2. Discussion and conclusion. This study indicated that innate immune sensors, TLR2 and NLRP3, could respond to foreign body particles in aseptic loose periprosthetic connective tissues. In this process, mRNA expression levels of TLR2 and NLRP3 in RAW264.7 were increased by phagocytosis of Ti particles, especially by LTA-coated Ti stimulation. Suppressed mRNA expression level of NLRP3 by knocked down of Irak2 indicated that TLR2 cascade could enhance activation NLRP3 cascade and/or free LTA may stimulate NLRP3 cascade directly. It may be possible that TLR2 and NLRP3 cascades in macrophages recognising PAMPs and/or DAMPs are activated each other and they play an important role of the pathogenesis of wear debris around loose hip joints

A randomised controlled trial was conducted using a rabbit model of a complex contaminated extremity war wound. Compared to saline soaked gauze dressings Inadine (iodine) and Acticoat (nanocrystalline silver) had significantly lower levels of Staphylococcus aureus after 7 days while Activon Tulle (Manuka honey) had significantly higher levels. Molecular level analysis of the wound was conducted. Plasma cytokines of interest were assayed using ELISA and levels of expression of relevant tissue genes measured using PCR following RNA extraction. Appreciable levels of Interleukins 4 and 6 and Tumour Necrosis Factor-α were identified in plasma with significantly higher levels of IL-4 and TNFα detected in the Activon Tulle group. In tissue TNFα, Matrix metalloproteinase-3 and the ratio of Matrix metalloproteinase-9 to Tissue Inhibitor of Matrix metalloproteinase-1 were significantly higher in tissue injured limbs than the uninjured limbs with no significant differences between groups. Interpretation of these results is challenging. IL-4 has been associated with transition from pathological inflammation to repair and TNFα with impaired healing. However, Activon Tulle had significantly higher levels of S. aureus and we found no differences in observational, histology, haematology or tissue gene expression outcomes over 7 days which would correlate with these molecular biology results

Introduction. The ability of activated platelets to induce cellular proliferation is well recognised. In a previous diffusion model, platelets combined with Tri-calcium phosphate (TCP) led to an osteoprogenitor mitogenic response followed by cellular differentiation. This study was designed to look at osteoprogenitor responses when cultured directly onto TCP granules combined with activated platelets. Method. Human osteoprogenitors were loaded onto TCP with activated platelets at a low seeding density and high seeding densities. Cellular proliferation was assessed using the pico-green DNA content analysis. Differentiation towards osteoblastic phenotype was assessed using an alkaline phosphatase assay. RNA extraction, reverse transcription and quantitative real-time polymerase chain reaction was used to assess gene expression for type 1 collagen and osteocalcin. Histological assessment for live/dead staining and alkaline phosphatase was used on cultured granule samples. Results. In the low seeding density, platelets induced an early proliferative response compared with controls. After 14 days of culture the cells had not differentiated to an osteoblastic phenotype. When seeded at high densities, cellular differentiation was induced by the activated platelets. Significant cellular proliferation was not observed after seeding at high density. Conclusions. This study demonstrates that osteoprogenitors respond to the local environment which is modulated by both cell-cell contact factors and inflammatory cytokines from the platelets. This study helps to define the concentration of progenitors and platelets needed for further on-growth studies. It may also help define the optimal conditions for seeding cells and platelets for clinical use of composite bone graft substitutes

Background. Current treatments for the prevention of thromboembolism include heparin and low-molecular weight heparins (LMWHs). A number of studies have suggested that long term administration of these drugs may adversely affect osteoblasts and therefore, bone metabolism. Xarelto(tm) (Rivaroxaban) is a new anti-thrombotic drug for the prevention of venous thromboembolism in adult patients undergoing elective hip and knee replacement surgery. The aim of this in vitro study was to investigate the possible effects of rivaroxaban on osteoblast proliferation, function, matrix mineralisation and gene expression compared to enoxaparin, a commonly used LMWH. Methods. Primary human osteoblast cultures were treated with varying concentrations of rivaroxaban (0.013, 0.13, 1.3 and 13 μg/ml) or enoxaparin (0.1, 1.0 and 10 international units/ml). The effect of each drug on osteoblast function and matrix mineralisation was evaluated by measuring alkaline phosphatase activity and calcium deposition, respectively. The MTS assay was used to assess the effect of drug treatments on cell proliferation. Changes in osteocalcin, Runx2 and BMP-2 messenger RNA (mRNA) expression following drug treatments were measured by real-time polymerase chain reaction (PCR). Results. Rivaroxaban and enoxaparin treatment did not adversely affect osteoblast proliferation. However, both drugs caused a significant reduction in osteoblast function, as measured by alkaline phosphatase activity, with a moderate reduction in calcium deposition also observed. This reduction in osteoblast function was associated with a reduction in the mRNA expression of the bone marker, osteocalcin, the transcription factor, Runx2, and the osteogenic factor, BMP-2. Conclusion. These data show that rivaroxaban treatment may negatively affect bone through a reduction in osteoblast function. The increased duration of recommended Rivaroxaban therapy (2 and 5 weeks) post-arthroplasty compared to Enoxaparin therapy (average one week) may have a more pronounced effect on bone homeostasis

Introduction. Osteoporosis is a common skeletal disorder characterised by a reduced bone mass and a progressive micro-architectural deterioration in bone tissue leading to bone fragility and susceptibility to fracture. With a progressively aging population, osteoporosis is becoming an increasingly important public health issue. The Wnt/β-catenin pathway is a major signalling cascade in bone biology, playing a key role in regulating bone development and remodelling, with aberrations in signalling resulting in disturbances in bone mass. Objectives. To assess the effects of silencing the expression of the Wnt antagonist Dickkopf-1 (Dkk1) on the bone profile of primary human osteoblasts exposed in vitro to 10-8M dexamethasone. Methods. Primary human osteoblasts (HOBs) were cultured in vitro and exposed to 10-8M dexamethasone over a time course of 4hr, 12hr and 24hr. Dkk1 expression was silenced using small interfering RNA (siRNA). Quantitative RT-PCR was performed to confirm gene knockdown. Control and Dex-treated phObs (silenced & non-silenced) were compared with respect to bone turnover. Markers of bone turnover analyzed included alkaline phosphatase activity, calcium deposition and osteocalcin expression as determined by pNPP assay, quantitative alizarine red staining and ELISA respectively. Results. Dkk1 expression in HOBs was increased in response to dexamethasone exposure with an associated reduction in alkaline phosphatase activity, calcium deposition and osteocalcin expression. Silencing of Dkk1 expression, as confirmed by quantitative RT-PCR, was associated with a rescue effect in dexamethasone-induced bone loss in vitro. Conclusions. Dkk1 is an antagonist of Wnt/β-catenin signalling and plays a key role in regulating bone development and remodelling. Silencing the expression of Dkk1 in primary human osteoblasts has been shown to rescue the effects of dexamethasone-induced bone loss in vitro. The pharmacological targeting of the Wnt/β-catenin signalling pathway offers an exciting opportunity for the development of novel anabolic bone agents to treat osteoporosis and disorders of bone mass

Introduction. Improvements in material properties of total joint prostheses and methods of fixation mean that arthroplasty is the most effective means of restoring mobility in osteoarthritic patients. Aseptic loosening is the major cause of long-term failure of prostheses. Cobalt particles may act directly on osteoblasts, decreasing bone formation and potentially playing a role in osteolysis and aseptic loosening. Objectives. To assess gene expression profiles of primary human osteoblasts exposed to cobalt ions in a temporal manner, and to identify gene clusters underpinning the osteoblast response to cobalt. Methods. Primary human osteoblasts were exposed to cobalt ions at a concentration of 10ppm. To determine gene expression profiles, comparisons were made between control and 12, 24, 48 and 72 hour time exposures. RNA isolation and cDNA synthesis were performed. Gene profiling was carried out using the Affymetrix Human Genome UI33 Plus 2.0 array. Data was normalised using RMA express and an average expression measure for each time point used to identify alterations in gene expression. Validation was achieved by performing quantitative real time RT-PCR on selected genes. Results. Oligonucleotide microarray profiling identified significant alterations in osteoblast gene expression in response to cobalt exposure. Distinct phase patterns were observed, with significant altered expression following 12, 24 and 48 hours cobalt ion exposure. Of the 22,233 gene sequences represented on the Affymetrix microarray, 4.8% (1077 genes), 4.1% (930 genes), and 2.13% (486 genes) were significantly altered. We identified dysregulation of key functional families in response to cobalt ions, including alterations in cellular proliferation, development and and inflammation. Conclusion. These data will provide novel avenues for exploration to further characterise the molecular mechanisms underpinning the initiation and progression of osteolysis