Introduction. The pathogenesis of rotator cuff disease (RCD) is complex and not fully understood. This systematic review set out to summarise the histological and molecular changes that occur throughout the spectrum of RCD. Methods. We conducted a systematic review of the scientific literature with specific inclusion and exclusion criteria. Results. A total of 101 studies met the inclusion criteria: 92 studies used human subjects exclusively, seven used animal overuse models, and the remaining two studies involved both humans and an animal overuse model. A total of 58 studies analysed supraspinatus tendon exclusively, 16 analysed subacromial bursal tissue exclusively, while the other studies analysed other tissue or varying combinations of tissue types including joint fluid and muscle. The molecular biomarkers that were altered in RCD included matrix substances, growth factors, enzymes and other proteins including certain neuropeptides. Conclusions. The pathogenesis of RCD is being slowly unravelled as a result of the significant recent advances in molecular medicine. Future research aimed at further unlocking these key molecular processes will be pivotal in developing new surgical interventions both in terms of the diagnosis and treatment of RCD

Aims. Osteoarthritis (OA) is a common degenerative joint disease worldwide, which is characterized by articular cartilage lesions. With more understanding of the disease, OA is considered to be a disorder of the whole joint. However, molecular communication within and between tissues during the disease process is still unclear. In this study, we used transcriptome data to reveal crosstalk between different tissues in OA. Methods. We used four groups of transcription profiles acquired from the Gene Expression Omnibus database, including articular cartilage, meniscus, synovium, and subchondral bone, to screen differentially expressed genes during OA. Potential crosstalk between tissues was depicted by ligand-receptor pairs. Results. During OA, there were 626, 97, 1,060, and 2,330 differentially expressed genes in articular cartilage, meniscus, synovium, and subchondral bone, respectively. Gene Ontology enrichment revealed that these genes were enriched in extracellular matrix and structure organization, ossification, neutrophil degranulation, and activation at different degrees. Through ligand-receptor pairing and proteome of OA synovial fluid, we predicted ligand-receptor interactions and constructed a crosstalk atlas of the whole joint. Several interactions were reproduced by transwell experiment in chondrocytes and synovial cells, including TNC-NT5E, TNC-SDC4, FN1-ITGA5, and FN1-NT5E. After lipopolysaccharide (LPS) or interleukin (IL)-1β stimulation, the ligand expression of chondrocytes and synovial cells was upregulated, and corresponding receptors of co-culture cells were also upregulated. Conclusion. Each tissue displayed a different expression pattern in transcriptome, demonstrating their specific roles in OA. We highlighted tissue molecular crosstalk through ligand-receptor pairs in OA pathophysiology, and generated a crosstalk atlas. Strategies to interfere with these candidate ligands and receptors may help to discover molecular targets for future OA therapy. Cite this article: Bone Joint Res 2022;11(12):862–872

The biological understanding for the disease progression osteoarthritis (OA) has uncovered specific biomarkers from either synovial fluid, articular chondrocytes or synoviocytes that can be used to diagnose the disease. Examples of these biomarkers include interleukin-1β (IL-1β) or collagen II fragments (1, 2). In parallel, isolation of chondrocytes or bone marrow derived mesenchymal stromal cells (MSCs) has yielded cell-based strategies that have shown long- term beneficial effects in a specific cohort of patients, specifically in traumatic cartilage lesions (2). This latter finding shows that patient stratification of OA is an important tool to both match patients for a specific treatment and to develop novel therapies, especially disease modifying drugs. In order to create disease stage specific therapies, the use of next generation analysis tools such as RNAseq and metabolomics, has the potential to decipher specific cellular and molecular endotypes. Alongside greater understanding of the clinical phenotype (e.g. imaging, pain, co- morbidities), therapies can be designed to alleviate the symptoms of OA at specific points of the disease in patients. This talk will outline the current biological understanding of OA and discuss how patient stratification could assist in the design of innovative therapies for the disease. Acknowledgements: This presentation was supported by the COST action, CA21110 – Building an open European Network on Osteoarthritis Research (NetwOArk)

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

Background. The molecular mechanisms underlying non-union bone fractures largely remain elusive. Recently, spatial transcriptomics approaches for musculoskeletal tissue samples have been developed requiring direct placement of histology sections on barcoded slides. However, Formalin-Fixed-Paraffin-Embedded (FFPE) bone sections have been associated with limited RNA quality and read depth compared to soft tissue. Here, we test spatial transcriptomics workflows based on transcriptomic probe transfer to characterize molecular features discriminating non-union and union bone fractures in mice. Method. Histological sections (n=8) used for spatial transcriptomics (Visium CytAssist FFPE; 10x Genomics, n=4 on glass slides, n=4 on hydrogel-coated slides) were obtained from a fracture healing study in female 20-week-old C57BL/6J mice receiving either a femur osteotomy (0.7mm) or a segmental defect (2.4mm) (license 22/2022, Grisons CH). Sequence alignment and manual segmentation of different tissues (bone, defect region/callus, bone marrow, muscle) were performed using SpaceRanger and LoupeBrowser (10x Genomics). Differential gene expression was performed using DESeq2 (Seurat) followed by Gene-Set-Enrichment-Analysis (GSEA) of Gene Ontology (ClusterProfiler). Group comparison of quality measures was done using a Welch's t-test. Results are given as mean±standard deviation. Result. The quality measures, mean counts, and genes per spot, were significantly ~10× higher for sections on hydrogel slides (counts: 4700±1796, genes: 2389±1170) compared to glass slides (counts: 463±415, genes: 250±223). In challenging tissues like cortical bone, we reached high counts+genes in comparison to published data. Direct comparison of a non-union and union section showed a total of 432 differentially regulated genes, 538 in the defect region/callus. GSEA revealed differential regulation of pathways involved in muscle organ morphogenesis, cartilage development and endochondral ossification. Conclusions. Optimized spatial transcriptomics workflows based on transcriptomic probe transfer enable for improved read depth in musculoskeletal tissue enabling the characterization of molecular features discriminating non-union and union bone fractures. Acknowledgements. AO Foundation (AOTRAUMA), SNSF (PhD salary)

Aim. Unexpected negative-cultures (UNC) are a common diagnostic problem in periprosthetic joint infection (PJI) of the hip and knee when using culture-based methods. A novel molecular approach (MC)1 based on the identification of the vast majority of bacterial species in a single assay using species-specific bacterial interspacing region length polymorphisms and phylum-specific 16S rDNA sequence polymorphisms has demonstrated clinical utility in PJI diagnostics (1). In addition, MC provides an estimate of the leukocyte concentration in the specimen analysed. The aim of this retrospective, blinded study was to evaluate the performance of MC in identifying the microbiological content and determining the leukocyte count in synovial fluid (SF) collected from hip and knee revision arthroplasty cases with UNC. It was also assessed whether antibiotic treatment would have been changed if the result from MC had been known. Method. A total of 89 SF samples from 70 patients (43 female; 27 male) who underwent revision arthroplasty (14 hip; 75 knee) were included. Using European and Bone Joint Infection Society (EBJIS) criteria, 82 cases were classified as infected (77 UNC and 5 septic culture-positive controls), five as non-infected (aseptic culture-negative controls), and two as likely infected, but infected by clinical observation. MC was performed and evaluated together with SF parameters. Antibiotic treatment, clinical outcome, patient demographics and surgical details were analysed. Results. Overall, 29.1% (23/79) of UNC had a positive yield by MC, of which 2/23 (8.7%) had two microorganisms detected simultaneously. Of the 25 microorganisms identified by MC, 12/25 (48%) were clinically relevant after re-evaluation of the patients’ microbiological history. The microorganisms detected were 5/25 (20%) Streptococcus pneumoniae/mitis, 4/25 (16%) Staphylococcus epidermidis, 3/25 (12%) Cutibacterium acnes, 3/25 (12%) Streptococcus agalactiae, 2/25 (8%) Streptococcus bovis, 2/25 (8%) Staphylococcus aureus, and 2/25 (8%) Haemophilus parainfluenzae. The prevalence of Enterococcus faecalis, Bacteroides fragillis, Staphylococcus lugdunensis, Corynebacterium striatum among all MC results was 1/25 (4%) each species. In total, 13/23 (56%) cases were associated with patients receiving antibiotic therapy at the time of SF collection. The yield for leukocyte counts provided the molecular technique was consistently much higher in the UNC and clearly septic groups than in the clearly aseptic group. Overall, 20/61 (32.8%) patients with UNC could have been managed differently and more accurately after MC assessment. Conclusions. MC shows clinical value in the diagnosis and management of PJI with UNC. The included leukocyte count shows promising results. Acknowledgments. This work was partially funded by Inbiome

Tendon is a bradytrophic and hypovascular tissue, hence, healing remains a major challenge. The molecular key events involved in successful repair have to be unravelled to develop novel strategies that reduce the risk of unfavourable outcomes such as non-healing, adhesion formation, and scarring. This review will consider the diverse pathophysiological features of tendon-derived cells that lead to failed healing, including misrouted differentiation (e.g. de- or transdifferentiation) and premature cell senescence, as well as the loss of functional progenitors. Many of these features can be attributed to disturbed cell-extracellular matrix (ECM) or unbalanced soluble mediators involving not only resident tendon cells, but also the cross-talk with immigrating immune cell populations. Unrestrained post-traumatic inflammation could hinder successful healing. Pro-angiogenic mediators trigger hypervascularization and lead to persistence of an immature repair tissue, which does not provide sufficient mechano-competence. Tendon repair tissue needs to achieve an ECM composition, structure, strength, and stiffness that resembles the undamaged highly hierarchically ordered tendon ECM. Adequate mechano-sensation and -transduction by tendon cells orchestrate ECM synthesis, stabilization by cross-linking, and remodelling as a prerequisite for the adaptation to the increased mechanical challenges during healing. Lastly, this review will discuss, from the cell biological point of view, possible optimization strategies for augmenting Achilles tendon (AT) healing outcomes, including adapted mechanostimulation and novel approaches by restraining neoangiogenesis, modifying stem cell niche parameters, tissue engineering, the modulation of the inflammatory cells, and the application of stimulatory factors. Cite this article: Bone Joint Res 2022;11(8):561–574

Aim. Bone and joint infections (BJIs) are serious infections requiring early optimized antimicrobial therapy. BJIs can be polymicrobial or caused by fastidious bacteria, and the patient may have received antibiotics prior to sampling, which may decrease the sensitivity of culture-based diagnosis. Furthermore, culture-based diagnosis can take up to 14 days. Molecular approaches can be useful to overcome these concerns. The BioFire® system performs syndromic multiplex PCR in 1 hour, with only a few minutes of sample preparation. The BioFire® Joint Infection (JI) panel (BF-JI), recently FDA-cleared, detects both Gram-positive (n=15) and Gram-negative bacteria (n=14), Candida, and eight antibiotic resistance genes directly from synovial fluids. The aim of this study was to evaluate its performance in acute JIs in real-life conditions. Method. BF-JI was performed on synovial fluid from patients with clinical suspicion of acute JI, either septic arthritis or periprosthetic JI, in 6 French centers. The results of BF-JI were compared with the results of culture of synovial fluid and other concomitantly collected osteoarticular samples obtained in routine testing in the clinical microbiology laboratory. Results. From July 2021 to May 2022, 319 patients (including 10 children < 5y and 136 periprosthetic infections) had been included in the study. The BF-JI test was invalid for one patient (not retested). Among the 318 remaining patients, overall concordance with comparative microbiology methods was 88% (280/318): 131 samples were negative with both BF-JI and culture, and 149 samples were positive with the same microorganisms using complementary techniques. In 33 cases (10.4%), BF-JI was negative while culture was positive: 18 microorganisms were not targeted by BF-JI (including Staphylococcus epidermidis, n=10, and Cutibacterium acnes, n=2); 15 microorganisms targeted by BF-JI were obtained in culture but not by the molecular test (false-negative 4.7%). In 20 cases, BF-JI was positive while culture was not: 12 patients had received antibiotics before sampling, and 7 cases involved fragile and fastidious bacteria (Kingella kingae, n=5; Neisseria gonorrhoeae, n=2). In 6 cases, both BF-JI and culture were positive, but no yielding the same bacteria (polymicrobial specimens). Conclusions. In acute JIs, the BF-JI panel shows a good concordance with culture for the microorganisms targeted by the panel. Therefore, this molecular tool may have a place in microbiological diagnosis of acute JIs in order to confirm JI faster than culture. Moreover, it allows easy detection of difficult-to-culture bacteria. Acknowledgements. study was supported by bioMérieux, who provided all reagents

Aims. The aim of this study was to compare the results of 16S/28S rRNA sequencing with the erythrocyte sedimentation rate (ESR), C-reactive protein (CRP) level, and synovial fluid analysis in the diagnosis of prosthetic joint infection (PJI). Patients and Methods. Between September 2015 and August 2016, 214 consecutive patients were enrolled. In the study population, there were 25 patients with a PJI and 189 controls. Of the PJI patients, 14 (56%) were women, and the mean age at the time of diagnosis was 65 years (38 to 83). The ESR and CRP levels were measured, and synovial fluid specimens were collected prospectively. Synovial fluid was subjected to reverse transcription polymerase chain reaction (RT-PCR)/sequence analysis targeting the 16S/28S rRNA, and to conventional culture. Laboratory personnel who were blind to the clinical information performed all tests. The diagnosis of PJI was based on the criteria of the Musculoskeletal Infection Society. Results. A total of 25 patients had a confirmed PJI. In 20 cases of monomicrobial PJI, the PCR products could be perfectly matched with the 16S/28S rRNA genes specific for different species of bacteria provided by sequence analysis. Of the five polymicrobial cases of PJI, 16S/28S rRNA PCR sequence analysis failed to identify the concordant bacteria species. In the 189 control patients, there was one false-positive RT-PCR result. The sensitivity and specificity of the molecular diagnosis method were 100% (95% confidence interval (CI) 85.7 to 100) and 99.5% (95% CI 97.1 to 99.9), respectively, whereas the positive and negative predictive values of PCR were 96.1% (95% CI 79.6 to 99.9) and 100% (95% CI 98.1 to 100), respectively. The PCR results were significantly better than serological diagnostic methods (p = 0.004 and p = 0.010 for ESR and CRP, respectively), the synovial fluid white blood cell (WBC) count (p = 0.036), and percentage of polymorphonuclear cells (PMN%) (p = 0.014). Conclusion. Stepwise RT-PCR and sequence analysis of the 16S/28S rRNA carried out under stringent laboratory conditions achieved highly sensitive and specific results for the differentiation between aseptic and septic joints undergoing arthroplasty. Sequence analysis successfully identified bacterial strains in monomicrobial infections but failed to identify molecular targets in polymicrobial infections. Further refinement of the protocols to identify the bacteria in polymicrobial infections is needed. Cite this article: Bone Joint J 2018;100-B:1345–51

Introduction and Objective. Chronic tendinopathy is a multifactorial disease and a common problem in both, athletes and the general population. Mechanical overload and in addition old age, adiposity, and metabolic disorders are among the risk factors for chronic tendinopathy but their role in the pathogenesis is not yet unequivocally clarified. Materials and Methods. Achilles tendons of young (10 weeks) and old (100 weeks) female rats bred for high (HCR) and low (LCR) intrinsic aerobic exercise capacity were investigated. Both Achilles tendons of 28 rats were included and groups were young HCR, young LCR, old HCR, and old LCR (n = 7 tendons per group/method). In this rat model, genetically determined aerobic exercise capacity is associated with a certain phenotype as LCR show higher body weight and metabolic dysfunctions in comparison to HCR. Quantitative real-time PCR (qPCR) was used to evaluate alterations in gene expression. For histological analysis, semi-automated image analysis and histological scoring were performed. Results. Age-related downregulation of tenocyte marker genes (Tenomodulin), genes related to matrix modelling and remodeling (Collagen type 1, Collagen type 3, Elastin, Biglycan, Fibronectin, Tenascin C), and Transforming growth factor beta 3 (Tgfb3) were detected in tendons from HCR and LCR. Furthermore, inflammatory marker Cyclooxygenase 2 (Cox2) was downregulated, while Microsomal prostaglandin E synthase 2 (Ptges2) was upregulated in tendons from old HCR and old LCR. No significant alteration was seen in Interleukin 6 (Il6), Interleukin 1 beta (Il1b), and Tumor necrosis factor alpha (Tnfa). Histological analysis revealed that Achilles tendons of old rats had fewer and more elongated tenocyte nuclei compared to young rats, indicating a reduced metabolic activity. Even though higher content of glycosaminoglycans as a sign of degeneration was found in tendons of old HCR and LCR, no further signs of tendinopathy were detectable in histological evaluation. Conclusions. Overall, aging seems to play a prominent role in molecular and structural alterations of Achilles tendon tissue, while low intrinsic exercise capacity did not cause any changes. Even though tendinopathy was not present in any of the groups, some of the shown age-related changes correspond to single characteristics of chronic tendon disease. This study gives an insight into tendon aging and its contribution to molecular and cellular changes in Achilles tendon tissue

R Appleyard, Murray Maxwell Biomechanics Lab, Royal North Shore Hospital, Sydney. The fundamental mechanisms that underlie tendon breakdown are ill understood. There is an emerging hypothesis that altered mechanical strain modulates the metabolism and/or phenotype of tenocytes, disrupting the balance of matrix synthesis and degradation, and that rupture then occurs through an abnormal tendon matrix. The critically regulated genes have not yet been determined. We have developed sheep model in sheep where both stress-deprived and over-stressed areas can be examined in the one tendon, to evaluate the pathological and molecular changes over time. We have also used ‘wild type’ and genetically modified mice to determine the role of specific enzymes and proteoglycans in tendon degeneration. Stress-deprived and over-stressed regions showed classical changes of increased cellularity and vascularity, rounded tenocytes and interfascicular matrix infiltration. These structural changes resolved for up to one year after injury. Resolution was more rapid in over-stressed regions. Irrespective of the initiating stress, proteoglycan staining and chondroid metaplasia increased in tendon with time. There were distinct molecular and temporal differences between regions, which are reviewed here. While tendon degeneration has traditionally been regarded as a single field of change, our studies show that at a molecular level, the injured tendon may be regarded as a number of distinct regions—overloaded and underloaded, adjacent to bone or adjacent to muscle. Each region manifests distinct molecular changes, driven by relevant gene expression. While collagen metabolism in pathological tendon has received much attention, accumulation of proteoglycan is also consistently induced by altered mechanical loading. We suggest that ADAMTS enzymes, which cleave aggrecan, versican and small proteoglycans, may play a significant role in tendon homeostasis and pathology. Regulating proteoglycan turnover may represent a novel target for treating tendon degeneration. We have initiated studies using mesenchymal stem cells (MSC), not to directly augment healing but to modify the molecular pathology in tendon resulting from altered loading. Preliminary data indicates that injection of MSC into an acute tendon defect significantly abrogates the increase in expression of aggrecan and collagen degrading metalloproteinases in the adjacent over-stressed tendon. This may decrease the resultant degeneration. The effects of MSC in treating tendon degeneration are reviewed here, as are the possible benefits of radiofrequency microtenotomy

Objectives. The biomembrane (induced membrane) formed around polymethylmethacrylate (PMMA) spacers has value in clinical applications for bone defect reconstruction. Few studies have evaluated its cellular, molecular or stem cell features. Our objective was to characterise induced membrane morphology, molecular features and osteogenic stem cell characteristics. Methods. Following Institutional Review Board approval, biomembrane specimens were obtained from 12 patient surgeries for management of segmental bony defects (mean patient age 40.7 years, standard deviation 14.4). Biomembranes from nine tibias and three femurs were processed for morphologic, molecular or stem cell analyses. Gene expression was determined using the Affymetrix GeneChip Operating Software (GCOS). Molecular analyses compared biomembrane gene expression patterns with a mineralising osteoblast culture, and gene expression in specimens with longer spacer duration (> 12 weeks) with specimens with shorter durations. Statistical analyses used the unpaired student t-test (two tailed; p < 0.05 was considered significant). Results. Average PMMA spacer in vivo time was 11.9 weeks (six to 18). Trabecular bone was present in 33.3% of the biomembrane specimens; bone presence did not correlate with spacer duration. Biomembrane morphology showed high vascularity and collagen content and positive staining for the key bone forming regulators, bone morphogenetic protein 2 (BMP2) and runt-related transcription factor 2 (RUNX2). Positive differentiation of cultured biomembrane cells for osteogenesis was found in cells from patients with PMMA present for six to 17 weeks. Stem cell differentiation showed greater variability in pluripotency for osteogenic potential (70.0%) compared with chondrogenic or adipogenic potentials (100% and 90.0%, respectively). Significant upregulation of BMP2 and 6, numerous collagens, and bone gla protein was present in biomembrane compared with the cultured cell line. Biomembranes with longer resident PMMA spacer duration (vs those with shorter residence) showed significant upregulation of bone-related, stem cell, and vascular-related genes. Conclusion. The biomembrane technique is gaining favour in the management of complicated bone defects. Novel data on biological mechanisms provide improved understanding of the biomembrane’s osteogenic potential and molecular properties. Cite this article: Dr H. E. Gruber. Osteogenic, stem cell and molecular characterisation of the human induced membrane from extremity bone defects. Bone Joint Res 2016;5:106–115. DOI: 10.1302/2046-3758.54.2000483

Aim. Community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) is commonly associated with serious cases of community-onset skin and musculoskeletal infections (Co-SMSI). Molecular epidemiology analysis of CA-MRSA recovered from skin and soft tissues specimens is lacking in Latin America. This study aimed to identify phenotypic and genotypic features of MRSA isolates recovered from patients presenting Co-SMSI. Methods. Consecutive MRSA isolates recovered from Co-SMSI of patients admitted from March 2022 to January 2023 in a Brazilian teaching hospital were tested for antimicrobial resistance and characterized by their genotypic features. Identification was carried out by automated method and through MALDI-TOF MS. Antimicrobial susceptibility was tested by disk diffusion, broth microdilution and E-test strips for determination of the minimal inhibitory concentration (MIC) according to recommendations from the Brazilian Committee on Antimicrobial Susceptibility Testing (BrCAST) and European Committee on Antimicrobial Susceptibility Testing (EUCAST). Gene mecA characterization and Sccmec typing were performed by multiplex polymerase chain reaction (PCR) assay, and gene lukF detection by single PCR. Patients were prospectively followed up for two months, in order to determine their clinical characteristics and outcomes. Results. Overall, 48 Staphylococcus aureus isolates were obtained from 68 samples recovered from patients with Co-SMSI. Twenty two (42%) were phenotypically characterized as MRSA, although mecA gene was only identified in 20 of those samples. Sccmec was untypable in 12 isolates, Sccmec was type II in 4 isolates and 2 were classified as type IVa. LukF gene was identified in 5 isolates. Antimicrobial resistance profile showed that all isolates were susceptible to linezolid and vancomycin with MIC = 1 and MIC = 2 in 66,7% and 33.3%, respectively. Susceptibility to quinolones was worryingly low and none of the isolates were sensitive to usual doses of ciprofloxacin and levofloxacin, and showed increased rates of resistance to increased exposure to these drugs, as well. Isolates were both susceptible to gentamicin and tetracycline in 85% and resistance to also Sulfamethoxazole/Trimethoprim occurred in only 2 isolates. Mortality rate evaluated within 1 month of the initial evaluation was 10% among MRSA isolates. Conclusions. Our results showed that CA-MRSA isolates causing Co-SMSI demonstrated an alarming pattern of multidrug resistance, including to β-lactam and quinolones, which have been commonly prescribed as empirical therapy for patients with skin, soft tissue and musculoskeletal infections

Thrombin has many biological properties similar to those of growth factors. In a previous study, we showed that thrombin improves healing of the rat tendo Achillis. Low molecular weight heparin (LMWH) inhibits the activity and the generation of thrombin. We therefore considered that LMWH at a thromboprophylactic dose might inhibit tendon repair. Transection of the tendo Achillis was carried out in 86 rats and the healing tested mechanically. Low molecular weight heparin (dalateparin) was either injected a few minutes before the operation and then given continuously with an osmotic mini pump for seven days, or given as one injection before the operation. In another experiment ,we gave LMWH or a placebo by injection twice daily. The anti-factor Xa activity was analysed. Continuous treatment with LMWH impaired tendon healing. After seven days, this treatment caused a 33% reduction in force at failure, a 20% reduction in stiffness and a 67% reduction in energy uptake. However, if injected twice daily, LMWH had no effect on tendon healing. Anti-factor Xa activity was increased by LMWH treatment, but was normal between intermittent injections. Low molecular weight heparin delays tendon repair if given continuously, but not if injected intermittently, probably because the anti-factor Xa activity between injections returns to normal, allowing sufficient thrombin stimulation for repair. These findings indicate the need for caution in the assessment of long-acting thrombin and factor Xa inhibitors

Prosthetic joint infection is one of the most challenging complications of joint alloplasty and the diagnosis remains difficult. The aim of the study was to investigate the bacterial flora in surgical samples from 22 prosthetic patients using a panel of culture-independent molecular methods including broad range 16S rRNA gene PCR, cloning, sequencing, phylogeny, quantitative PCR (qPCR), and fluorescence in situ hybridization (FISH). Concomitant samples were cultured by standard methods. Molecular methods detected presence of bacteria in samples from 12 of 22 patients. Using clone libraries a total of 40 different bacterial species were identified including known pathogens and species not previously described in association with prosthetic joint infections. The predominant species were Propionibacterium acnes and Staphylococcus epidermidis; polymicrobial infections were found in 9 patients. Culture-based methods showed bacterial growth in 8 cases with the predominant species being S. epidermidis. Neither anaerobic bacteria (including P. acnes) nor any of the species not previously described in implant infections were isolated. Additionally, 7 of the 8 culture positive cases were monomicrobial. Overall, the results of culture-based and molecular methods showed concordance in 11 cases (hereof 9 negative by both methods) and discrepancy in 6 cases. In the remaining 5 cases, culture-based methods identified only one species or a group of bacteria (e.g., coagulase negative staphylococci or coryneform rods), while culture-independent molecular methods were able to detect several distinct bacterial species including a species within the group identified by culture. A qPCR assay was developed to assess the abundance of Propionibacterium while S. aureus was quantified by a published S. aureus qPCR assay. These quantifications confirmed the findings from the clone library approach and showed the potential of qPCR for fast detection of bacteria in orthopedic samples. Additionally, both single cells and microcolonies were visualized using FISH and confocal scanning laser microscopy. In conclusion, the molecular methods detected a more diverse bacterial flora in prosthetic joint infections than revealed by standard culture-based methods, and polymicrobial infections were more frequently observed. The pathogenesis of these microorganisms and their role in implant-associated infections needs to be determined

Purpose: Compartment syndrome is a severe complication of skeletal trauma. Intravital microscopy (IVVM) has demonstrated an inflammatory response to compartment syndrome (CS). The molecular mechanisms underlying this inflammatory response are unknown. The purpose of this study was threefold. First, a broad inflammatory cytokine profile was examined to determine the molecules responsible for white cell recruitment. As well, skeletal muscle expression of white cell adhesion molecules including P-Selectin, E-Selectin, Mac-1 and ICAM-1 were examined to assess the extent of white cell activation in target tissues. Finally, skeletal muscle apoptosis was measured to determine the magnitude of cell death. Method: Normal and neutropenic rats were randomised to either compartment syndrome or control groups. CS Animals were treated with 45 minutes of elevated intra-compartmental pressure (EICP) of the hindlimb. Fasciotomy was then performed, followed by 60 minutes of reperfusion. Control animals experienced no EICP. Blood was collected from carotid arterial lines used for pressure monitoring. Skeletal muscle tissue samples were collected from the EDL following reperfusion. Blood samples were obtained from carotid arterial lines and skeletal muscle was collected following reperfusion. A Multiplex assay was used to examine serum levels of 24 proinflammatory cytokines/chemokines. Skeletal muscle mRNA levels of P-Selectin, E-Selectin, Mac-1 and ICAM-1 were evaluated using real-time PCR. Finally, skeletal muscle apoptosis was measured by DNA laddering and a caspase-3 assay. Results: Neutropenic CS animals demonstrated a continuous increase in TNF-alpha levels, peaking at 700+/−350pg/ml by 60 minutes of reperfusion. TNF-alpha values for other groups did not increase. A 104-fold increase in ICAM-1 mRNA levels was observed in neutropenic CS rats while other groups showed no significant increase. There was no significant increase in any group for P-Selectin, E-Selectin, or Mac-1. Conclusion: This study is the first to attempt to describe the molecular inflammatory response in CS. Neutropenic CS animals demonstrated an upregulation in TNF-alpha and ICAM-1 mRNA levels. This likely represents an attempt to generate an inflammatory response in the neutropenic animals. Additional data at incremental timepoints is necessary to further characterize the molecular mechanisms. However, both TNF-alpha and ICAM-1 appear to be important in the mechanism of inflammatory activation in compartment syndrome

While stable long-term clinical results have been achieved in total joint arthroplasty, periprosthetic joint infection (PJI) has been actualized as difficult issue in this decade. For accurate diagnosis, it is important to establish standard criteria such as MSIS criteria, and it is prevailing now. As an issue involving PJI, however, the existence of viable, but non-culturable (VNC) bacteria must be noticed. It is difficult to identify the VNC state infection, because microbiologic culture result shows negative and other markers tend to be negative. Here, molecular diagnosis based on polymerase chain reaction (PCR) has certain role as potential diagnostic tools for such VNC infection. We have applied a real-time PCR system for the diagnosis of PJI, which is able to detect methicillin-resistant Staphylococcus (MRS) and distinguish gram-positive from gram-negative bacteria. The prominent advantage is that PCR is the singular way to identify MRS in such culture negative cases. Recent development of full-automatic PCR system may improve the time efficiency for routine application. In this presentation, we will show the overall sensitivity and specificity of our PCR system for diagnosing PJI and discuss the current problem and future prospect

Tendon pathologies represent an unresolved clinical challenge where the patients suffer from pain and impaired mobility. One of the most frequently ruptured tendons is the Achilles tendon and primarily seen in recreational and professional athletes. A study from Sweden reported a significant increase in the incidence of Achilles tendon ruptures of 17% in men and 22% in women due to the demographic changes and the higher sportive activity of older adults (Huttunen TT Am J Sports Med 2014). The re-rupture rate is between 2–10%, and the patients suffer from an impairment over a long time accompanied with incapability to work. The healing process results in the formation of a mechanically insufficient scar tissue. A detailed knowledge on the cellular and molecular processes underlying human Achilles tendon healing is necessary to develop new treatment strategies and judge therapeutic success. The analysis of human Achilles tendon samples at different time points post rupture and the comparison to intact and degenerated tendon tissue provides important information on the healing process

Introduction. Aseptic loosening is a major cause of revision of total joint arthroplasty (TJA). Although crosslinked Ultra-high molecular weight polyethylene (UHMWPE) have improved wear resistance, residual radicals remaining in the material have a possibility to increase bio-reactivity of particles [2]. In this study, we attempt to evaluate the effects of irradiation and residual radicals on bio-reactivity of the material with a new method called the inverse culture method [1]. Material and methods. UHMWPE particles (10µm diameter in average, Mitsui chemicals Co., LTD) along with irradiated particles (RAD, 300kGy electron irradiation) and particles annealed after the irradiation (RAD+ANN, 100°C 72 hours) are co-incubated with mouse macrophage cell line RAW264 using the inverse culture method. The amount of TNF-α was measured with ELISA. Results and discussion. The amount of TNF-α released by macrophages reacting with virgin UHMWPE, RAD and RAD+ANN is shown in Figure 1. The horizontal axis represents the total surface area of the particles. The coefficient of determination and inclination of the approximate curve are calculated to analyze the result. The coefficient of determination suggested that cytokines released from macrophage is dose-dependent to the surface area of polyethylene particles, which was consistent with the result of our former study[1]. We use the inclination of the approximation curve in Figure 1 as an index to evaluate the bio-reactivity. The values of the index of virgin, RAD and RAD+ANN were 21×10. -4. gLm. -2. , 100×10. -4. gLm. -2. and 59×10. -4. gLm. -2. The inclination of the approximation line of RAD is significantly larger than that of virgin (test for the difference of regression line angle). These suggest that the irradiation to UHMWPE particles increases their bio-reactivity possibly due to radicals. The increased reactivity cannot be eliminated by annealing (100°C 72 hours) completely. Conclusion. Although electron irradiation increases the bio-reactivity of UHMWPE particles, annealing after the irradiation can decrease it, but cannot restore to original reactivity. For any figures or tables, please contact authors directly

Objectives. Ischaemic preconditioning (IPC) is a phenomenon whereby tissues develop an increased tolerance to ischaemia and subsequent reperfusion if first subjected to sublethal periods of ischaemia. Despite extensive investigation of IPC, the molecular mechanism remains largely unknown. Our aim was to show genetic changes that occur in skeletal muscle cells in response to IPC. Methods. We established an in-vitro model of IPC using a human skeletal muscle cell line. Gene expression of both control and preconditioned cells at various time points was determined. The genes examined were HIF-1?, EGR1, JUN, FOS, and DUSP1. HIF-1? is a marker of hypoxia. EGR1, JUN, FOS and DUSP1 are early response genes and may play a role in the protective responses induced by IPC. Secondly, the expression of HSP22 was examined in a cohort of preconditioned total knee arthroplasty patients. Results. HIF-1? was upregulated following 1 and 2 hours of simulated ischaemia (p = 0.076 and 0.841 respectively) verifying that hypoxic conditions were met. Expression of EGR1, FOS and DUSP1 were upregulated and peaked after 1 hour of hypoxia (p = 0.001, < 0.00, and 0.038 respectively). cFOS was upregulated at 2 and 3 hours of hypoxia. IPC prior to simulated hypoxia resulted in a greater level of upregulation of EGR1, JUN and FOS genes (p = < 0.00, 0.047, and < 0.00 respectively). HSP22 was not significantly upregulated following IPC using the hypoxic model. It was, however, upregulated on an mRNA level in total knee arthroplasty patients (p = 0.15). Conclusion. This study has validated the use of our hypoxic model for studying IPC in-vitro. IPC results in a greater upregulation of protective genes in skeletal muscle cells exposed to hypoxia than in control cells. We have demonstrated hitherto unknown molecular mechanisms of IPC in cell culture and in patients undergoing TKA