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

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Osteoarthritis (OA) is a common degenerative disease. PA28γ is a member of the 11S proteasome activator and is involved in the regulation of several important cellular processes, including cell proliferation, apoptosis, and inflammation. This study aimed to explore the role of PA28γ in the occurrence and development of OA and its potential mechanism.

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)

Introduction. Identification of the causative pathogen in musculoskeletal infection is critical as it directs further treatment. Fracture-related infection is often associated with ‘no growth’ in standard culture. We investigated the efficiency of two alternate methods to identify the causative pathogen, namely extended bacterial culture and 16Sr RNA gene sequence analysis with next generation sequencing (NGS) in ‘culture negative’ fracture related infections. Method. Patients were diagnosed as having fracture related infection based on the MSIS criteria (n=120). All patients had samples taken for culture and sensitivity. All samples which were culture negative by standard culture methods formed the study group. These samples were subjected to further extended culture in both aerobic and anaerobic medium for 14 days to improve recovery of pathogens. Further, DNA isolated from implants from a sub-group of these culture negative patients were subjected to 16SrRNA gene amplification followed by Sanger sequencing. Subsequent sequencing analysis was performed using the Illumina NGS platform which identified and detected the most abundant genera/species present in those samples more precisely. Result. 57 culture negative samples formed the study group. Eight samples (14%) converted to positivity after 14 days of culture. Bacteroides fragilis was the commonest yield. 14 samples underwent 16SrRNA gene amplification followed by Sanger sequencing. Acinetobacter baumannii, Enterococcus faecalis, Pseudomonas aeruginosa were identified as common pathogens. Next generation sequencing (n=12) not only identified common pathogens like as Staphylococcus, Acinetobacter baumannii, but also many uncultivable species. Conclusion. Positive results from extended bacterial culture are about 15%. The delay in definite identification of pathogens in extended culture may be critical in certain clinical situations. Molecular methods are quicker and have additional yield in culture negative infections. The exact role of all microorganisms identified by molecular methods in the pathogenesis of infection is unknown

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This study aimed to define the histopathology of degenerated humeral head cartilage and synovial inflammation of the glenohumeral joint in patients with omarthrosis (OmA) and cuff tear arthropathy (CTA). Additionally, the potential of immunohistochemical tissue biomarkers in reflecting the degeneration status of humeral head cartilage was evaluated.

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This study examined the relationship between obesity (OB) and osteoporosis (OP), aiming to identify shared genetic markers and molecular mechanisms to facilitate the development of therapies that target both conditions simultaneously.

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This study aimed to demonstrate the promoting effect of elastic fixation on fracture, and further explore its mechanism at the gene and protein expression levels.

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Rotator cuff tear (RCT) is the leading cause of shoulder pain, primarily associated with age-related tendon degeneration. This study aimed to elucidate the potential differential gene expressions in tendons across different age groups, and to investigate their roles in tendon degeneration.

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

The August 2024 Research Roundup³⁶⁰ looks at: Effect of vitamin D deficiency on periprosthetic joint infection and complications after primary total joint replacement; Postoperative angiotensin receptor blocker use associated with decreased rates of manipulation under anaesthesia in patients undergoing total knee arthroplasty; Central sensitization: the missing link between psychological distress and poor outcome following primary total knee arthroplasty; Thromboprophylaxis for the trauma and orthopaedic surgeon; Life expectancy after treatment of metastatic bone disease: an international trend analysis.

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The antidiabetic agent metformin inhibits fibrosis in various organs. This study aims to elucidate the effects of hyperglycaemia and metformin on knee joint capsule fibrosis in mice.

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Adenosine, lidocaine, and Mg²⁺ (ALM) therapy exerts differential immuno-inflammatory responses in males and females early after anterior cruciate ligament (ACL) reconstruction (ACLR). Our aim was to investigate sex-specific effects of ALM therapy on joint tissue repair and recovery 28 days after surgery.

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Current diagnostic tools are not always able to effectively identify periprosthetic joint infections (PJIs). Recent studies suggest that circulating microRNAs (miRNAs) undergo changes under pathological conditions such as infection. The aim of this study was to analyze miRNA expression in hip arthroplasty PJI patients.

Aims. This study aimed to determine the expression and clinical significance of a cartilage protein, cartilage oligomeric matrix protein (COMP), in knee osteoarthritis (OA) patients. Methods. A total of 270 knee OA patients and 93 healthy controls were recruited. COMP messenger RNA (mRNA) and protein levels in serum, synovial fluid, synovial tissue, and fibroblast-like synoviocytes (FLSs) of knee OA patients were determined using enzyme-linked immunosorbent assay, real-time polymerase chain reaction, and immunohistochemistry. Results. COMP protein levels were significantly elevated in serum and synovial fluid of knee OA patients, especially those in the advanced stages of the disease. Serum COMP was significantly correlated with radiological severity as well as measures of body composition, physical performance, knee pain, and disability. Receiver operating characteristic curve analysis unveiled a diagnostic value of serum COMP as a biomarker of knee OA (41.64 ng/ml, area under the curve (AUC) = 1.00), with a sensitivity of 99.6% and a specificity of 100.0%. Further analysis uncovered that COMP mRNA expression was markedly upregulated in the inflamed synovium of knee OA, consistent with immunohistochemical staining revealing localization of COMP protein in the lining and sub-lining layers of knee OA inflamed synovium. Most notably, relative COMP mRNA expression in knee OA synovium was positively associated with its protein levels in serum and synovial fluid of knee OA patients. In human knee OA FLSs activated with tumour necrosis factor-alpha, COMP mRNA expression was considerably up-regulated in a time-dependent manner. Conclusion. All results indicate that COMP might serve as a supportive diagnostic marker for knee OA in conjunction with the standard diagnostic methods. Cite this article: Bone Joint Res 2024;13(6):261–271

Aims. In this investigation, we administered oxidative stress to nucleus pulposus cells (NPCs), recognized DNA-damage-inducible transcript 4 (DDIT4) as a component in intervertebral disc degeneration (IVDD), and devised a hydrogel capable of conveying small interfering RNA (siRNA) to IVDD. Methods. An in vitro model for oxidative stress-induced injury in NPCs was developed to elucidate the mechanisms underlying the upregulation of DDIT4 expression, activation of the reactive oxygen species (ROS)-thioredoxin-interacting protein (TXNIP)-NLRP3 signalling pathway, and nucleus pulposus pyroptosis. Furthermore, the mechanism of action of small interfering DDIT4 (siDDIT4) on NPCs in vitro was validated. A triplex hydrogel named siDDIT4@G5-P-HA was created by adsorbing siDDIT4 onto fifth-generation polyamidoamine (PAMAM) dendrimer using van der Waals interactions, and then coating it with hyaluronic acid (HA). In addition, we established a rat puncture IVDD model to decipher the hydrogel’s mechanism in IVDD. Results. A correlation between DDIT4 expression levels and disc degeneration was shown with human nucleus pulposus and needle-punctured rat disc specimens. We confirmed that DDIT4 was responsible for activating the ROS-TXNIP-NLRP3 axis during oxidative stress-induced pyroptosis in rat nucleus pulposus in vitro. Mitochondria were damaged during oxidative stress, and DDIT4 contributed to mitochondrial damage and ROS production. In addition, siDDIT4@G5-P-HA hydrogels showed good delivery activity of siDDIT4 to NPCs. In vitro studies illustrated the potential of the siDDIT4@G5-P-HA hydrogel for alleviating IVDD in rats. Conclusion. DDIT4 is a key player in mediating pyroptosis and IVDD in NPCs through the ROS-TXNIP-NLRP3 axis. Additionally, siDDIT4@G5-P-HA hydrogel has been found to relieve IVDD in rats. Our research offers an innovative treatment option for IVDD. Cite this article: Bone Joint Res 2024;13(5):247–260

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To assess the alterations in cell-specific DNA methylation associated with chondroitin sulphate response using peripheral blood collected from Kashin-Beck disease (KBD) patients before initiation of chondroitin sulphate treatment.

Tendinopathy can commonly occur around the foot and ankle resulting in isolated rupture, debilitating pain and degenerative foot deformity. The pathophysiology and key cells involved are not fully understood. This is partly because the dense collagen matrix that surrounds relatively few resident cells limits the ability of previous techniques to identify and target those cells of interest. In this study, we apply novel single cell RNA sequencing (CITE-Seq) techniques to healthy and tendinopathic foot/ankle tendons. For the first time we have identified multiple sub-populations of cells in human tendons. These findings challenge the view that there is a single principal tendon cell type and open new avenues for further study. Healthy tendon samples were obtained from patients undergoing tendon transfer procedures; including tibialis posterior and FHL. Diseased tendon samples were obtained during debridement of intractable Achilles and peroneal tendinopathy, and during fusion of degenerative joints. Single cell RNA sequencing with surface proteomic analysis identified 10 sub-populations of human tendon derived cells. These included groups expressing genes associated with fibro-adipogenic progenitors (FAPs) as well as ITGA7+VCAM1- recently described in mouse muscle but, as yet, not human tendon. In addition we have identified previously unrecognised sub-classes of collagen type 1 associated tendon cells. Each sub-class expresses a different set of extra-cellular matrix genes suggesting they each play a unique role in maintaining the structural integrity of normal tendon. Diseased tendon harboured a greater proportion of macrophages and cytotoxic lymphocytes than healthy tendon. This inflammatory response is potentially driven by resident tendon fibroblasts which show increased expression of pro-inflammatory cytokines. Finally, identification of a previously unknown sub-population of cells found predominantly in tendinopathic tissue offers new insight into the underlying pathophysiology. Further work aims to identify novel proteins targets for possible therapeutic pathways

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Pigment epithelium-derived factor (PEDF) is known to induce several types of tissue regeneration by activating tissue-specific stem cells. Here, we investigated the therapeutic potential of PEDF 29-mer peptide in the damaged articular cartilage (AC) in rat osteoarthritis (OA).

Aims. Osteoarthritis (OA) is the most common chronic pathema of human joints. The pathogenesis is complex, involving physiological and mechanical factors. In previous studies, we found that ferroptosis is intimately related to OA, while the role of Sat1 in chondrocyte ferroptosis and OA, as well as the underlying mechanism, remains unclear. Methods. In this study, interleukin-1β (IL-1β) was used to simulate inflammation and Erastin was used to simulate ferroptosis in vitro. We used small interfering RNA (siRNA) to knock down the spermidine/spermine N1-acetyltransferase 1 (Sat1) and arachidonate 15-lipoxygenase (Alox15), and examined damage-associated events including inflammation, ferroptosis, and oxidative stress of chondrocytes. In addition, a destabilization of the medial meniscus (DMM) mouse model of OA induced by surgery was established to investigate the role of Sat1 inhibition in OA progression. Results. The results showed that inhibition of Sat1 expression can reduce inflammation, ferroptosis changes, reactive oxygen species (ROS) level, and lipid-ROS accumulation induced by IL-1β and Erastin. Knockdown of Sat1 promotes nuclear factor-E2-related factor 2 (Nrf2) signalling. Additionally, knockdown Alox15 can alleviate the inflammation-related protein expression induced by IL-1β and ferroptosis-related protein expression induced by Erastin. Furthermore, knockdown Nrf2 can reverse these protein expression alterations. Finally, intra-articular injection of diminazene aceturate (DA), an inhibitor of Sat1, enhanced type II collagen (collagen II) and increased Sat1 and Alox15 expression. Conclusion. Our results demonstrate that inhibition of Sat1 could alleviate chondrocyte ferroptosis and inflammation by downregulating Alox15 activating the Nrf2 system, and delaying the progression of OA. These findings suggest that Sat1 provides a new approach for studying and treating OA. Cite this article: Bone Joint Res 2024;13(3):110–123

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Continuous local antibiotic perfusion (CLAP) has recently attracted attention as a new drug delivery system for orthopaedic infections. CLAP is a direct continuous infusion of high-concentration gentamicin (1,200 μg/ml) into the bone marrow. As it is a new system, its influence on the bone marrow is unknown. This study aimed to examine the effects of high-concentration antibiotics on human bone tissue-derived cells.