Osteoarthritis (OA) is a degenerative disease resulting from progressive joint destruction caused by many factors. Its pathogenesis is complex and has not been elucidated to date. Advanced glycation end products (AGEs) are a series of irreversible and stable macromolecular complexes formed by reducing sugar with protein, lipid, and nucleic acid through a non-enzymatic glycosylation reaction (Maillard reaction). They are an important indicator of the degree of ageing. Currently, it is considered that AGEs accumulation in vivo is a molecular basis of age-induced OA, and AGEs production and accumulation in vivo is one of the important reasons for the induction and acceleration of the pathological changes of OA. In recent years, it has been found that AGEs are involved in a variety of pathological processes of OA, including extracellular matrix degradation, chondrocyte apoptosis, and autophagy. Clearly, AGEs play an important role in regulating the expression of OA-related genes and maintaining the chondrocyte phenotype and the stability of the intra-articular environment. This article reviews the latest research results of AGEs in a variety of pathological processes of OA, to provide a new direction for the study of OA pathogenesis and a new target for prevention and treatment. Cite this article: Bone Joint Res 2022;11(5):292–300

Aims. This study aimed to explore the biological and clinical importance of dysregulated key genes in osteoarthritis (OA) patients at the cartilage level to find potential biomarkers and targets for diagnosing and treating OA. Methods. Six sets of gene expression profiles were obtained from the Gene Expression Omnibus database. Differential expression analysis, weighted gene coexpression network analysis (WGCNA), and multiple machine-learning algorithms were used to screen crucial genes in osteoarthritic cartilage, and genome enrichment and functional annotation analyses were used to decipher the related categories of gene function. Single-sample gene set enrichment analysis was performed to analyze immune cell infiltration. Correlation analysis was used to explore the relationship among the hub genes and immune cells, as well as markers related to articular cartilage degradation and bone mineralization. Results. A total of 46 genes were obtained from the intersection of significantly upregulated genes in osteoarthritic cartilage and the key module genes screened by WGCNA. Functional annotation analysis revealed that these genes were closely related to pathological responses associated with OA, such as inflammation and immunity. Four key dysregulated genes (cartilage acidic protein 1 (CRTAC1), iodothyronine deiodinase 2 (DIO2), angiopoietin-related protein 2 (ANGPTL2), and MAGE family member D1 (MAGED1)) were identified after using machine-learning algorithms. These genes had high diagnostic value in both the training cohort and external validation cohort (receiver operating characteristic > 0.8). The upregulated expression of these hub genes in osteoarthritic cartilage signified higher levels of immune infiltration as well as the expression of metalloproteinases and mineralization markers, suggesting harmful biological alterations and indicating that these hub genes play an important role in the pathogenesis of OA. A competing endogenous RNA network was constructed to reveal the underlying post-transcriptional regulatory mechanisms. Conclusion. The current study explores and validates a dysregulated key gene set in osteoarthritic cartilage that is capable of accurately diagnosing OA and characterizing the biological alterations in osteoarthritic cartilage; this may become a promising indicator in clinical decision-making. This study indicates that dysregulated key genes play an important role in the development and progression of OA, and may be potential therapeutic targets. Cite this article: Bone Joint Res 2024;13(2):66–82

Osteoarthritis (OA), one of the most common motor system disorders, is a degenerative disease involving progressive joint destruction caused by a variety of factors. At present, OA has become the fourth most common cause of disability in the world. However, the pathogenesis of OA is complex and has not yet been clarified. Long non-coding RNA (lncRNA) refers to a group of RNAs more than 200 nucleotides in length with limited protein-coding potential, which have a wide range of biological functions including regulating transcriptional patterns and protein activity, as well as binding to form endogenous small interference RNAs (siRNAs) and natural microRNA (miRNA) molecular sponges. In recent years, a large number of lncRNAs have been found to be differentially expressed in a variety of pathological processes of OA, including extracellular matrix (ECM) degradation, synovial inflammation, chondrocyte apoptosis, and angiogenesis. Obviously, lncRNAs play important roles in regulating gene expression, maintaining the phenotype of cartilage and synovial cells, and the stability of the intra-articular environment. This article reviews the results of the latest research into the role of lncRNAs in a variety of pathological processes of OA, in order to provide a new direction for the study of OA pathogenesis and a new target for prevention and treatment. Cite this article: Bone Joint Res 2021;10(2):122–133

Charcot neuroarthropathy is a rare but serious complication of diabetes, causing progressive destruction of the bones and joints of the foot leading to deformity, altered biomechanics and an increased risk of ulceration. Management is complicated by a lack of consensus on diagnostic criteria and an incomplete understanding of the pathogenesis. In this review, we consider recent insights into the development of Charcot neuroarthropathy. It is likely to be dependent on several interrelated factors which may include a genetic pre-disposition in combination with diabetic neuropathy. This leads to decreased neuropeptides (nitric oxide and calcitonin gene-related peptide), which may affect the normal coupling of bone formation and resorption, and increased levels of Receptor activator of nuclear factor kappa-B ligand, potentiating osteoclastogenesis. Repetitive unrecognized trauma due to neuropathy increases levels of pro-inflammatory cytokines (interleukin-1β, interleukin-6, tumour necrosis factor α) which could also contribute to increased bone resorption, in combination with a pre-inflammatory state, with increased autoimmune reactivity and a profile of monocytes primed to transform into osteoclasts - cluster of differentiation 14 (CD14). Increased blood glucose and loss of circulating Receptor for Advanced Glycation End-Products (AGLEPs), leading to increased non-enzymatic glycation of collagen and accumulation of AGLEPs in the tissues of the foot, may also contribute to the pathological process. An understanding of the relative contributions of each of these mechanisms and a final common pathway for the development of Charcot neuroarthropathy are still lacking. Cite this article: S. E. Johnson-Lynn, A. W. McCaskie, A. P. Coll, A. H. N. Robinson. Neuroarthropathy in diabetes: pathogenesis of Charcot arthropathy. Bone Joint Res 2018;7:373–378. DOI: 10.1302/2046-3758.75.BJR-2017-0334.R1

This review discusses the pathogenesis and surgical treatment of tears of the rotator cuff

Osteoarthritis (OA) is mainly caused by ageing, strain, trauma, and congenital joint abnormalities, resulting in articular cartilage degeneration. During the pathogenesis of OA, the changes in subchondral bone (SB) are not only secondary manifestations of OA, but also an active part of the disease, and are closely associated with the severity of OA. In different stages of OA, there were microstructural changes in SB. Osteocytes, osteoblasts, and osteoclasts in SB are important in the pathogenesis of OA. The signal transduction mechanism in SB is necessary to maintain the balance of a stable phenotype, extracellular matrix (ECM) synthesis, and bone remodelling between articular cartilage and SB. An imbalance in signal transduction can lead to reduced cartilage quality and SB thickening, which leads to the progression of OA. By understanding changes in SB in OA, researchers are exploring drugs that can regulate these changes, which will help to provide new ideas for the treatment of OA. Cite this article: Bone Joint Res 2023;12(9):536–545

Discogenic low back pain is a common cause of disability, but its pathogenesis is poorly understood. We collected 19 specimens of lumbar intervertebral discs from 17 patients with discogenic low back pain during posterior lumbar interbody fusion, 12 from physiologically ageing discs and ten from normal control discs. We investigated the histological features and assessed the immunoreactive activity of neurofilament (NF200) and neuropeptides such as substance P (SP) and vasoactive-intestinal peptide (VIP) in the nerve fibres. The distinct histological characteristic of the painful disc was the formation of a zone of vascularised granulation tissue from the nucleus pulposus to the outer part of the annulus fibrosus along the edges of the fissures. SP-, NF- and VIP-immunoreactive nerve fibres in the painful discs were more extensive than in the control discs. Growth of nerves deep into the annulus fibrosus and nucleus pulposus was observed mainly along the zone of granulation tissue in the painful discs. This suggests that the zone of granulation tissue with extensive innervation along the tears in the posterior part of the painful disc may be responsible for causing the pain of discography and of discogenic low back pain

We examined the pathogenesis of Schmorl’s nodes, correlating the histological findings from 12 lumbar vertebrae with the corresponding conventional radiographs, tomographs, MR images and CT scans. The last revealed round, often multiple cystic lesions with indistinct sclerotic margins beneath the cartilaginous endplate. The appearances are similar to the typical CT changes of osteonecrosis. Histological examination of en-bloc slices through Schmorl’s nodes gave clear evidence of subchondral osteonecrosis. Beneath the cartilage endplate, we found fibrosis within the marrow cavities with the disappearance of fat cells. Osteocytes within bone trabeculae were either dead or had disappeared. We suggest that Schmorl’s nodes are the end result of ischaemic necrosis beneath the cartilaginous endplate and that herniation into the body of the vertebra is secondary

Aims. Acquired heterotopic ossification (HO) is a debilitating disease characterized by abnormal extraskeletal bone formation within soft-tissues after injury. The exact pathogenesis of HO remains unknown. It was reported that BRD4 may contribute to osteoblastic differentiation. The current study aims to determine the role of BRD4 in the pathogenesis of HO and whether it could be a potential target for HO therapy. Methods. Achilles tendon puncture (ATP) mouse model was performed on ten-week-old male C57BL/6J mice. One week after ATP procedure, the mice were given different treatments (e.g. JQ1, shMancr). Achilles tendon samples were collected five weeks after treatment for RNA-seq and real-time quantitative polymerase chain reaction (RT-qPCR) analysis; the legs were removed for micro-CT imaging and subsequent histology. Human bone marrow mesenchymal stem cells (hBMSCs) were isolated and purified bone marrow collected during surgeries by using density gradient centrifugation. After a series of interventions such as knockdown or overexpressing BRD4, Alizarin red staining, RT-qPCR, and Western Blot (Runx2, alkaline phosphatase (ALP), Osx) were performed on hBMSCs. Results. Overexpression of BRD4 enhanced while inhibition of Brd4 suppressed the osteogenic differentiation of hBMSCs in vitro. Overexpression of Brd4 increased the expression of mitotically associated long non-coding RNA (Mancr). Downregulation of Mancr suppressed the osteoinductive effect of BRD4. In vivo, inhibition of BRD4 by JQ1 significantly attenuated pathological bone formation in the ATP model (p = 0.001). Conclusion. BRD4 was found to be upregulated in HO and Brd4-Mancr-Runx2 signalling was involved in the modulation of new bone formation in HO. Cite this article: Bone Joint Res 2021;10(10):668–676

Aims. This study aimed to explore the role of small colony variants (SCVs) of Staphylococcus aureus in intraosseous invasion and colonization in patients with periprosthetic joint infection (PJI). Methods. A PJI diagnosis was made according to the MusculoSkeletal Infection Society (MSIS) for PJI. Bone and tissue samples were collected intraoperatively and the intracellular invasion and intraosseous colonization were detected. Transcriptomics of PJI samples were analyzed and verified by polymerase chain reaction (PCR). Results. SCVs can be isolated from samples collected from chronic PJIs intraoperatively. Transmission electron microscopy (TEM) and immunofluorescence (IF) showed that there was more S. aureus in bone samples collected from chronic PJIs, but much less in bone samples from acute PJIs, providing a potential mechanism of PJI. Immunofluorescence results showed that SCVs of S. aureus were more likely to invade osteoblasts in vitro. Furthermore, TEM and IF also demonstrated that SCVs of S. aureus were more likely to invade and colonize in vivo. Cluster analysis and principal component analysis (PCA) showed that there were substantial differences in gene expression profiles between chronic and acute PJI. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that these differentially expressed genes were enriched to chemokine-related signal pathways. PCR also verified these results. Conclusion. Our study has shown that the S. aureus SCVs have a greater ability to invade and colonize in bone, resulting in S. aureus remaining in bone tissues long-term, thus explaining the pathogenesis of chronic PJI. Cite this article: Bone Joint Res 2022;11(12):843–853

A clinical, cadaveric, biomechanical and radiological investigation of the pathogenesis of idiopathic scoliosis indicates that biplanar asymmetry is the essential lesion. Many normal children have coronal plane asymmetry (an inconsequential lateral curvature of the spine), and certainly all have vertebral body asymmetry in the transverse plane, but when median plane asymmetry (flattening or more usually reversal of the normal thoracic kyphosis at the apex of the scoliosis) is superimposed during growth, a progressive idiopathic scoliosis occurs. Idiopathic kyphoscoliosis cannot and does not exist, from the mildest cases in the community to the most severe cases in pathology museums. Median plane asymmetry is crucial for progression and the lateral profile of the spine must be carefully scrutinised. Increased anterior vertebral height at the apex of the curve with posterior end-plate irregularity characterises the median plane asymmetry and suggests that idiopathic scoliosis is the reverse of Scheuermann's disease

Aims. The aim of this study was to explore the genetic correlation and causal relationship between blood plasma proteins and rheumatoid arthritis (RA). Methods. Based on the genome-wide association studies (GWAS) summary statistics of RA from European descent and the GWAS summary datasets of 3,622 plasma proteins, we explored the relationship between RA and plasma proteins from three aspects. First, linkage disequilibrium score regression (LD score regression) was applied to detect the genetic correlation between RA and plasma proteins. Mendelian randomization (MR) analysis was then used to evaluate the causal association between RA and plasma proteins. Finally, GEO2R was used to screen the differentially expressed genes (DEGs) between patients with RA and healthy controls. Results. We found that seven kinds of plasma proteins had genetic correlations with RA, such as Soluble Receptor for Advanced Glycation End Products (sRAGE) (correlation coefficient = 0.2582, p = 0.049), vesicle transport protein USE1 (correlation coefficient = 0.1337, p = 0.018), and spermatogenesis-associated protein 20 (correlation coefficient = 0.3706, p = 0.018). There was a significant causal relationship between sRAGE and RA. By comparing the genes encoding seven plasma proteins, we found that only USE1 was a DEG associated with RA. Conclusion. Our study identified a set of candidate plasma proteins that showed signals correlated with RA. Since the results of this study need further experimental verification, they should be interpreted with caution. However, we hope that this paper will provide new insights for the discovery of pathogenic genes and RA pathogenesis in the future. Cite this article: Bone Joint Res 2022;11(2):134–142

Specimens of tissue from haemophilic synovium and articular cartilage were collected from 39 patients during reconstructive surgery. They were studied by histochemistry, electron microscopy and microprobe analysis. The detailed findings are presented and discussed. It is suggested that haemophilic arthropathy is the result of a number of mechanisms affecting the synovial lining which becomes progressively fibrotic and the hyaline cartilage which disintegrates and is eventually lost. Mechanical and chemical processes cause degeneration of cells but enzymatic processes appear to be primarily responsible for the degradation of the matrix of the articular cartilage.

The clinical, radiological and pathological features of hallux rigidus affecting nine toes (in seven patients) are described. Characteristic chondral and osteochondral lesions are seen to occur at a specific site on the metatarsal head, and account for the limitation of dorsiflexion but relatively unrestricted plantarflexion typical of hallux rigidus. Radiologically these lesions are often missed because they are mainly cartilaginous and are later obscured by secondary degenerative changes. Histological evidence indicates a traumatic aetiology and a mechanism of injury is suggested.

It has been shown that in the puppy, two infarcts separated by an interval of four weeks produce a disorder of long duration which results in flattening and broadening of the femoral head and which reproduces the radiological changes seen in Perthes' disease in man. The histological appearances produced by two infarcts are characteristic. In this study the histological appearance of fifty-seven femoral head biopsy specimens in Perthes' disease in man have been studied. In 51 per cent of hips histopathological changes characteristic of double infarction were present, and there were grounds for postulating that double infarction might eventually occur in all cases. The findings support the concept that the deformation of the femoral head and the chronicity of Perthes' disease in man may be due at least as much or even more to repeated episodes of infarction and the ensuing abnormalities of growth as to mechanical factors related to weight-bearing.

Aims

The metabolic variations between the cartilage of osteoarthritis (OA) and Kashin-Beck disease (KBD) remain largely unknown. Our study aimed to address this by conducting a comparative analysis of the metabolic profiles present in the cartilage of KBD and OA.

Aims

A higher failure rate has been reported in haematogenous periprosthetic joint infection (PJI) compared to non-haematogenous PJI. The reason for this difference is unknown. We investigated the outcome of haematogenous and non-haematogenous PJI to analyze the risk factors for failure in both groups of patients.

From a total of 1571 surgically excised menisci, 112 (7.1 per cent) were found by gross and microscopic examination to contain one or more cysts. All of these cysts were associated with tears, either primarily horizontal or with a horizontal component. Tracks were often demonstrable leading from the tear to the cysts, and in some cases of osteoarthritis, detritus of bone could be found in their periphery. It is concluded that the cysts are fuelled by synovial fluid. The relationship of cysts to "myxoid" change of the meniscus is discussed.

This article reviews the current knowledge of the intervertebral disc (IVD) and its association with low back pain (LBP). The normal IVD is a largely avascular and aneural structure with a high water content, its nutrients mainly diffusing through the end plates. IVD degeneration occurs when its cells die or become dysfunctional, notably in an acidic environment. In the process of degeneration, the IVD becomes dehydrated and vascularised, and there is an ingrowth of nerves. Although not universally the case, the altered physiology of the IVD is believed to precede or be associated with many clinical symptoms or conditions including low back and/or lower limb pain, paraesthesia, spinal stenosis and disc herniation.

New treatment options have been developed in recent years. These include biological therapies and novel surgical techniques (such as total disc replacement), although many of these are still in their experimental phase. Central to developing further methods of treatment is the need for effective ways in which to assess patients and measure their outcomes. However, significant difficulties remain and it is therefore an appropriate time to be further investigating the scientific basis of and treatment of LBP.

We studied 16 club feet and 27 normal feet from spontaneously aborted human fetuses in the second trimester of gestation and measured the length of the spring ligament, and the declination angle and size of the talus. We also studied the cellular characteristics of the spring ligament and the immunohistochemical features of the medial ankle ligaments using monoclonal antibodies against type-III collagen, desmin, vimentin, and smooth muscle actin. Histomorphometric results indicated that the talar deformity was not the primary lesion. Histological and immunohistochemical findings showed that the cells and collagen fibres of the medial ankle ligaments of club feet appeared to be the site of the earliest changes, in that they had lost their spatial orientation and had contracted. In severe club feet before the third trimester of gestation, myofibroblast-like cells seemed to create a disorder of the ligaments resembling fibromatosis. This led to contraction and resulted in typical club-foot deformity.

There is an evolving body of evidence that demonstrates the role of epigenetic mechanisms, such as DNA-methylation in the pathogenesis of OA. This systematic review aims to summarize the current evidence of DNA methylation and its influence on the pathogenesis of OA. A pre-defined protocol in alignment with the PRISMA guidelines was employed to systematically review eight bibliographic databases, to identify associations between DNA-methylation of articular chondrocytes and osteoarthritis. A search of Medline (Ovid), Embase, Web-of-Science, Scopus, PubMed, Cinahl (EBSCOhost), Cochrane Central and Google Scholar was performed between 1st January 2015 to 31st January 2021. Data extraction was performed by two independent reviewers. During the observation period, we identified 15 gene specific studies and 24 genome wide methylation analyses. The gene specific studies mostly focused on the expression of pro-inflammatory markers, such as IL8 and MMP13 which are overexpressed in OA chondrocytes. DNA hypomethylation in the promoter region resulted in overexpression, whereas hypermethylation was seen in non-OA chondrocytes. Others reported on the association between OA risk genes and the DNA methylation pattern close to RUNX2, which is an important OA signal. The genome wide methylation studies reported mostly on differentially methylated regions comparing OA chondrocytes and non-OA chondrocytes. Clustering of the regions identified genes that are involved in skeletal morphogenesis and development. Differentially methylated regions were seen in hip OA and knee OA chondrocytes, and even within different regions of an OA affected knee joint, differentially methylated regions were identified depending on the disease stage. This systematic review demonstrates the growing evidence of epigenetic mechanisms, such as DNA methylation, in the pathogenesis of OA. In recent years, there has been a focus on the interplay between OA risk genes and DNA methylation changes which revealed a reactivation of genes responsible for endochondral ossification during development. These are important findings and may help to identify eventual future therapeutic targets. However, the current body of literature is mostly showing the differences in DNA methylation of OA chondrocytes and non-OA chondrocytes, but a true longitudinal analysis demonstrating the DNA methylation changes actually happening is still not available

Osteoarthritis, the most common degenerative joint disease, significantly impairs life quality and labor capability of patients. Synovial inflammation, initiated by HMGB1 (High mobility group box 1)-induced activation of macrophage, precedes other pathological changes. As an upstream regulator of NF-κB (nuclear factor-kappa B) and MAPK (mitogen-activated protein kinase) signaling pathway, TAK1 (TGF-β activated kinase 1) participates in macrophage activation, while its function in osteoarthritis remains unveiled. This study aims to investigate the role of TAK1 in the pathogenesis of osteoarthritis via both in vitro and in vivo approaches. We performed immunohistochemical staining for TAK1 in synovial tissue, both in osteoarthritis patients and healthy control. Besides, immunofluorescence staining for F4/80 as macrophage marker and TAK1 were conducted as well. TAK1 expression was examined in RAW264.7 macrophages stimulated by HMGB1 via qPCR (Quantitative polymerase chain reaction) and Western blotting, and the effect of TAK1 inhibitor (5z-7 oxozeaenol) on TNF-α production was evaluated by immunofluorescence staining. Further, we explored the influence of intra-articular shRNA (short hairpin RNA) targeting TAK1 on collagenase-induced osteoarthritis in mice. Immunohistochemical staining confirmed significant elevation of TAK1 in osteoarthritic synovium, and immunofluorescence staining suggested macrophages as predominant residence of TAK1. In HMGB1-stimulated RAW264.7 macrophages, TAK1 expression was up-regulated both in mRNA and protein level. Besides, TAK1 inhibitor significantly impairs the production of TNF-α by macrophages upon HMGB1 stimulation. Moreover, intra-articular injection of lentivirus loaded with shRNA targeting TAK1 (sh-TAK1) reduced peri-articular osteophyte formation in collagenase-induced osteoarthritis in mice. TAK1 exerts a potent role in the pathogenesis of osteoarthritis by mediating the activation of macrophages

Osteoarthritis (OA) of the equine distal interphalangeal joint (DIPJ) is a common cause of lameness. MicroRNAs (miRNAs) from biofluids such as plasma and synovial fluid make promising biomarker and therapeutic candidates. The objectives of this study are (1) Identify differentially expressed (DE) miRNAs in mild and severe equine DIPJ OA synovial fluid samples and (2) Determine the effects of DE miRNAs on equine chondrocytes in monolayer culture. Synovial fluid samples from five horses with mild and twelve horses with severe DIPJ OA were submitted for RNA-sequencing; OA diagnosis was made from MRI T2 mapping, macroscopic and histological evaluation. Transfection of equine chondrocytes (n=3) was performed using the Lipofectamine® RNAiMAX system with a negative control and a miR-92a mimic and inhibitor. qPCR was used to quantify target mRNA genes. RNA-seq showed two miRNAs (miR-16 and miR-92a) were significantly DE (p<0.05). Ingenuity Pathway Analysis (IPA) identified important downstream targets of miR-92a involved in the pathogenesis of osteoarthritis and so this miRNA was used to transfect equine chondrocytes from three donor horses diagnosed with OA. Transfection was successfully demonstrated by a 1000-20000 fold increase in miR-92a expression in the equine chondrocytes. There was a significant (p<0.05) increase in COMP, COL3A1 and Sox9 in the miR-92a mimic treatment and there was no difference in ADAMTS-5 expression between the miR-92 mimic and inhibitor treatment. RNA-seq demonstrated miR-92a was downregulated in severe OA synovial fluid samples which has not previously been reported in horses, however miR-92a is known to play a role in the pathogenesis of OA in other species. Over expression of miR-92a in equine chondrocytes led to significantly increased COMP and Sox9 expression, consistent with a chondrogenic phenotype which has been identified in human and murine chondrocytes

Aims. Dupuytren’s contracture is characterized by increased fibrosis of the palmar aponeurosis, with eventual replacement of the surrounding fatty tissue with palmar fascial fibromatosis. We hypothesized that adipocytokines produced by adipose tissue in contact with the palmar aponeurosis might promote fibrosis of the palmar aponeurosis. Methods. We compared the expression of the adipocytokines adiponectin and leptin in the adipose tissue surrounding the palmar aponeurosis of male patients with Dupuytren’s contracture, and of male patients with carpal tunnel syndrome (CTS) as the control group. We also examined the effects of adiponectin on fibrosis-related genes and proteins expressed by fibroblasts in the palmar aponeurosis of patients with Dupuytren’s contracture. Results. Adiponectin expression in the adipose tissue surrounding the palmar aponeurosis was significantly lower in patients with Dupuytren’s contracture than in those with CTS. The expression of fibrosis-related genes and proteins, such as types 1 and 3 collagen and α-smooth muscle actin, was suppressed in a concentration-dependent manner by adding AdipoRon, an adiponectin receptor agonist. The expression of fibrosis-related genes and proteins was also suppressed by AdipoRon in the in vitro model of Dupuytren’s contracture created by adding TGF-β to normal fibroblasts collected from patients with CTS. Conclusion. Fibrosis of the palmar aponeurosis in Dupuytren’s contracture in males may be associated with adiponectin expression in the adipose tissue surrounding the palmar aponeurosis. Although fibroblasts within the palmar aponeurosis are often the focus of attention when elucidating the pathogenesis of Dupuytren’s contracture, adiponectin expression in adipose tissues warrants closer attention in future research. Cite this article: Bone Joint Res 2023;12(8):486–493

Aims. Currently, the effect of drug treatment for osteoporosis is relatively poor, and the side effects are numerous and serious. Melatonin is a potential drug to improve bone mass in postmenopausal women. Unfortunately, the mechanism by which melatonin improves bone metabolism remains unclear. The aim of this study was to further investigate the potential mechanism of melatonin in the treatment of osteoporosis. Methods. The effects of melatonin on mitochondrial apoptosis protein, bmal1 gene, and related pathway proteins of RAW264.7 (mouse mononuclear macrophage leukaemia cells) were analyzed by western blot. Cell Counting Kit-8 was used to evaluate the effect of melatonin on cell viability. Flow cytometry was used to evaluate the effect of melatonin on the apoptosis of RAW264.7 cells and mitochondrial membrane potential. A reactive oxygen species (ROS) detection kit was used to evaluate the level of ROS in osteoclast precursors. We used bmal1-small interfering RNAs (siRNAs) to downregulate the Bmal1 gene. We established a postmenopausal mouse model and verified the effect of melatonin on the bone mass of postmenopausal osteoporosis in mice via micro-CT. Bmal1 lentiviral activation particles were used to establish an in vitro model of overexpression of the bmal1 gene. Results. Melatonin promoted apoptosis of RAW264.7 cells and increased the expression of BMAL1 to inhibit the activation of ROS and phosphorylation of mitogen-activated protein kinase (MAPK)-p38. Silencing the bmal1 gene weakened the above effects of melatonin. After that, we used dehydrocorydaline (DHC) to enhance the activation of MAPK-p38, and the effects of melatonin on reducing ROS levels and promoting apoptosis of RAW264.7 cells were also blocked. Then, we constructed a mouse model of postmenopausal osteoporosis and administered melatonin. The results showed that melatonin improves bone loss in ovariectomized mice. Finally, we established a model of overexpression of the bmal1 gene, and these results suggest that the bmal1 gene can regulate ROS activity and change the level of the MAPK-p38 signalling pathway. Conclusion. Our study confirmed that melatonin promotes the apoptosis of RAW264.7 cells through BMAL1/ROS/MAPK-p38, and revealed the therapeutic effect and mechanism of melatonin in postmenopausal osteoporosis. This finding enriches BMAL1 as a potential target for the treatment of osteoporosis and the pathogenesis of postmenopausal osteoporosis. Cite this article: Bone Joint Res 2023;12(11):677–690

Aims. Transcription factor nuclear factor kappa B (NF-κB) plays a major role in the pathogenesis of chronic inflammatory diseases in all organ systems. Despite its importance, NF-κB targeted drug therapy to mitigate chronic inflammation has had limited success in preclinical studies. We hypothesized that sex differences affect the response to NF-κB treatment during chronic inflammation in bone. This study investigated the therapeutic effects of NF-κB decoy oligodeoxynucleotides (ODN) during chronic inflammation in male and female mice. Methods. We used a murine model of chronic inflammation induced by continuous intramedullary delivery of lipopolysaccharide-contaminated polyethylene particles (cPE) using an osmotic pump. Specimens were evaluated using micro-CT and histomorphometric analyses. Sex-specific osteogenic and osteoclastic differentiation potentials were also investigated in vitro, including alkaline phosphatase, Alizarin Red, tartrate-resistant acid phosphatase staining, and gene expression using reverse transcription polymerase chain reaction (RT-PCR). Results. Local delivery of NF-κB decoy ODN in vivo increased osteogenesis in males, but not females, in the presence of chronic inflammation induced by cPE. Bone resorption activity was decreased in both sexes. In vitro osteogenic and osteoclastic differentiation assays during inflammatory conditions did not reveal differences among the groups. Receptor activator of nuclear factor kappa Β ligand (Rankl) gene expression by osteoblasts was significantly decreased only in males when treated with ODN. Conclusion. We demonstrated that NF-κB decoy ODN increased osteogenesis in male mice and decreased bone resorption activity in both sexes in preclinical models of chronic inflammation. NF-κB signalling could be a therapeutic target for chronic inflammatory diseases involving bone, especially in males. Cite this article: Bone Joint Res 2024;13(1):28–39

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

Aims. Circular RNA (circRNA) S-phase cyclin A-associated protein in the endoplasmic reticulum (ER) (circSCAPER, ID: hsa_circ_0104595) has been found to be highly expressed in osteoarthritis (OA) patients and has been associated with the severity of OA. Hence, the role and mechanisms underlying circSCAPER in OA were investigated in this study. Methods. In vitro cultured human normal chondrocyte C28/I2 was exposed to interleukin (IL)-1β to mimic the microenvironment of OA. The expression of circSCAPER, microRNA (miR)-140-3p, and enhancer of zeste homolog 2 (EZH2) was detected using quantitative real-time polymerase chain reaction and Western blot assays. The extracellular matrix (ECM) degradation, proliferation, and apoptosis of chondrocytes were determined using Western blot, cell counting kit-8, and flow cytometry assays. Targeted relationships were predicted by bioinformatic analysis and verified using dual-luciferase reporter and RNA immunoprecipitation (RIP) assays. The levels of phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) pathway-related protein were detected using Western blot assays. Results. CircSCAPER was highly expressed in OA cartilage tissues and IL-1β-induced chondrocytes. Knockdown of circSCAPER reduced IL-1β-evoked ECM degradation, proliferation arrest, and apoptosis enhancement in chondrocytes. Mechanistically, circSCAPER directly bound to miR-140-3p, and miR-140-3p inhibition reversed the effects of circSCAPER knockdown on IL-1β-induced chondrocytes. miR-140-3p was verified to target EZH2, and overexpression of miR-140-3p protected chondrocytes against IL-1β-induced dysfunction via targeting EZH2. Additionally, we confirmed that circSCAPER could regulate EZH2 through sponging miR-140-3p, and the circSCAPER/miR-140-3p/EZH2 axis could activate the PI3K/AKT pathway. Conclusion. CircSCAPER promoted IL-1β-evoked ECM degradation, proliferation arrest, and apoptosis enhancement in chondrocytes via regulating miR-140-3p/EZH2 axis, which gained a new insight into the pathogenesis of OA. Cite this article: Bone Joint Res 2022;11(2):61–72

Aims. To assess the effect of physical exercise (PE) on the histological and transcriptional characteristics of proteoglycan-induced arthritis (PGIA) in BALB/c mice. Methods. Following PGIA, mice were subjected to treadmill PE for ten weeks. The tarsal joints were used for histological and genetic analysis through microarray technology. The genes differentially expressed by PE in the arthritic mice were obtained from the microarray experiments. Bioinformatic analysis in the DAVID, STRING, and Cytoscape bioinformatic resources allowed the association of these genes in biological processes and signalling pathways. Results. Arthritic mice improved their physical fitness by 42.5% after PE intervention; it induced the differential expression of 2,554 genes. The bioinformatic analysis showed that the downregulated genes (n = 1,371) were significantly associated with cellular processes that mediate the inflammation, including Janus kinase-signal transducer and activator of transcription proteins (JAK-STAT), Notch, and cytokine receptor interaction signalling pathways. Moreover, the protein interaction network showed that the downregulated inflammatory mediators interleukin (IL) 4, IL5, IL2 receptor alpha (IL2rα), IL2 receptor beta (IL2rβ), chemokine ligand (CXCL) 9, and CXCL12 were interacting in several pathways associated with the pathogenesis of arthritis. The upregulated genes (n = 1,183) were associated with processes involved in the remodelling of the extracellular matrix and bone mineralization, as well as with the processes of aerobic metabolism. At the histological level, PE attenuated joint inflammatory infiltrate and cartilage erosion. Conclusion. Physical exercise influences parameters intimately linked to inflammatory arthropathies. Research on the effect of PE on the pathogenesis process of arthritis is still necessary for animal and human models. Cite this article:Bone Joint Res. 2020;9(1):36–48

Syndesmotic ankle lesions involve disruption of the osseous tibiofibular mortise configuration as well as ligamentous structures stabilizing the ankle joint. Incomplete diagnosis and maltreatment of these injuries is frequent, resulting in chronic pain and progressive instability thus promoting development of ankle osteoarthritis in the long term. Although the pathogenesis is not fully understood, abnormal mechanics has been implicated as a principal determinant of ankle joint degeneration after syndesmotic ankle lesions. Therefore, the focus of this presentation will be on our recent development of a computationally efficient algorithm to calculate the contact pressure distribution in patients with a syndesmotic ankle lesion, enabling us to stratify the risk of OA development in the long term and thereby guiding patient treatment

The aim of this study is to clarify the implication of ciliary pathway on the onset of the spinal curvature that occurs in Adolescent Idiopathic Scoliosis (AIS) patients through functional studies of two genes: POC5 and TTLL11. Since the genetic implication for AIS is accepted, many association and candidate gene analysis revealed the implication of ciliary genes. The characterisation of these two proteins was assessed by qPCR, WB and immunofluorescence in vitro using control cells and cells derived from AIS patients. The impact of genetic modification of these genes on the functionality of the proteins in vitro and in vivo was analysed in zebrafish model created by CRISPR/Cas9 using microCT and histologic analysis. Our study revealed that mutant cells, for both gene, were less ciliated and the primary cilia was significantly shorter compared to control cells. We also observed a default in cilia glutamylation by immunofluorescence and Western Blot. Moreover, we observed in both zebrafish model, a 3D spine curvature similar to the spinal deformation in AIS. Interestingly, our preliminary results of immunohistology showed a retinal defect, especially at the cone cell layer level. This study strongly supports the implication of the ciliary pathway in the onset of AIS and this is the first time that a mechanism is described for AIS. Indeed, we show that shorter cilia could be less sensitive to environmental factors due to lower glutamylation and result in altered signalling pathway. Identifying the biological mechanism involved is crucial for elucidating AIS pathogenesis

To determine whether spinal facet osteoblasts at the curve apex display a different phenotype to osteoblasts from outside the curve in patients with adolescent idiopathic scoliosis (AIS). Intrinsic differences in the phenotype of spinal facet bone tissue and in spinal osteoblasts have been implicated in the pathogenesis of AIS. However, no study has compared the phenotype of facet osteoblasts at the curve apex with the facet osteoblasts from outside the curve in patients with AIS. Facet bone tissue was collected from three sites, the concave and convex side at the curve apex and from outside the curve from three female patients with AIS (aged 13–16 years). Micro-CT analysis was used to determine the density and trabecular structure. Osteoblasts were then cultured from the sampled bone. Osteoblast phenotype was investigated by assessing cellular proliferation (MTS assay), cellular metabolism (alkaline phosphatase and Seahorse Analyser), bone nodule mineralisation (Alizarin red assay), and the mRNA expression of Wnt signalling genes (quantitative RT-PCR). Convex bone showed greater bone mineral density and trabecular thickness than did concave bone. The convex side of the curve apex exhibited a significantly higher proliferative and metabolic phenotype and a greater capacity to form mineralised bone nodules than did concave osteoblasts. mRNA expression of SKP2 was significantly greater in both concave and convex osteoblasts than in non-curve osteoblasts. The expression of SFRP1 was significantly downregulated in convex osteoblasts compared with either concave or non-curve. Intrinsic differences that affect osteoblast function are exhibited by spinal facet osteoblasts at the curve apex in patients with AIS

Aims. The Chopart joint complex is a joint between the midfoot and hindfoot. The static and dynamic support system of the joint is critical for maintaining the medial longitudinal arch of the foot. Any dysfunction leads to progressive collapsing flatfoot deformity (PCFD). Often, the tibialis posterior is the primary cause; however, contrary views have also been expressed. The present investigation intends to explore the comprehensive anatomy of the support system of the Chopart joint complex to gain insight into the cause of PCFD. Methods. The study was conducted on 40 adult embalmed cadaveric lower limbs. Chopart joint complexes were dissected, and the structures supporting the joint inferiorly were observed and noted. Results. The articulating bones exhibit features like a cuboid shelf and navicular beak, which appear to offer inferior support to the joint. The expanse of the spring ligament complex is more medial than inferior, while the superomedial part is more extensive than the intermediate and inferoplantar parts. The spring ligament is reinforced by the tendons in the superomedial part (the main tendon of tibialis posterior), the inferomedial part (the plantar slip of tibialis posterior), and the master knot of Henry positioned just inferior to the gap between the inferomedial and inferoplantar bundles. Conclusion. This study highlights that the medial aspect of the talonavicular articulation has more extensive reinforcement in the form of superomedial part of spring ligament and tibialis posterior tendon. The findings are expected to prompt further research in weightbearing settings on the pathogenesis of flatfoot. Cite this article: Bone Jt Open 2024;5(4):335–342

Aims. Long non-coding RNAs (lncRNAs) act as crucial regulators in osteoporosis (OP). Nonetheless, the effects and potential molecular mechanism of lncRNA PCBP1 Antisense RNA 1 (PCBP1-AS1) on OP remain largely unclear. The aim of this study was to explore the role of lncRNA PCBP1-AS1 in the pathogenesis of OP. Methods. Using quantitative real-time polymerase chain reaction (qRT-PCR), osteogenesis-related genes (alkaline phosphatase (ALP), osteocalcin (OCN), osteopontin (OPN), and Runt-related transcription factor 2 (RUNX2)), PCBP1-AS1, microRNA (miR)-126-5p, group I Pak family member p21-activated kinase 2 (PAK2), and their relative expression levels were determined. Western blotting was used to examine the expression of PAK2 protein. Cell Counting Kit-8 (CCK-8) assay was used to measure cell proliferation. To examine the osteogenic differentiation, Alizarin red along with ALP staining was used. RNA immunoprecipitation assay and bioinformatics analysis, as well as a dual-luciferase reporter, were used to study the association between PCBP1-AS1, PAK2, and miR-126-5p. Results. The expression of PCBP1-AS1 was pre-eminent in OP tissues and decreased throughout the development of human bone marrow-derived mesenchymal stem cells (hBMSCs) into osteoblasts. PCBP1-AS1 knockdown and overexpression respectively promoted and suppressed hBMSC proliferation and osteogenic differentiation capacity. Mechanistically, PCBP1-AS1 sponged miR-126-5p and consequently targeted PAK2. Inhibiting miR-126-5p significantly counteracted the beneficial effects of PCBP1-AS1 or PAK2 knockdown on hBMSCs’ ability to differentiate into osteoblasts. Conclusion. PCBP1-AS1 is responsible for the development of OP and promotes its progression by inducing PAK2 expression via competitively binding to miR-126-5p. PCBP1-AS1 may therefore be a new therapeutic target for OP patients. Cite this article: Bone Joint Res 2023;12(6):375–386

Osteoarthritis (OA) affects the whole joint and leads to chronic pain. The sympathetic nervous system (SNS) seems to be involved in OA pathogenesis, as indicated by in vitro studies as well as by our latest work demonstrating that sympathectomy in mice results in increased subchondral bone volume in the OA knee joint. We assume that chronic stress may lead to opposite effects, such as an increased bone loss in OA due to an elevated sympathetic tone. Therefore, we analyzed experimental OA progression in mice exposed to chronic stress. OA was induced in male C57BL/6J mice by surgical destabilization of the medial meniscus (DMM) and Sham as well as non-operated mice served as controls. Half of these groups were exposed to chronic unpredictable mild stress (CUMS). After 12 weeks, chronic stress efficiency was assessed using behavioral tests. In addition to measuring body weight and length, changes in subchondral bone were analyzed by μCT. Dynamic Weight Bearing system was used to monitor OA-related pain. Histological scoring will be conducted to investigate the severity cartilage degeneration and synovial inflammation. CUMS resulted in increased anxiety and significant decrease in body weight gain in all CUMS groups compared to non-CUMS groups. CUMS also increased serum corticosterone in healthy mice, with even higher levels in CUMS mice after DMM surgery. CUMS had no significant effect on subchondral bone, but subarticular bone mineral density and trabecular thickness were increased. Moreover, CUMS resulted in significant potentiation of DMM-associated pain. Our results suggest that the autonomic imbalance with increased sympathetic nervous activity induced by chronic stress exacerbates the severity of OA pain perception. We expect significantly increased cartilage degeneration as well as more severe synovial inflammation in CUMS DMM mice compared to DMM mice

Introduction. Gram-negative prosthetic joint infections (GN-PJI) present unique challenges in management due to their distinct pathogenesis of biofilm formation on implant surfaces. The purpose of this study is to establish a clinically representative GN-PJI model that can reliably recapitulate biofilm formation on titanium implant surface in vivo. We hypothesized that biofilm formation on an implant surface will affect its ability to osseointegrate. Methods. The model was developed using 3D-printed titanium hip implants, to replace the femoral head of male Sprague-Dawley rats. GN-PJI was induced using two bioluminescent Pseudomonas aeruginosa strains: a reference strain (PA14-lux) and a mutant biofilm-defective strain (ΔflgK-lux). Infection was monitored in real-time using the in vivo imaging system (IVIS) and Magnetic Resonance Imaging (MRI). Bacterial loads on implant surface and in periprosthetic tissues were quantified utilizing viable-colony-count. Field-emission scanning-electron-microscopy of the explanted implants was used to visualize the biofilm formation at the bone-implant-interface. The implant stability, as an outcome, was directly assessed by quantifying the osseointegration in vitro using microCT scan, and indirectly assessed by identifying the gait pattern changes using DigiGait. TM. system in vivo. Results. Localized infection was established within the hip joint and was followed by IVIS in real-time. There was a quantitative and qualitative difference in the bacterial load and biofilm formation between PA14-lux and ΔflgK-lux. This difference in the ability to persist in the model between the two strains was reflected in the gait pattern and implant osseointegration. Conclusions. We developed a novel uncemented hip hemiarthroplasty, GN-PJI rat model. To date, the proposed in vivo biofilm-based model is the most clinically representative for GN-PJI since animals can bear weight on the implant and poor osseointegration correlates with biofilm formation. In addition, localized PJI was detected by various modalities. Clinical Relevance. The proposed in vivo GN-PJI model will allow for more reliable testing of novel biofilm-targeting therapeutics

We have developed a novel technique to analyse bone, using imaging mass cytometry (IMC) without the constraints of using immunofluorescent histochemistry. IMC can measure the expression of over 40 proteins simultaneously, without autofluorescence. We analysed mitochondrial respiratory chain (RC) protein deficiencies in human bone which are thought to contribute to osteoporosis with increasing age. Osteoporosis is characterised by reduced bone mineral density (BMD) and fragility fractures. Humans accumulate mitochondrial mutations and RC deficiency with age and this has been linked to the changing phenotype in advancing age and age-related disease. Mitochondrial mutations are detectable from the age of 30 onwards, coincidently the age BMD begins to decline. Mitochondria contain their own genome which accumulates somatic variants at around 10 times the rate of nuclear DNA. Once these mutations exceed a threshold, RC deficiency and cellular dysfunction occur. The PolgD257A/D257A mouse model expresses a proof-reading deficient version of PolgA, a mtDNA polymerase. These mice accumulate mutations 3-5 times higher than wild-type mice showing enhanced levels of age-related osteoporosis and RC deficiency in osteoblasts. Bone samples were analysed from young and old patients, developing a protocol and analysis framework for IMC in bone tissue sections to analyse osteoblasts in-situ for RC deficiency. Samples from the femoral neck of 10 older healthy volunteers aged 40 – 85 were compared with samples from young patients aged 1-19. We have identified RC complex I defect in osteoblasts from 6 of the older volunteers, complex II defects in 2 of the older volunteers, complex IV defect in just 1 older volunteer, and complex V defect in 4 of the older volunteers. These observations are consistent with the PolgD257A/D257A mouse-model and suggest that RC deficiency, due to age-related pathogenic mitochondrial DNA mutations, may play a significant role in the pathogenesis of human age-related osteoporosis

Nuclear factor erythroid 2–related factor 2 (Nrf2)/antioxidant response element (ARE) pathway is key in maintaining redox homeostasis and the pathogenesis of osteoarthritis (OA) involves oxidative distress. We thus investigated whether Nrf2/ARE signaling may control expression of key chondrogenic differentiation and hyaline cartilage maintenance factor SOX9. In human C-28/I2 chondrocytes SOX9 expression was measured by RT–qPCR after shRNA-mediated knockdown of Nrf2 or its antagonist the Kelch-like erythroid cell-derived protein with cap “n” collar homology-associated protein 1 (Keap1). Putative ARE-binding sites in the proximal SOX9 promoter region were inactivated, cloned into pGL3, and co-transfected with phRL–TK for dual-luciferase assays to verify whether Nrf2 transcriptionally regulates SOX9. SOX9 promoter activity without and with Nrf2-inducer methysticin were analyzed. Sox9 expression in articular chondrocytes was correlated to cartilage thickness and degeneration in wild-type (WT) and Nrf2-knockout mice. Data were analyzed by one-way ANOVA, a Student's t-test, or Wilcoxon rank-sum test, according to the normal distribution. Statistical significance was set to p < 0.05. While Keap1-specific RNAi increased SOX9 expression, Nrf2-specific RNAi significantly decreased it. Putative ARE sites (ARE. 1. , ARE. 2. ) were identified in the SOX9 promoter region. ARE. 2. mutagenesis significantly reduced SOX9 promoter activity, while truncation of ARE. 1. did not. A functional ARE. 2. site was thus essential for methysticin-mediated induction of SOX9 promoter activity. Knee cartilage of young Nrf2-knockout mice further revealed significantly fewer Sox9-positive chondrocytes as compared to old Nrf2-knockout animals, which further showed thinner cartilage and more severe cartilage erosion. Our data suggest that SOX9 expression in articular cartilage is directly Nrf2-dependent and that pharmacological Nrf2 activation may hold potential to diminish age-dependent osteoarthritic changes in knee cartilage through improving protective SOX9 expression

Joint tissues release extracellular vesicles (EVs) that potentially sustain joint homeostasis and contribute to osteoarthritis (OA) pathogenesis. EVs are putative novel therapeutics for OA, and transport biologically active molecules (including small non-coding RNAs (SNCRNAs)) between cells. This study identified altering SNCRNA cargo in EVs in OA which may act as early diagnostic markers and treatment targets. OA was surgically induced in four skeletally mature Standardbred horses using an osteochondral fragment model in the left middle carpal joint. The right joint underwent sham surgery. Synovial fluid (SF) and plasma were obtained weekly throughout the 70-day study. EVs were isolated using size exclusion chromatography and characterised using nanoparticle tracking (Nanosight), and exosome fluorescence detection and tetraspanin phenotyping (Exoview). RNA was extracted from EVs derived from SF (sham and OA joints) and plasma collected at days 10, 35, 42, 49, 56, 63, and subjected to small RNA sequencing on a NovaSeq SP100 flow cell (Illumina). Nanosight-derived EV characteristics of size and concentration were not significantly different following disease induction. The diameter of the temporal population of plasma and SF-derived exosomes changed significantly for CD9 and CD81 following OA induction with significant temporal, and disease-related changes in CD63 and CD81 protein expressin in plasma and SF. In SF and plasma-derived EVs snoRNAs, snRNAs, tRNAs, lncRNA, y-RNA, piRNAs and scRNA were found. Following pairwise analysis of all-time points we identified 27 miRs DE in plasma and 45 DE miRs in SF. Seven were DE in plasma and SF; miR-451, miR-25, miR-215, miR-92a, miR-let-7c, miR-486-5p, miR-23a. In plasma and SF 35 and 21 snoRNAs were DE with four DE in plasma and SF; U3, snord15, snord46, snord58. This work has identified alterations to OA EV sncRNAs in plasma and SF providing a greater understanding of the role of EVs in early OA

Osteoarthritis (OA) is the leading cause of pain and disability worldwide and is characterized by the degenerative changes of articular cartilage. Joint loading is required for cartilage maintenance; however, hyper-physiologic loading is a risk factor for OA. Mechanosensitive ion channels Piezo1 and Piezo2 synergistically transduce hyper-physiologic compression of chondrocytes, leading to chondrocyte death and onset of OA. This injury response is inhibited by Piezo channel loss of function, however the mechanistic role of Piezo channels in vivo is unknown. We examined the hypothesis that deletion of Piezo in chondrocytes will protect mice from joint damage and pain-related behaviors following a surgical destabilization of the medial meniscus (DMM), investigating a key mechanistic and mechanobiological role of these channels in the pathogenesis of OA. Aggrecan-Cre Piezo1 and Piezo1/2 knockout mice ((Agc)1-CRE. ERT2. ;Piezo1. fl/fl. Piezo2. fl/fl. ) were generated and given a 5-day Tamoxifen regimen at 12-weeks of age (n=6–12/group/sex). Cre-negative mice served as controls. At 16-weeks, mice received DMM surgery on the left knee. 12-weeks following DMM prior to sacrifice, activity and hyperalgesia were measured using spontaneous running wheels and a small animal algometer. Structural changes in bone, cartilage, and synovium were characterized using microCT, histology, and Modified Mankin Score criteria. Knockout of Piezo1/2 channels was chondroprotective in both sexes following DMM surgery as demonstrated by reduced Modified Mankin Score compared to control animals. Piezo1 KO was chondroprotective in only female mice, indicating a sexually dimorphic response. Piezo1 and Piezo1/2 KO was protective against pain in male mice, while females displayed no differences compared to controls. No changes were observed in bone morphology. Chondrocyte-specific Piezo1/2 knockout protects the knee joint from structural damage, hyperalgesia and functional deficits in a surgical model of PTOA in male and female mice, illustrating the importance of Piezo channels in response to injury in vivo. Future work aims to interrogate potential sexually dimorphic responses to cartilage damage and investigating Piezo2 KO mice

Aim. Patients with late acute periprosthetic joint infections (PJI) and treated with surgical debridement have a high failure rate. Previous studies have shown that rheumatoid arthritis (RA) is an independent risk factor for treatment failure. We conducted a case-control study to identify predictors for failure in late acute PJI treatment in RA patients. We hypothesize that patients with RA have a higher failure rate compared to controls due to the use of immunosuppressive drugs. Method. Data of an international multicenter retrospective observational study was used. Late acute PJI was defined as a sudden onset of symptoms and signs of a PJI, more than 3 months after implantation. Failure of treatment was defined as persistent signs of infection, relapse with the same or reinfection with a different micro-organism, need for prosthesis removal or death. Cases with RA were matched with cases without RA based on the affected joint. A Cox survival analyses, stratified for RA, was used to calculate hazard ratio's (HR) for failure. Subgroup analyses were used to explore other predictors for treatment failure in RA patients. Results. A total of 40 patients with RA and 80 controls without RA were included. Treatment failure occurred in 65% patients with RA compared to 45% for controls (p= .052). 68% of patients with RA used immunosuppressive drugs at time of PJI diagnosis. The use or continuation of immunosuppressive drugs in PJI was not associated with a higher failure rate; neither were the duration of symptoms and causative microorganism. The time between implantation of the prosthetic joint and diagnosis of infection was longer in RA patients: median 110 (IQR 41-171) vs 29 months (IQR 7.5–101.25). Exchange of mobile components was associated with a lower risk of treatment failure (HR 0.489, 95% CI 0.242–0.989, p-value .047). Conclusions. The use of immunosuppressive drugs does not seem to be associated with a higher failure rate in patients with RA. Mobile exchange in RA patients is associated with a lower risk of failure. This might be due to the significantly older age of the prosthesis in RA patients. Future studies are needed to explore these associations and its underlying pathogenesis

Gram-negative prosthetic joint infections (GN-PJI) present unique challenges in management due to their distinct pathogenesis of biofilm formation on implant surfaces. To date, there are no animal models that can fully recapitulate how a biofilm is challenged in vivo in the setting of GN-PJI. The purpose of this study is to establish a clinically representative GN-PJI in vivo model that can reliably depict biofilm formation on titanium implant surface. We hypothesized that the biofilm formation on the implant surface would affect the ability of the implant to be osseointegrated. The model was developed using a 3D-printed, medical-grade titanium (Ti-6Al-4V), monoblock, cementless hemiarthroplasty hip implant. This implant was used to replace the femoral head of a Sprague-Dawley rat using a posterior surgical approach. To induce PJI, two bioluminescent Pseudomonas aeruginosa (PA) strains were utilized: a reference strain (PA14-lux) and a mutant strain that is defective in biofilm formation (DflgK-lux). PJI development and biofilm formation was quantitatively assessed in vivo using the in vivo imaging system (IVIS), and in vitro using the viable colony count of the bacterial load on implant surface. Magnetic Resonance Imaging (MRI) was acquired to assess the involvement of periprosthetic tissue in vivo, and the field emission scanning electron microscopy (FE-SEM) of the explanted implants was used to visualize the biofilm formation at the bone-implant interface. The implant stability, as an outcome, was directly assessed by quantifying the osseointegration using microCT scans of the extracted femurs with retained implants in vitro, and indirectly assessed by identifying the gait pattern changes using DigiGaitTM system in vivo. A localized prosthetic infection was reliably established within the hip joint and was followed by IVIS in real-time. There was a quantitative and qualitative difference in the bacterial load and biofilm formation between PA14 and DflgK. This difference in the ability to persist in the model between the two strains was reflected on the gait pattern and implant osseointegration. We developed a novel uncemented hip hemiarthroplasty GN-PJI rat model. This model is clinically representative since animals can bear weight on the implant. PJI was detected by various modalities. In addition, biofilm formation correlated with implant function and stability. In conclusion, the proposed in vivo GN-PJI model will allow for more reliable testing of novel biofilm-targeting therapetics

Aim. Osteomyelitis (OM) is a debilitating infection of the bone that originates from hematogenous spreading of microbes or contamination after surgery/fracture. OM is mainly caused by the opportunistic bacterium Staphylococcus aureus (SA), which can evade the host immune response, acquire antibiotic resistance and chronically colonize the musculoskeletal tissue . 1,2. , yet the underlying molecular and cellular processes are largely unclear. This study aimed to characterize the pathogenetic mechanisms of SA-OM with a focus on the long pentraxin 3 (PTX3), a soluble pattern recognition molecule and bone tissue component that is emerging as a new player in osteoimmunology . 3. and a diagnostic marker of periprosthetic joint infections, a common form of OM. 4. . Method. A murine model of OM based on intra-bone injection of SA was developed that closely mimicked surgery/trauma-related OM in humans and allowed addressing the role of PTX3 in gene-modified (Ptx3-/-) animals. Local and systemic infection and inflammation were assessed via microbiology, flow cytometry, histochemistry and microCT techniques. Results. SA-injected mice developed chronic infection with measurable levels of viable bone-resident bacteria up until 30 days from microbial challenge. The infection was confined to the treated limbs only and accompanied by extensive tissue remodelling. The bacterial load was higher in WT than Ptx3. -/-. animals at 6 and 14 days from SA injection. Accordingly, WT mice had enhanced systemic inflammation with expanded innate immune compartment in the spleen and increased serum levels of inflammatory cytokines and chemokines. PTX3 levels were higher in SA- than vehicle (PBS)-injected WT animals both in the serum and bone tissue. Furthermore, administration of a PTX3-targeting antibody reduced the bacterial burden in the bones of SA-injected WT mice. Conclusions. In a mouse model of SA-OM, genetic deficiency of PTX3 protected from infection and inflammation, pointing to this pentraxin as a crucial player in OM pathogenesis and a novel therapeutic target in bone infections. The study was approved by the Italian Ministry of Health (approval n. 520/2019-PR issued on 19/07/2019) and supported by Fondazione Beppe and Nuccy Angiolini

TGF-β/Smad2 signaling is considered to be one of the important pathways involved in osteoarthritis (OA) and protein phosphatase magnesium-dependent 1A (PPM1A) functions as an exclusive phosphatase of Smad2 and regulates TGF-β signaling, here, we investigated the functional role of PPM1A in OA pathogenesis. PPM1A expressions in both human OA cartilage and experimental OA mice chondrocytes were analyzed immunohistochemically. Besides, the mRNA and protein expression of PPM1A induced by IL-1β treatment were also detected by q-PCR and immunofluorescence in vitro. OA was induced in PPM1A knockout (KO) mice by destabilization of the medial meniscus (DMM), and histopathological examination was performed. OA was also induced in wild-type (WT) mice, which were then treated with an intra-articular injection of a selective PPM1A inhibitor for 8 weeks. PPM1A protein expressions were increased in both human OA cartilage and experimental OA mice chondrocytes. We also found that treatment with IL-1β in mouse primary chondrocytes significantly increased both mRNA and protein expression of PPM1A in vitro. Importantly, our data showed that PPM1A deletion could substantially protect against surgically induced OA. Concretely, the average OARSI score and quantification of BV/TV of subchondral bone in KO mice were significantly lower than that in WT mice 8 weeks after DMM surgery. Besides, TUNEL staining revealed a significant decrease in apoptotic chondrocytes in PPM1A-KO mice with DMM operation. With OA induction, the rates of chondrocytes positive for Mmp-13 and Adamts-5 in KO mice were also significantly lower than those in WT mice. Moreover, compared with WT mice, the phosphorylation of Smad2 in chondrocytes was increased in KO mice underwent DMM surgery. However, articular-injection with SD-208, a selective inhibitor of TGF-β/Smad2 signaling could significantly abolish the chondroprotective phenotypes in PPM1A-KO mice. Additionally, both cartilage degeneration and subchondral bone subchondral bone sclerosis in DMM model were blunted following intra-articular injection with BC-21, a small-molecule inhibitor for PPM1A. Our study demonstrated that PPM1A inhibition attenuates OA by regulating TGF-β/Smad2 signaling. Furthermore, PPM1A is a potential target for OA treatment and BC-21 may be employed as alternative therapeutic agents for the management of OA

Abstract. Introduction. Synovitis impacts osteoarthritis symptomatology and progression. The transcription factors controlling synovial gene expression have not been described. This study analyses gene expression in synovium samples from 16 patients with osteoarthritis with 9 undergoing arthroscopic and 8 knee trauma surgery for non-arthritic pathologies. Methodology. Intra-operative synovial biopsies were immersed in RNAlater at 4oC before storage at -80oC. Total RNA was extracted using RNAeasy. After purification, RT-PCR and quality assessment, cDNA was applied to Affymetrix Clariom D microarray gene chips. Bioinformatics analyses were performed. Linear models were prepared in limma with gender and BMI factors incorporated sequentially for each pathology comparison, generating 12 models of probes differentially expressed at FDR p<0.05 and Bayes number, B>0. Data analysis of differently expressed genes utilized Ingenuity Pathway Analysis and Cytoscape with Cluego and Cytohubba plug-ins. Results. Amongst the 2084 genes with significantly differential expression (DEG), 135 had transcription regulator capabilities and 121 a nuclear location. IPA analysis of OATKR and arthroscopic tissue comparison DEG identified 12 nuclear transcription factors linked to 31 DEG whose encoded proteins located within cytoplasmic and cell membrane compartments. All 12 were significantly up-regulated and acting in pathways up-regulating transcription of DNA and RNA, cell survival and angiogenesis while down-regulating senescence and apoptosis. NFE2L2, integral to the TGF-beta signalling pathway, was identified as a bottleneck gene. Conclusion. This analysis indicates the complexity of synovial gene expression regulation and offers target genes and pathways for evaluation during osteoarthritis pathogenesis

Abstract. OBJECTIVES. Staphylococcus aureus is one of the most common pathogens in orthopaedic biomaterial-associated infections. The transition of planktonic S. aureus to its biofilm phenotype is critical in the pathogenesis of biomaterial-associated infections and the development of antimicrobial tolerance, which leads to ineffective eradication in clinical practice. This study sought to elucidate the effect of non-lethal dispersion on antimicrobial tolerance in S. aureus biofilms. METHODS. Using a methicillin-sensitive S. aureus reference strain, the effect of non-lethal dispersion on gentamicin tolerance, cellular activity, and the intracellular metabolome of biofilm-associated bacteria were examined. Gentamicin tolerance was estimated using the dissolvable bead biofilm assay. Cellular activity was estimated using the triphenyltetrazolium chloride assay. Metabolome analysis was performed using tandem high-performance liquid chromatography and mass spectrometry. RESULTS. Non-lethal dispersion of biofilm-associated S. aureus was associated with a four-fold reduction in gentamicin tolerance and a 25% increase in cellular respiration of both dispersed and adherent cells. Metabolome analysis found non-lethal dispersion reduced intracellular levels of L-ornithine and L-proline, with increased levels of cyclic nucleotides (p<0.05) in both liberated cells and the remaining biofilm-associated bacteria. These metabolomic changes have previously been shown to be associated with inactivation of the carbon catabolite repression mechanism, which is a key regulatory gatekeeper in the cellular resuscitation of dormant S. aureus cells. CONCLUSION. The metabolomic pipeline described in this study presents a valuable tool in the elucidation of molecular mechanistic pathways in biofilm pathogenesis. Kreb's cycle reactivation, through the carbon catabolite repression regulatory mechanism, has been shown to be associated with the reversal of biofilm-associated gentamicin tolerance. Understanding of the biosynthetic changes associated with the biofilm state will assist in the discovery of novel therapeutic targets in the management of biomaterial-related infections. Declaration of Interest. (b) declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported:I declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research project

Our knowledge of primary bone marrow edema (BME) of the knee is still limited. A major contributing factor is that it shares several radiological findings with a number of vascular, traumatic, and inflammatory conditions having different histopathological features and etiologies. BME can be primary or secondary. The most commonly associated conditions are osteonecrosis, osteochondritis dissecans, complex regional pain syndrome, mechanical strain such as bone contusion/bruising, micro-fracture, stress fracture, osteoarthritis, and tumor. The etiology and pathogenesis of primary BME are unclear. Conservative treatment includes analgesics, non-steroidal anti-inflammatory drugs, weight-bearing limitations, physiotherapy, pulsed electromagnetic fields, prostacyclin, and bisphosphonates. Surgical treatment, with simple perforation, fragment stabilization, combined scraping and perforation, and eventually osteochondral or chondrocyte transplant, is reserved for the late stages. This retrospective study of a cohort of patients with primary BME of the knee was undertaken to describe their clinical and demographic characteristics, identify possible risk factors, and assess treatment outcomes. We reviewed the records of 48 patients with primary BME of the knee diagnosed on MRI by two radiologists and two orthopedists. History, medications, pain type, leisure activities, smoking habits, allergies, and environmental factors were examined. Analysis of patients’ characteristics highlighted that slightly overweight middle-aged female smokers with a sedentary lifestyle are the typical patients with primary BME of the knee. In all patients, the chief symptom was intractable day and night pain (mean value, 8.5/10 on the numerical rating scale) with active as well as passive movement, regardless of BME extent. Half of the patients suffered from thyroid disorders; indeed, the probability of having a thyroid disorder was higher in our patients than in two unselected groups of patients, one referred to our orthopedic center (odds ratio, 18.5) and another suffering from no knee conditions (odds ratio, 9.8). Before pain onset, 56.3% of our cohort had experienced a stressful event (mourning, dismissal from work, concern related to the COVID-19 pandemic). After conservative treatment, despite the clinical improvement and edema resolution on MRI, 93.8% of patients described two new symptoms: a burning sensation in the region of the former edema and a reduced ipsilateral patellar reflex. These data suggest that even though the primary BME did resolve on MRI, the knee did not achieve full healing

Aim. One of the surgical therapeutic options for periprosthetic joint infection (PJI) includes debridement, antibiotics, and implant retention (DAIR). Prognostically favorable criteria for DAIR include short duration of symptoms, stable implant, pathogen susceptible to a ‘biofilm-active’ antimicrobial agent, and intact soft-tissue conditions. Despite this, there is a proportion of failures after DAIR, possibly because the duration of infection is underestimated. With the hypothesis that the duration of infection correlates with the bacterial load, and hence, the bacterial load is associated with failure after DAIR, we aimed to investigate the association of bacterial load in the sonication fluid of mobile parts and clinical outcome after DAIR. Method. From our PJI cohort (2010–2021), patients with DAIR (both palliative and curative approaches) were reviewed retrospectively. Patients with hip, knee or shoulder arthroplasties fulfilling infection definition, available sonication results, and ≥2 years follow-up were included. Sonication results were categorized in ≤ or >1000 cfu/mL. Univariate analysis was performed to identify predictors for DAIR failure. Results. Out of 209 PJIs, we identified 96 patients (100 PJIs, 47.8%) with DAIR. In 67 (69.8%) patients with 71 PJIs, there was a follow-up of ≥2 years. The mean age was 72.7 (SD 12.99) years, 50% were male. The infection affected 36 hips (50.7%), 32 knees (45.1%) and 3 shoulders (4.2%). At follow-up, there were 29 (40.8%) cured and 42 (59.2%) failed cases. When comparing failed and cured cases, we found no difference in comorbidities and previously defined risk factors for PJI, ASA score, Charlson score, anatomic location, no. of previous surgeries, pathogenesis of infection or laboratory values. The proportion of patients with high bacterial load on mobile parts (i.e. >1000 cfu/mL) was significantly higher in the failed DAIR group than it was in the cured group (61.9% vs 20.7%, p<0.001). Conclusions. In this study, a high bacterial load in sonication fluid of mobile parts was associated with failure after DAIR in patients with PJI. Sonication may help to differentiate acute hematogenous seeding to the implant and late reactivation of a previously silent implant-associated infection

Aims. Dystrophic calcification (DC) is the abnormal appearance of calcified deposits in degenerating tissue, often associated with injury. Extensive DC can lead to heterotopic ossification (HO), a pathological condition of ectopic bone formation. The highest rate of HO was found in combat-related blast injuries, a polytrauma condition with severe muscle injury. It has been noted that the incidence of HO significantly increased in the residual limbs of combat-injured patients if the final amputation was performed within the zone of injury compared to that which was proximal to the zone of injury. While aggressive limb salvage strategies may maximize the function of the residual limb, they may increase the possibility of retaining non-viable muscle tissue inside the body. In this study, we hypothesized that residual dead muscle tissue at the zone of injury could promote HO formation. Methods. We tested the hypothesis by investigating the cellular and molecular consequences of implanting devitalized muscle tissue into mouse muscle pouch in the presence of muscle injury induced by cardiotoxin. Results. Our findings showed that the presence of devitalized muscle tissue could cause a systemic decrease in circulating transforming growth factor-beta 1 (TGF-β1), which promoted DC formation following muscle injury. We further demonstrated that suppression of TGF-β signalling promoted DC in vivo, and potentiated osteogenic differentiation of muscle-derived stromal cells in vitro. Conclusion. Taken together, these findings suggest that TGF-β1 may play a protective role in dead muscle tissue-induced DC, which is relevant to understanding the pathogenesis of post-traumatic HO. Cite this article: Bone Joint Res 2020;9(11):742–750