Objective: The aim of this study was to characterize the genome‐wide DNA methylation profile of chondrocytes from knee and hip cartilage obtained from patients with osteoarthritis (OA) and hip cartilage obtained from patients with femoral neck fracture, providing the first comparison of DNA methylation between OA and non‐OA hip cartilage, and between OA hip and OA knee cartilage.Methods: The study was performed using the Illumina Infinium HumanMethylation450 BeadChip array, which allows the annotation of ∼480,000 CpG sites. Genome‐wide methylation was assessed in chondrocyte DNA extracted from 23 hip OA patients, 73 knee OA patients, and 21 healthy hip control patients with femoral neck fracture.Results: Analysis revealed that chondrocytes from the hip cartilage of OA patients and healthy controls have unique methylation profiles, with 5,322 differentially methylated loci (DMLs) identified between the 2 groups. In addition, a comparison between hip and knee OA chondrocytes revealed 5,547 DMLs between the 2 groups, including DMLs in several genes known to be involved in the pathogenesis of OA. Hip OA samples were found to cluster into 2 groups. A total of 15,239 DMLs were identified between the 2 clusters, with an enrichment of genes involved in inflammation and immunity. Similarly, we confirmed a previous report of knee OA samples that also clustered into 2 groups.Conclusion: We demonstrated that global DNA methylation using a high‐density array can be a powerful tool in the characterization of OA at the molecular level. Identification of pathways enriched in DMLs between OA and OA‐free cartilage highlight potential etiologic mechanisms that are involved in the initiation and/or progression of the disease and that could be therapeutically targeted.

Osteoarthritis (OA) is a leading cause of disability, globally. Despite an emerging role for synovial inflammation in OA pathogenesis, attempts to target inflammation therapeutically have had limited success. A better understanding of the cellular and molecular processes occurring in the OA synovium is needed to develop novel therapeutics. We investigated macrophage phenotype and gene expression in synovial tissue of OA and inflammatory-arthritis (IA) patients. Compared with IA, OA synovial tissue contained higher but variable proportions of macrophages ( P < 0.001). These macrophages exhibited an activated phenotype, expressing folate receptor-2 and CD86, and displayed high phagocytic capacity. RNA sequencing of synovial macrophages revealed 2 OA subgroups. Inflammatory-like OA (iOA) macrophages are closely aligned to IA macrophages and are characterized by a cell proliferation signature. In contrast, classical OA (cOA) macrophages display cartilage remodeling features. Supporting these findings, when compared with cOA, iOA synovial tissue contained higher proportions of macrophages ( P < 0.01), expressing higher levels of the proliferation marker Ki67 ( P < 0.01). These data provide new insight into the heterogeneity of OA synovial tissue and suggest distinct roles of macrophages in pathogenesis. Our findings could lead to the stratification of OA patients for suitable disease-modifying treatments and the identification of novel therapeutic targets.

Matrix metalloproteinase‐13 (MMP‐13) is a uniquely important collagenase that promotes the irreversible destruction of cartilage collagen in osteoarthritis (OA). Collagenase activation is a key control point for cartilage breakdown to occur, yet our understanding of the proteinases involved in this process is limited. Neutrophil elastase (NE) is a well‐described proteoglycan‐degrading enzyme which is historically associated with inflammatory arthritis, but more recent evidence suggests a potential role in OA. In this study, we investigated the effect of neutrophil elastase on OA cartilage collagen destruction and collagenase activation. Neutrophil elastase induced significant collagen destruction from human OA cartilage ex vivo, in an MMP‐dependent manner. In vitro, neutrophil elastase directly and robustly activated pro‐MMP‐13, and N‐terminal sequencing identified cleavage close to the cysteine switch at ⁷²MKKPR, ultimately resulting in the fully active form with the neo‐N terminus of ⁸⁵YNVFP. Mole‐per‐mole, activation was more potent than by MMP‐3, a classical collagenase activator. Elastase was detectable in human OA synovial fluid and OA synovia which displayed histologically graded evidence of synovitis. Bioinformatic analyses demonstrated that, compared with other tissues, control cartilage exhibited remarkably high transcript levels of the major elastase inhibitor, (AAT) alpha‐1 antitrypsin (gene name SERPINA1), but these were reduced in OA. AAT was located predominantly in superficial cartilage zones, and staining enhanced in regions of cartilage damage. Finally, active MMP‐13 specifically inactivated AAT by removal of the serine proteinase cleavage/inhibition site. Taken together, this study identifies elastase as a novel activator of pro‐MMP‐13 that has relevance for cartilage collagen destruction in OA patients with synovitis.

Lack of suitable in vitro culture conditions for primary acute lymphoblastic leukaemia (ALL) cells severely impairs their experimental accessibility and the testing of new drugs on cell material reflecting clonal heterogeneity in patients. We show that Nestin-positive human mesenchymal stem cells (MSC) support expansion of a range of biologically and clinically distinct patient-derived ALL samples. Adherent ALL cells showed an increased accumulation in the S-phase of the cell cycle and diminished apoptosis when compared to cells in the suspension fraction. Moreover, surface expression of adhesion molecules CD34, CDH2 and CD10 increased several folds. About 20% of the ALL cells were in G0 phase of the cell cycle suggesting that MSCs may support quiescent ALL cells. Cellular barcoding demonstrated long-term preservation of clonal abundance. Expansion of ALL cells for more than 3 months compromised neither feeder dependence nor cancer initiating ability as judged by their engraftment potential in immunocompromised mice. Finally, we demonstrate the suitability of this co-culture approach for the investigation of drug combinations with luciferase-expressing primograft ALL cells. Taken together, we have developed a preclinical platform with patient-derived material that will facilitate the development of clinically effective combination therapies for ALL.

Osteosarcoma (OS) is the most common primary bone cancer in children and, unfortunately, is associated with poor survival rates. OS most commonly arises around the knee joint, and was traditionally treated with amputation until surgeons began to favour limb-preserving surgery in the 1990s. Whilst improving functional outcomes, this was not without problems, such as implant failure and limb length discrepancies. OS can also arise in areas such as the pelvis, spine, head, and neck, which creates additional technical difficulty given the anatomical complexity of the areas. We reviewed the literature and summarised the recent advances in OS surgery. Improvements have been made in many areas; developments in pre-operative imaging technology have allowed improved planning, whilst the ongoing development of intraoperative imaging techniques, such as fluorescent dyes, offer the possibility of improved surgical margins. Technological developments, such as computer navigation, patient specific instruments, and improved implant design similarly provide the opportunity to improve patient outcomes. Going forward, there are a number of promising avenues currently being pursued, such as targeted fluorescent dyes, robotics, and augmented reality, which bring the prospect of improving these outcomes further.