Background: Osteoarthritis (OA) is a whole joint disease that affects all joint tissues, with changes in the articular cartilage (AC), subchondral bone and synovium. Pathologies in menisci and ligaments, however, are rarely analysed, although both are known to play vital roles in the mechanical stability of the joint. The aim of our study was to describe the pathological changes in menisci and ligament during disease development in murine spontaneous and post-traumatic surgically induced OA and to quantify tissue mineralisation in the joint space using microcomputed tomography (μCT) imaging during OA progression. Methods: Knees of Str/ort mice (spontaneous OA model; 26-40 weeks) and C57CBA F1 mice following destabilisation of medial meniscus (DMM) surgery (post-traumatic OA model; 8 weeks after DMM), were used to assess histological meniscal and ligament pathologies. Joint space mineralised tissue volume was quantified by μCT. Results: Meniscal pathological changes in Str/ort mouse knees were associated with articular cartilage lesion severity. These meniscal changes included ossification, hyperplasia, cell hypertrophy, collagen type II deposition and Sox9 expression in the fibrous region near the attachment to the knee joint capsule. Anterior cruciate ligaments exhibited extracellular matrix changes and chondrogenesis particularly at the tibial attachment site, and ossification was seen in collateral ligaments. Similar changes were confirmed in the post-traumatic DMM model. μCT analysis showed increased joint space mineralised tissue volume with OA progression in both the post-traumatic and spontaneous OA models. Conclusions: Modifications in meniscal and ligament mineralisation and chondrogenesis are seen with overt AC degeneration in murine OA. Although the aetiology and the consequences of such changes remain unknown, they will influence stability and load transmission of the joint and may therefore contribute to OA progression. In addition, these changes may have important roles in movement restriction and pain, which represent major human clinical symptoms of OA. Description of such soft tissue changes, in addition to AC degradation, should be an important aspect of future studies in mouse models in order to furnish a more complete understanding of OA pathogenesis.

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Tendon transcriptomics is a rapidly growing field in musculoskeletal biology. The ultimate aim of many current tendon transcriptomic studies is characterization of in vitro, ex vivo, or in vivo, healthy, and diseased tendon microenvironments to identify the underlying pathways driving human tendon pathology. The transcriptome interfaces between genomic, proteomic, and metabolomic signatures of the tendon cellular niche and the response of this niche to stimuli. Some of the greatest bottlenecks in tendon transcriptomics relate to the availability and quality of human tendon tissue, hence animal tissues are frequently used even though human tissue is most translationally relevant. Here, we review the variability associated with human donor and procurement factors, such as whether the tendon is cadaveric or a clinical remnant, and how these variables affect the quality and relevance of the transcriptomes obtained. Moreover, age, sex, and health demographic variables impact the human tendon transcriptome. Tendons present tissue‐specific challenges for cell, nuclei, and RNA extraction that include a dense extracellular matrix, low cellularity, and therefore low RNA yield of variable quality. Consideration of these factors is particularly important for single‐cell and single‐nuclei resolution transcriptomics due to the necessity for unbiased and representative cell or nuclei populations. Different cell, nuclei, and RNA extraction methods, library preparation, and quality control methods are used by the tendon research community and attention should be paid to these when designing and reporting studies. We discuss the different components and challenges of human tendon transcriptomics, and propose pipelines, quality control, and reporting guidelines for future work in the field.

CCN2 is a matricellular protein involved in several crucial biological processes. In particular, CCN2 is involved in cartilage development and in osteoarthritis. Ccn2 null mice exhibit a range of skeletal dysmorphisms, highlighting its importance in regulating matrix formation during development; however, its role in adult cartilage remains unclear. The aim of this study was to determine the role of CCN2 in postnatal chondrocytes in models of post-traumatic osteoarthritis (PTOA). Ccn2 deletion was induced in articular chondrocytes of male transgenic mice at 8 weeks of age. PTOA was induced in knees either surgically or non-invasively by repetitive mechanical loading at 10 weeks of age. Knee joints were harvested, scanned with micro-computed tomography and processed for histology. Sections were stained with Toluidine Blue and scored using the Osteoarthritis Research Society International (OARSI) grading system. In the non-invasive model, cartilage lesions were present in the lateral femur, but no significant differences were observed between wild-type (WT) and Ccn2 knockout (KO) mice 6 weeks post-loading. In the surgical model, severe cartilage degeneration was observed in the medial compartments, but no significant differences were observed between WT and Ccn2 KO mice at 2, 4 and 8 weeks post-surgery. We conclude that Ccn2 deletion in chondrocytes does not modify the development of PTOA in mice, suggesting that chondrocyte expression of CCN2 in adults is not a crucial factor in protecting cartilage from the degeneration associated with PTOA.This article has an associated First Person interview with the first author of the paper.

statement:This manuscript describes histological changes in mouse knee joints in two models of osteoarthritis and correlates joint space mineralised tissue volume measured by µCT with disease severity.

The molecular and cellular basis of health in human tendons remains poorly understood. Amongst human tendons, the hamstrings are the least likely to be injured or degenerate, providing a prototypic healthy tendon reference. The aim of this study was to define the transcriptome and location of all cell types in healthy hamstring tendon. We profiled the transcriptomes of 10,533 nuclei from 4 healthy donors using single-nucleus RNA sequencing (snRNA-seq) and identified 12 distinct cell types. We confirmed the presence of two fibroblast cell types, endothelial cells, mural cells, and immune cells, and revealed the presence of cell types previously unreported for tendon sites, including different skeletal muscle cell types, satellite cells, adipocytes, and nerve cells, which are undefined nervous system cells. Location of these cell types within tendon was defined using spatial transcriptomics and imaging, and transcriptional networks and cell-cell interactions were identified. We demonstrate that fibroblasts have a high number of potential cell-cell interactions, are present throughout the whole tendon tissue, and play an important role in the production and organisation of extracellular matrix, thus confirming their role as key regulators of hamstring tendon tissue homeostasis. Overall, our findings highlight the highly complex cellular networks underpinning tendon function and underpins the importance of fibroblasts as key regulators of hamstring tendon tissue homeostasis.

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The collection of (human) tissue is essential for many aspects of biomedical research. In order to maintain tissue quality and achieve reproducible and representative results, it is critical that the tissue is collected in a consistent manner, especially when tissue is collected by different people, at different locations, and/or over a long period of time. This is particularly important for snap-frozen tissue, which can be stored for long periods of time before use in a range of experiments including bulk and single nucleus RNA sequencing and imaging. Therefore, this protocol for the collection of snap-frozen tissue provides a set of reproducible steps, including recommendations for pictures to be taken and specific details to be noted down, to ensure that consistent tissue collection, such that the collected tissue can be optimally used in the future.