Objective Single-cell RNA-sequencing (scRNA-seq) has advanced our understanding of the heterogeneity of synovial fibroblasts and their roles in tissue repair and osteoarthritis. Here, we compared the fibroblast responses to different types and duration of joint injury in mice. Design Published scRNA-seq data of synovial fibroblasts from a model of joint surface injury (JSI, day 6) and two post-traumatic osteoarthritis models, destabilisation of the medial meniscus (DMM, day 7 and 2 months) and anterior cruciate ligament rupture (ACLR, day 7 and day 28), were integrated and analysed using clustering, DEG, gene-set enrichment, regulon, pseudotime trajectory and cell-cycle analyses. Results The response to JSI was comparatively dominated by lining progenitor and fibroblast-like synoviocyte (FLS) expansion, while DMM and ACLR induced transient expansion of perturbed-state fibroblasts (PSF) containing a myofibroblast subset, followed by FLS expansion over time. Early injury responses were underpinned by proliferating cells that directly supplied new PSF and FLS. Molecular pathway analysis revealed rapid and sustained injury-induced transcriptomic shifts in both lining and sublining fibroblasts, with post-traumatic OA models in particular showing marked upregulation of matrix-related genes indicative of myofibroblast activity and tissue remodeling. Activity of regulons associated with the PSF response, including Hes6, Hif1α, Zfp354c, Runx1 and Foxp1, showed model and stage-dependent upregulation. Finally, co-expression of lineage-specifying transcription factors Sox9, Runx2 and Scx, indicative of multilineage potential, was a common feature of PSF in all injury models. Conclusions This study uncovers similarities and differences in synovial fibroblast responses between injury models, guiding future research on joint repair and osteoarthritis.

Damage to articular cartilage, tendons, ligaments and entheses as a result of trauma, degeneration or inflammation in rheumatic diseases is prevalent. Regenerative medicine offers promising strategies for repairing damaged tissues, with the aim of restoring both their structure and function. While these strategies have traditionally relied on tissue engineering approaches using exogenous cells, interventions based on the activation of endogenous repair mechanisms are an attractive alternative. Key to advancing such approaches is a comprehensive understanding of the diversity of the stem and progenitor cells that reside in the adult synovial joint and how they function to repair damaged tissues. Advances in developmental biology have provided a lens through which to understand the origins, identities and functions of these cells, and insights into the roles of stem and progenitor cells in joint tissue repair, as well as their complex relationship with fibroblasts, have emerged. Integration of knowledge obtained through studies using advanced single-cell technologies will be crucial to establishing unified models of cell populations, lineage hierarchies and their molecular regulation. Ultimately, a more complete understanding of how cells repair tissues in adult life will guide the development of innovative pro-regenerative drugs, which are poised to enter clinical practice in musculoskeletal medicine. Stem and progenitor cells that contribute to tissue repair reside within various niches in the synovial joints of adult mammals. Understanding of the origins, identities and functions of these cells will guide the development of pro-regenerative therapeutic strategies.

Immunohistochemistry (IHC) is a routinely used technique in clinical diagnosis of pathological conditions and in basic and translational research. It combines anatomical, immunological, and biochemical methods and relies on the specific binding of an antibody to an antigen. Using the technique with mineralized tissues is more challenging than with soft tissues. Demineralizing the samples allows embedding in paraffin wax, and also facilitates cryosectioning. This chapter describes methods for IHC on formaldehyde-fixed, demineralized, paraffin-embedded, or frozen sections to detect antigens in skeletal tissues. We include a protocol using a streptavidin-based amplification method for immunofluorescence staining.

Articular cartilage, an avascular, matrix-rich tissue, is thought to have limited repair, thereby contributing to osteoarthritis (OA), the common degenerative disease of joints. Cartilage regeneration does occur, however, in OA joints that are mechanically off-loaded. Here we show that mechanical stress, through release of matrix-bound growth factors, reprogrammes chondrocytes, the primary cells of cartilage, into ‘mechano-activated chondroprogenitors’ (MACs). Studying OA joint fluid before and after mechanical off-loading, reveals evidence of chronic MAC activity, which switches back to a chondrogenic one when mechanical stress is removed. Taken together, we conclude that OA is a disease of ‘arrested repair’ in which mechanical stress signals need to be switched off before full repair can occur. This novel paradigm uncovers exciting new treatment opportunities. ### Competing Interest Statement T.L.V. has received grant support for STEpUP OA from Pfizer, Novartis, UCB, Fidia, Biosplice, Galapagos and received ad hoc personal consultancy fees from Zoetis. C.D.B. and A.J.R. have received research grant funding through the institution from Biosplice Therapeutics (formerly Samumed LLC). C.D.B. has received consultancy fees from UCB and Galapagos. Centre for OA Pathogenesis Versus Arthritis Grants, 20205, 21621 Versus Arthritis, 20783 German Academic Exchange Service, https://ror.org/039djdh30, DAAD Dutch Arthritis Society, LLP-9 ZonMW-NWO talent program, VENI UKRI Future Leaders Fellowship, MR/S016538, MR/Y003470/1 Reumafonds grant, ISP14-3-301/16-1-404 Tissue Engineering and Regenerative Therapies Centre Versus Arthritis grant, 21156 Versus Arthritis project grant, 20775

Regulation of neutrophil activation is critical for disease control. Neutrophil extracellular traps (NETs), which are web-like structures composed of DNA and neutrophil-derived proteins, are formed following pro-inflammatory signals; however, if this process is uncontrolled, NETs contribute to disease pathogenesis, exacerbating inflammation and host tissue damage1,2. Here we show that myeloid inhibitory C-type lectin-like (MICL), an inhibitory C-type lectin receptor, directly recognizes DNA in NETs; this interaction is vital to regulate neutrophil activation. Loss or inhibition of MICL functionality leads to uncontrolled NET formation through the ROS–PAD4 pathway and the development of an auto-inflammatory feedback loop. We show that in the context of rheumatoid arthritis, such dysregulation leads to exacerbated pathology in both mouse models and in human patients, where autoantibodies to MICL inhibit key functions of this receptor. Of note, we also detect similarly inhibitory anti-MICL autoantibodies in patients with other diseases linked to aberrant NET formation, including lupus and severe COVID-19. By contrast, dysregulation of NET release is protective during systemic infection with the fungal pathogen Aspergillus fumigatus. Together, we show that the recognition of NETs by MICL represents a fundamental autoregulatory pathway that controls neutrophil activity and NET formation.

Giant cell arteritis (GCA), the most common systemic vasculitis, is characterised by aberrant interactions between infiltrating and resident cells of the vessel wall. Ageing and breach of tolerance are prerequisites for GCA development, resulting in dendritic and T-cell dysfunction. Inflammatory cytokines polarise T-cells, activate resident macrophages and synergistically enhance vascular inflammation, providing a loop of autoreactivity. These events originate in the adventitia, commonly regarded as the biological epicentre of the vessel wall, with additional recruitment of cells that infiltrate and migrate towards the intima. Thus, GCA-vessels exhibit infiltrates across the vascular layers, with various cytokines and growth factors amplifying the pathogenic process. These events activate ineffective repair mechanisms, where dysfunctional vascular smooth muscle cells and fibroblasts phenotypically shift along their lineage and colonise the intima. While high-dose glucocorticoids broadly suppress these inflammatory events, they cause well known deleterious effects. Despite the emerging targeted therapeutics, disease relapse remains common, affecting >50% of patients. This may reflect a discrepancy between systemic and local mediators of inflammation. Indeed, temporal arteries and aortas of GCA-patients can show immune-mediated abnormalities, despite the treatment induced clinical remission. The mechanisms of persistence of vascular disease in GCA remain elusive. Studies in other chronic inflammatory diseases point to the fibroblasts (and their lineage cells including myofibroblasts) as possible orchestrators or even effectors of disease chronicity through interactions with immune cells. Here, we critically review the contribution of immune and stromal cells to GCA pathogenesis and analyse the molecular mechanisms by which these would underpin the persistence of vascular disease.

OBJECTIVE:Obesity increases osteoarthritis (OA) risk due to adipose tissue dysfunction with associated metabolic syndrome and excess weight. Lipodystrophy syndromes exhibit systemic metabolic and inflammatory abnormalities similar to obesity without biomechanical overloading. Here, we used lipodystrophy mouse models to investigate the effects of systemic versus intra-articular adipose tissue dysfunction on the knee. METHODS:Intra-articular adipose tissue development was studied using reporter mice. Mice with selective lipodystrophy of intra-articular adipose tissue were generated by conditional knockout (cKO) of Bscl2 in Gdf5-lineage cells, and compared with whole-body Bscl2 knockout (KO) mice with generalised lipodystrophy and associated systemic metabolic dysfunction. OA was induced by surgically destabilising the medial meniscus (DMM) and obesity by high-fat diet (HFD). Gene expression was analysed by quantitative RT-PCR and tissues were analysed histologically. RESULTS:The infrapatellar fat pad (IFP), in contrast to overlying subcutaneous adipose tissue, developed from a template established from the Gdf5-expressing joint interzone during late embryogenesis, and was populated shortly after birth by adipocytes stochastically arising from Pdgfrα-expressing Gdf5-lineage progenitors. While female Bscl2 KO mice with generalised lipodystrophy developed spontaneous knee cartilage damage, Bscl2 cKO mice with intra-articular lipodystrophy did not, despite the presence of synovial hyperplasia and inflammation of the residual IFP. Furthermore, male Bscl2 cKO mice showed no worse cartilage damage after DMM. However, female Bscl2 cKO mice showed increased susceptibility to the cartilage-damaging effects of HFD-induced obesity. CONCLUSION:Our findings emphasise the prevalent role of systemic metabolic and inflammatory effects in impairing cartilage homeostasis, with a modulatory role for intra-articular adipose tissue.

Background: Lorecivivint (LOR) is a potential disease-modifying osteoarthritis (OA) drug in clinical development. It is a novel small-molecule inhibitor of Cdc2-Like Kinases (CLKs) and Dual-Specificity Tyrosine Phosphorylation-Regulated Kinases (DYRKs), intra-nuclear kinases involved in alternative splicing. Lorecivivint is thought to modulate Wnt/ inflammatory pathways and has demonstrated chondrogenic properties in vitro. It is not known whether LOR promotes cartilage regeneration in vivo. Objectives: To investigate whether LOR promotes cartilage regeneration in vivo in a mouse model of joint surface injury that is permissive for endogenous healing. Methods: Female mice carrying Gdf5-Cre and Cre-inducible tdTomato transgenes to label and trace endogenous joint reparative cells underwent unilateral surgical joint surface injury at 8-9 weeks of age. Mice were randomly divided into 3 groups. The first group (control) received 1 µl vehicle applied to the defect at time of surgery and 10 µl vehicle via intra-articular injection 1 week after surgery. The second group (1x LOR) received 1 µl vehicle at time of surgery and 10 µl of 9 µg/ml LOR via intra-articular injection 1 week after surgery. The third group (2x LOR) received 1 µl of 90 µg/ml LOR applied to the defect during surgery, and 10 µl of 9 µg/ml LOR via intra-articular injection 1 week after surgery. Cartilage repair was assessed histologically at 8 weeks after surgery by staining with Safranin O and Fast Green. Sections for staining were selected at set intervals and an average of 9 sections per knee (range: 5-13) were included in the analysis based on anatomical landmarks. Scoring of the repair tissue was performed independently by 3 researchers using an adaptation of the Wakitani scoring system with the following parameters: thickness of repair tissue (0-4), cell morphology (0-3), matrix staining (0-3), surface regularity (0-2), and integration (0-2), yielding a maximum summed score of 14 indicating poorest repair. Significant discrepancies between scorers were reviewed, and following discussion, amended as appropriate. Data from the 3 scorers were averaged, and expressed as the average of the 3 lowest (i.e. best repair) summed scores for each knee. All individuals involved in the study were blinded to treatment groups throughout all experimental procedures and analyses. Results: Vehicle-treated joint surface defects (control group) typically showed a partial repair, with a predominantly fibrous-like tissue overlying bony tissue, as expected in this model. Analysis of summed repair scores identified a single statistical outlier in the 1x LOR group, based on Grubb’s outlier test at P<0.01, with a summed repair score of 13.8. After removal of the outlier, there was no statistically significant difference in summed repair scores between groups (P=0.20, Kruskal-Wallis test). However, mice receiving LOR showed a modest dose-dependent improvement in repair, with a summed repair score in the control group of 7.1 (95% CI 6.4 to 7.9, n=15), the 1x LOR group of 6.6 (95% CI 5.8 to 7.4, n=13), and the 2x LOR group of 5.9 (95% CI 4.9 to 7.0, n=16), which did not reach statistical significance (P=0.14 for 2x LOR versus control, Dunn’s post-test). Analysis of individual parameters showed that modest and statistically non-significant improvements in thickness, cell morphology, matrix staining and surface regularity contributed to the overall improved summed repair score in the 2x LOR compared to control group. The 2x LOR group included three mice with the best summed repair score (1.7, 3.1 and 3.2), showing cartilage-like repair tissue, while the best repair score in the 1x LOR group was 4.8 and in the control group was 5.0. Conclusion: These findings indicate that LOR may promote cartilage repair, which could contribute to its OA-protective activity. Further studies with different dosing regimens are needed to confirm. REFERENCES: NIL. Acknowledgements: NIL. Disclosure of Interests: Anke J Roelofs Biosplice Therapeutics Inc. (formerly Samumed LLC)., Susan M Clark: None declared, Jessica J McClure: None declared, Rebecca A Symons: None declared, Iain Cunningham: None declared, Megan Robertson: None declared, Alison Richmond: None declared, Fraser L Collins: None declared, Sarah Kennedy Biosplice Therapeutics Inc., Biosplice Therapeutics Inc., Jeyanesh R S Tambiah Biosplice Therapeutics Inc., Biosplice Therapeutics Inc., Cosimo De Bari UCB, Galapagos and Celltrion Healthcare, Biosplice Therapeutics Inc. (formerly Samumed LLC)

Lipids play a crucial role in signaling and metabolism, regulating the development and maintenance of the skeleton. Membrane lipids have been hypothesized to act as intermediates upstream of orphan phosphatase 1 (PHOSPHO1), a major contributor to phosphate generation required for bone mineralization. Here, we spatially resolve the lipid atlas of the healthy mouse knee and demonstrate the effects of PHOSPHO1 ablation on the growth plate lipidome. Lipids spanning 17 subclasses were mapped across the knee joints of healthy juvenile and adult mice using matrix-assisted laser desorption ionization imaging mass spectrometry (MALDI-IMS), with annotation supported by shotgun lipidomics. Multivariate analysis identified 96 and 80 lipid ions with differential abundances across joint tissues in juvenile and adult mice, respectively. In both ages, marrow was enriched in phospholipid platelet activating factors (PAFs) and related metabolites, cortical bone had a low lipid content, whereas lysophospholipids were strikingly enriched in the growth plate, an active site of mineralization and PHOSPHO1 activity. Spatially-resolved profiling of PHOSPHO1-knockout (KO) mice across the resting, proliferating, and hypertrophic growth plate zones revealed 272, 306, and 296 significantly upregulated, and 155, 220, and 190 significantly downregulated features, respectively, relative to wild-type (WT) controls. Of note, phosphatidylcholine, lysophosphatidylcholine, sphingomyelin, lysophosphatidylethanolamine, and phosphatidylethanolamine derived lipid ions were upregulated in PHOSPHO1-KO versus WT. Our imaging pipeline has established a spatially-resolved lipid signature of joint tissues and has demonstrated that PHOSPHO1 ablation significantly alters the growth plate lipidome, highlighting an essential role of the PHOSPHO1-mediated membrane phospholipid metabolism in lipid and bone homeostasis. © 2023 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

OBJECTIVE:To explore the significance of BMP signaling in osteoarthritis (OA) etiology, and thereafter propose a disease-modifying therapy for OA.METHODS:To examine the role of the BMP signaling in pathogenesis of OA, an Anterior Cruciate Ligament Transection (ACLT) surgery was performed to incite OA in C57BL/6J mouse line at postnatal day 120 (P120). Thereafter, to investigate whether activation of BMP signaling is necessary and sufficient to induce OA, we have used conditional gain- and loss-of-function mouse lines in which BMP signaling can be activated or depleted, respectively, upon intraperitoneal injection of tamoxifen. Finally, we locally inhibited BMP signaling through intra-articular injection of LDN-193189 pre- and post-onset surgically induced OA. The majority of the investigation has been conducted using micro-CT, histological staining, and immuno histochemistry to assess the disease etiology.RESULTS:Upon induction of OA, depletion of SMURF1-an intra-cellular BMP signaling inhibitor in articular cartilage coincided with the activation of BMP signaling, as measured by pSMAD1/5/9 expression. In mouse articular cartilage, the BMP gain-of-function mutation is sufficient to induce OA even without surgery. Further, genetic, or pharmacological BMP signaling suppression also prevented pathogenesis of OA. Interestingly, inflammatory indicators were also significantly reduced upon LDN-193189 intra-articular injection which inhibited BMP signaling and slowed OA progression post onset.CONCLUSION:Our findings showed that BMP signaling is crucial to the etiology of OA and inhibiting BMP signaling locally can be a potent strategy for alleviating OA.

Great progress continues to be made in our understanding of the multiple facets of osteoarthritis (OA) biology. Here, we review the major advances in this field and progress towards therapy development over the past year, highlighting a selection of relevant published literature from a PubMed search covering the year from the end of April 2022 to the end of April 2023. The selected articles have been arranged in themes. These include 1) molecular regulation of articular cartilage and implications for OA, 2) mechanisms of subchondral bone remodelling, 3) role of synovium and inflammation, 4) role of age-related changes including cartilage matrix stiffening, cellular senescence, mitochondrial dysfunction, metabolic dysfunction, and impaired autophagy, and 5) peripheral mechanisms of OA pain. Progress in the understanding of the cellular and molecular mechanisms responsible for the multiple aspects of OA biology is unravelling novel therapeutic targets for disease modification.

Purpose: Obesity is a major risk factor in the pathogenesis of osteoarthritis (OA). The infrapatellar fat pad (IFP) within the knee has been implicated in contributing to knee OA development by the secretion of inflammatory mediators. Here, we developed a novel transgenic mouse model of localised lipodystrophy of intra-articular adipose tissue to investigate whether alterations in adipose tissue in the knee can affect joint homeostasis and OA development.

Objective We aimed to understand the role of the transcriptional co-factor Yes-associated protein (Yap) in the molecular pathway underpinning the pathogenic transformation of synovial fibroblasts (SF) in rheumatoid arthritis (RA) to become invasive and cause joint destruction. Methods Synovium from patients with RA and mice with antigen-induced arthritis (AIA) was analysed by immunostaining and qRT-PCR. SF were targeted using Pdgfrα-CreER and Gdf5-Cre mice, crossed with fluorescent reporters for cell tracing and Yap-flox mice for conditional Yap ablation. Fibroblast phenotypes were analysed by flow cytometry, and arthritis severity was assessed by histology. Yap activation was detected using Yap–Tead reporter cells and Yap–Snail interaction by proximity ligation assay. SF invasiveness was analysed using matrigel-coated transwells. Results Yap, its binding partner Snail and downstream target connective tissue growth factor were upregulated in hyperplastic human RA and in mouse AIA synovium, with Yap detected in SF but not macrophages. Lineage tracing showed polyclonal expansion of Pdgfrα -expressing SF during AIA, with predominant expansion of the Gdf5 -lineage SF subpopulation descending from the embryonic joint interzone. Gdf5 -lineage SF showed increased expression of Yap and adopted an erosive phenotype (podoplanin+Thy-1 cell surface antigen−), invading cartilage and bone. Conditional ablation of Yap in Gdf5 -lineage cells or Pdgfrα -expressing fibroblasts ameliorated AIA. Interleukin (IL)-6, but not tumour necrosis factor alpha (TNF-α) or IL-1β, Jak-dependently activated Yap and induced Yap–Snail interaction. SF invasiveness induced by IL-6 stimulation or Snail overexpression was prevented by Yap knockdown, showing a critical role for Yap in SF transformation in RA. Conclusions Our findings uncover the IL-6–Yap–Snail signalling axis in pathogenic SF in inflammatory arthritis.

Purpose: The synovium, a specialised connective tissue, encapsulates synovial joints providing a barrier between the joint space and surrounding tissues. The healthy synovium consists of two layers, the sub-lining, composed of fibroblasts and other cell types, and the synovial lining that contains a specialised tissue-resident fibroblast known as the fibroblast-like synoviocyte (FLS). The FLS and sub-lining fibroblasts play a critical role in joint health and osteoarthritis. In the healthy joint, FLS produce essential joint lubricants such as hyaluronic acid and lubricin.

ObjectivesFibroblasts in synovium include fibroblast-like synoviocytes (FLS) in the lining andThy1+ connective-tissue fibroblasts in the sublining. We aimed to investigate their developmental origin and relationship with adult progenitors.MethodsTo discriminate betweenGdf5-lineage cells deriving from the embryonic joint interzone and otherPdgfrα-expressing fibroblasts and progenitors, adultGdf5-Cre;Tom;Pdgfrα-H2BGFPmice were used and cartilage injury was induced to activate progenitors. Cells were isolated from knees, fibroblasts and progenitors were sorted by fluorescence-activated cell-sorting based on developmental origin, and analysed by single-cell RNA-sequencing. Flow cytometry and immunohistochemistry were used for validation. Clonal-lineage mapping was performed usingGdf5-Cre;Confettimice.ResultsIn steady state,Thy1+ sublining fibroblasts were of mixed ontogeny. In contrast,Thy1-Prg4+ lining fibroblasts predominantly derived from the embryonic joint interzone and includedPrg4-expressing progenitors distinct from molecularly defined FLS. Clonal-lineage tracing revealed compartmentalisation ofGdf5-lineage fibroblasts between lining and sublining. Following injury, lining hyperplasia resulted from proliferation and differentiation ofPrg4-expressing progenitors, with additional recruitment of non-Gdf5-lineage cells, into FLS. Consistent with this, a second population of proliferating cells, enriched near blood vessels in the sublining, supplied activated multipotent cells predicted to give rise toThy1+ fibroblasts, and to feed into the FLS differentiation trajectory. Transcriptional programmes regulating fibroblast differentiation trajectories were uncovered, identifying Sox5 and Foxo1 as key FLS transcription factors in mice and humans.ConclusionsOur findings blueprint a cell atlas of mouse synovial fibroblasts and progenitors in healthy and injured knees, and provide novel insights into the cellular and molecular principles governing the organisation and maintenance of adult synovial joints.

Articular cartilage (AC) has limited capacity for repair. The first attempt to repair cartilage using tissue engineering was reported in 1977. Since then, cell-based interventions have entered clinical practice in orthopaedics, and several tissue engineering approaches to repair cartilage are in the translational pipeline towards clinical application. Classically, these involve a scaffold, substrate or matrix to provide structure, and cells such as chondrocytes or mesenchymal stromal cells to generate the tissue. We discuss the advantages and drawbacks of the use of various cell types, natural and synthetic scaffolds, multiphasic or gradient-based scaffolds, and self-organizing or self-assembling scaffold-free systems, for the engineering of cartilage constructs. Several challenges persist including achieving zonal tissue organization and integration with the surrounding tissue upon implantation. Approaches to improve cartilage thickness, organization and mechanical properties include mechanical stimulation, culture under hypoxic conditions, and stimulation with growth factors or other macromolecules. In addition, advanced technologies such as bioreactors, biosensors and 3D bioprinting are actively being explored. Understanding the underlying mechanisms of action of cell therapy and tissue engineering approaches will help improve and refine therapy development. Finally, we discuss recent studies of the intrinsic cellular and molecular mechanisms of cartilage repair that have identified novel signals and targets and are inspiring the development of molecular therapies to enhance the recruitment and cartilage reparative activity of joint-resident stem and progenitor cells. A one-fits-all solution is unrealistic, and identifying patients who will respond to a specific targeted treatment will be critical.