Disentangling the detrimental effects of local from systemic adipose tissue dysfunction on articular cartilage in the knee.

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.

Recognition and control of neutrophil extracellular trap formation by MICL.

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.

Vascular disease persistence in giant cell arteritis: are stromal cells neglected?

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.

Osteoarthritis year in review 2023: Biology.

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.

BMP signaling: A significant player and therapeutic target for osteoarthritis.

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.

Towards disease modification in osteoarthritis.

In December 2022, Gerwin et al published in Nature Medicine that the C-terminal portion of angiopoietin-like 3, called LNA043, has chondroprotective and cartilage-regenerative properties. Molecular data from an experimental medicine phase I study suggested potential efficacy in humans. Here, we respond to and complement a commentary from Vincent and Conaghan and discuss unresolved issues and the potential of this molecule as a disease-modifying osteoarthritis drug.

Spatial Lipidomic Profiling of Mouse Joint Tissue Demonstrates the Essential Role of PHOSPHO1 in Growth Plate Homeostasis.

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).

Taxonomy of fibroblasts and progenitors in the synovial joint at single-cell resolution.

OBJECTIVES: Fibroblasts in synovium include fibroblast-like synoviocytes (FLS) in the lining and Thy1+ connective-tissue fibroblasts in the sublining. We aimed to investigate their developmental origin and relationship with adult progenitors. METHODS: To discriminate between Gdf5-lineage cells deriving from the embryonic joint interzone and other Pdgfrα-expressing fibroblasts and progenitors, adult Gdf5-Cre;Tom;Pdgfrα-H2BGFP mice 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 using Gdf5-Cre;Confetti mice. RESULTS: In steady state, Thy1+ sublining fibroblasts were of mixed ontogeny. In contrast, Thy1-Prg4+ lining fibroblasts predominantly derived from the embryonic joint interzone and included Prg4-expressing progenitors distinct from molecularly defined FLS. Clonal-lineage tracing revealed compartmentalisation of Gdf5-lineage fibroblasts between lining and sublining. Following injury, lining hyperplasia resulted from proliferation and differentiation of Prg4-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 to Thy1+ 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. CONCLUSIONS: Our 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.

A cholinergic neuroskeletal interface promotes bone formation during postnatal growth and exercise.

The autonomic nervous system is a master regulator of homeostatic processes and stress responses. Sympathetic noradrenergic nerve fibers decrease bone mass, but the role of cholinergic signaling in bone has remained largely unknown. Here, we describe that early postnatally, a subset of sympathetic nerve fibers undergoes an interleukin-6 (IL-6)-induced cholinergic switch upon contacting the bone. A neurotrophic dependency mediated through GDNF-family receptor-α2 (GFRα2) and its ligand, neurturin (NRTN), is established between sympathetic cholinergic fibers and bone-embedded osteocytes, which require cholinergic innervation for their survival and connectivity. Bone-lining osteoprogenitors amplify and propagate cholinergic signals in the bone marrow (BM). Moderate exercise augments trabecular bone partly through an IL-6-dependent expansion of sympathetic cholinergic nerve fibers. Consequently, loss of cholinergic skeletal innervation reduces osteocyte survival and function, causing osteopenia and impaired skeletal adaptation to moderate exercise. These results uncover a cholinergic neuro-osteocyte interface that regulates skeletogenesis and skeletal turnover through bone-anabolic effects.

Targeting the IL-6-Yap-Snail signalling axis in synovial fibroblasts ameliorates inflammatory arthritis.

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-1ß, 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.

Human Mesenchymal Stromal Cells Enhance Cartilage Healing in a Murine Joint Surface Injury Model.

Human umbilical cord (hUC)- or bone marrow (hBM)-derived mesenchymal stromal cells (MSCs) were evaluated as an allogeneic source of cells for cartilage repair. We aimed to determine if they could enhance healing of chondral defects with or without the recruitment of endogenous cells. hMSCs were applied into a focal joint surface injury in knees of adult mice expressing tdTomato fluorescent protein in cells descending from Gdf5-expressing embryonic joint interzone cells. Three experimental groups were used: (i) hUC-MSCs, (ii) hBM-MSCs and (iii) PBS (vehicle) without cells. Cartilage repair was assessed after 8 weeks and tdTomato-expressing cells were detected by immunostaining. Plasma levels of pro-inflammatory mediators and other markers were measured by electrochemiluminescence. Both hUC-MSC (n = 14, p = 0.009) and hBM-MSC (n = 13, p = 0.006) treatment groups had significantly improved cartilage repair compared to controls (n = 18). While hMSCs were not detectable in the repair tissue at 8 weeks post-implantation, increased endogenous Gdf5-lineage cells were detected in repair tissue of hUC-MSC-treated mice. This xenogeneic study indicates that hMSCs enhance intrinsic cartilage repair mechanisms in mice. Hence, hMSCs, particularly the more proliferative hUC-MSCs, could represent an attractive allogeneic cell population for treating patients with chondral defects and perhaps prevent the onset and progression of osteoarthritis.

Evaluation of adjunctive mycophenolate for large vessel giant cell arteritis.

OBJECTIVES: GCA patients with large vessel involvement (LV-GCA) experience greater CS requirements and higher relapse rates compared with classical cranial GCA. Despite the distinct disease course, interventions in LV-GCA have yet to be investigated specifically. This study aimed to evaluate the CS-sparing effect and tolerability of first-line mycophenolate in LV-GCA. METHODS: A retrospective cohort study was conducted in patients with LV-GCA identified from a regional clinical database between 2005 and 2019. All cases were prescribed mycophenolate derivatives (MYC; MMF or mycophenolic acid) at diagnosis and were followed up for ≥2 years. The primary outcome was the cumulative CS dose at 1 year. Secondary outcomes included MYC tolerance, relapse rates and CRP levels at 1 and 2 years. RESULTS: A total of 37 patients (65% female; mean age 69.4 years, SD 7.9 years) were identified. All cases demonstrated large vessel involvement via CT/PET (n = 34), CT angiography (n = 5) or magnetic resonance angiography (n = 2). After 2 years, 31 patients remained on MYC, whereas 6 had switched to MTX or tocilizumab owing to significant disease relapse. The mean (±SD) cumulative prednisolone dose at 1 year was 4960 (±1621) mg. Relapse rates at 1 and 2 years were 16.2 and 27%, respectively, and CRP levels at 1 and 2 years were 4 [interquartile range (IQR) 4-6] and 4 (IQR 4-4) mg/l, respectively. CONCLUSION: To our knowledge, this is the first attempt to assess the effectiveness of any specific agent in LV-GCA. MYC might be both effective in reducing CS exposure and well tolerated in this subpopulation. A future randomized controlled trial is warranted.

Identification of the skeletal progenitor cells forming osteophytes in osteoarthritis.

OBJECTIVES: Osteophytes are highly prevalent in osteoarthritis (OA) and are associated with pain and functional disability. These pathological outgrowths of cartilage and bone typically form at the junction of articular cartilage, periosteum and synovium. The aim of this study was to identify the cells forming osteophytes in OA. METHODS: Fluorescent genetic cell-labelling and tracing mouse models were induced with tamoxifen to switch on reporter expression, as appropriate, followed by surgery to induce destabilisation of the medial meniscus. Contributions of fluorescently labelled cells to osteophytes after 2 or 8 weeks, and their molecular identity, were analysed by histology, immunofluorescence staining and RNA in situ hybridisation. Pdgfrα-H2BGFP mice and Pdgfrα-CreER mice crossed with multicolour Confetti reporter mice were used for identification and clonal tracing of mesenchymal progenitors. Mice carrying Col2-CreER, Nes-CreER, LepR-Cre, Grem1-CreER, Gdf5-Cre, Sox9-CreER or Prg4-CreER were crossed with tdTomato reporter mice to lineage-trace chondrocytes and stem/progenitor cell subpopulations. RESULTS: Articular chondrocytes, or skeletal stem cells identified by Nes, LepR or Grem1 expression, did not give rise to osteophytes. Instead, osteophytes derived from Pdgfrα-expressing stem/progenitor cells in periosteum and synovium that are descendants from the Gdf5-expressing embryonic joint interzone. Further, we show that Sox9-expressing progenitors in periosteum supplied hybrid skeletal cells to the early osteophyte, while Prg4-expressing progenitors from synovial lining contributed to cartilage capping the osteophyte, but not to bone. CONCLUSION: Our findings reveal distinct periosteal and synovial skeletal progenitors that cooperate to form osteophytes in OA. These cell populations could be targeted in disease modification for treatment of OA.

Stratified glucocorticoid monotherapy is safe and effective for most cases of giant cell arteritis.

OBJECTIVES: High-dose glucocorticoids anchor standard care in GCA but are associated with significant toxicity. We aimed to evaluate the safety and effectiveness of a stratified approach to glucocorticoid tapering. The strategy aggressively reduced glucocorticoid doses in those manifesting an adequate early response to treatment, with a view to minimizing glucocorticoid complications. METHODS: A retrospective, population-based study of GCA was performed. All cases were confirmed by temporal artery biopsy between November 2010 and November 2015. Baseline and outcome data were extracted from secondary and primary care records at diagnosis and 1 year follow-up. The primary outcome was loss of vision. Secondary outcomes included remission and relapse rates and CS-related complications. RESULTS: The cohort consisted of 73 patients (76% female; mean age 73.5 years, s.d. 7.6 years). At presentation, a reduction in visual acuity was recorded in 17 patients (22.3%). The median CRP at diagnosis was 69.5 mg/l [interquartile range (IQR) 40.5-101 mg/l], with a median ESR of 80 mm/h (IQR 60-91 mm/h). At 1 year, remission was achieved in 64 patients (87.7%), whereas 10 patients (13.7%) relapsed. A single patient sustained visual loss after initiation of therapy. The median CRP at 1 year was 4 mg/l (IQR 4-9.5 mg/l) and the mean prednisolone dose was 5.4 mg (0-15 mg). CS-related complications were observed in 10 patients (13.7%). CONCLUSION: A stratified approach to CS tapering appeared safe and effective in GCA. It was associated with a high rate of remission and promisingly low rates of relapse at 1 year follow-up. These real-world data indicate that glucocorticoid exposure can be minimized safely in some patients with GCA.

Agrin induces long-term osteochondral regeneration by supporting repair morphogenesis.

Cartilage loss leads to osteoarthritis, the most common cause of disability for which there is no cure. Cartilage regeneration, therefore, is a priority in medicine. We report that agrin is a potent chondrogenic factor and that a single intraarticular administration of agrin induced long-lasting regeneration of critical-size osteochondral defects in mice, with restoration of tissue architecture and bone-cartilage interface. Agrin attracted joint resident progenitor cells to the site of injury and, through simultaneous activation of CREB and suppression of canonical WNT signaling downstream of ß-catenin, induced expression of the chondrogenic stem cell marker GDF5 and differentiation into stable articular chondrocytes, forming stable articular cartilage. In sheep, an agrin-containing collagen gel resulted in long-lasting regeneration of bone and cartilage, which promoted increased ambulatory activity. Our findings support the therapeutic use of agrin for joint surface regeneration.

Regulation of Gdf5 expression in joint remodelling, repair and osteoarthritis.

Growth and Differentiation Factor 5 (GDF5) is a key risk locus for osteoarthritis (OA). However, little is known regarding regulation of Gdf5 expression following joint tissue damage. Here, we employed Gdf5-LacZ reporter mouse lines to assess the spatiotemporal activity of Gdf5 regulatory sequences in experimental OA following destabilisation of the medial meniscus (DMM) and after acute cartilage injury and repair. Gdf5 expression was upregulated in articular cartilage post-DMM, and was increased in human OA cartilage as determined by immunohistochemistry and microarray analysis. Gdf5 expression was also upregulated during cartilage repair in mice and was switched on in injured synovium in prospective areas of cartilage formation, where it inversely correlated with expression of the transcriptional co-factor Yes-associated protein (Yap). Indeed, overexpression of Yap suppressed Gdf5 expression in chondroprogenitors in vitro. Gdf5 expression in both mouse injury models required regulatory sequence downstream of Gdf5 coding exons. Our findings suggest that Gdf5 upregulation in articular cartilage and synovium is a generic response to knee injury that is dependent on downstream regulatory sequence and in progenitors is associated with chondrogenic specification. We propose a role for Gdf5 in tissue remodelling and repair after injury, which may partly underpin its association with OA risk.

The burden of metabolic syndrome on osteoarthritic joints.

BACKGROUND: The prevalence of osteoarthritis (OA) increases with obesity, with up to two thirds of the elderly obese population affected by OA of the knee. The metabolic syndrome (MetS), frequently associated with central obesity and characterised by elevated waist circumference, raised fasting plasma glucose concentration, raised triglycerides, reduced high-density lipoproteins, and/or hypertension, is implicated in the pathogenesis of OA. This narrative review discusses the mechanisms involved in the influence of MetS on OA, with a focus on the effects on macrophages and chondrocytes. MAIN TEXT: A skewing of macrophages towards a pro-inflammatory M1 phenotype within synovial and adipose tissues is thought to play a role in OA pathogenesis. The metabolic perturbations typical of MetS are important drivers of pro-inflammatory macrophage polarisation and activity. This is mediated via alterations in the levels and activities of the cellular nutrient sensors 5' adenosine monophosphate-activated protein kinase (AMPK) and mammalian target of rapamycin complex 1 (mTORC1), intracellular accumulation of metabolic intermediates such as succinate and citrate, and increases in free fatty acids (FFAs) and hyperglycaemia-induced advanced glycation end-products (AGEs) that bind to receptors on the macrophage surface. Altered levels of adipokines, including leptin and adiponectin, further influence macrophage polarisation. The metabolic alterations in MetS also affect the cartilage through direct effects on chondrocytes by stimulating the production of pro-inflammatory and catabolic factors and possibly by suppressing autophagy and promoting cellular senescence. CONCLUSIONS: The influence of MetS on OA pathogenesis involves a wide range of metabolic alterations that directly affect macrophages and chondrocytes. The relative burden of intra-articular versus systemic adipose tissue in the MetS-associated OA remains to be clarified. Understanding how altered metabolism interacts with joints affected by OA is crucial for the development of further strategies for treating this debilitating condition, such as supplementing existing therapies with metformin and utilising ω-3 fatty acid derivatives to restore imbalances in ω-3 and ω-6 fatty acids.

Immunostaining of Skeletal Tissues.

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 for 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.

Targeting anti-chondrogenic factors for the stimulation of chondrogenesis: A new paradigm in cartilage repair.

Trauma and age-related cartilage disorders represent a major global cause of morbidity, resulting in chronic pain and disability in patients. A lack of effective therapies, together with a rapidly aging population, creates an impressive clinical and economic burden on healthcare systems. In this scenario, experimental therapies based on transplantation or in situ stimulation of skeletal Mesenchymal Stem/progenitor Cells (MSCs) have raised great interest for cartilage repair. Nevertheless, the challenge of guiding MSC differentiation and preventing cartilage hypertrophy and calcification still needs to be overcome. While research has mostly focused on the stimulation of cartilage anabolism using growth factors, several issues remain unresolved prompting the field to search for novel solutions. Recently, inhibition of anti-chondrogenic regulators has emerged as an intriguing opportunity. Anti-chondrogenic regulators include extracellular proteins as well as intracellular transcription factors and microRNAs that act as potent inhibitors of pro-chondrogenic signals. Suppression of these inhibitors can enhance MSC chondrogenesis and production of cartilage matrix. We here review the current knowledge concerning different types of anti-chondrogenic regulators. We aim to highlight novel therapeutic targets for cartilage repair and discuss suitable tools for suppressing their anti-chondrogenic functions. Further effort is needed to unveil the therapeutic perspectives of this approach and pave the way for effective treatment of cartilage injuries in patients. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res.

Stem cell-based therapeutic strategies for cartilage defects and osteoarthritis.

The gold standard cell therapy for repair of articular cartilage defects is autologous chondrocyte implantation, with good outcomes long-term. Mesenchymal stromal/stem cells (MSCs) from bone marrow or connective tissues such as fat are being pursued as alternatives for cartilage repair, and are trialled via intra-articular administration in patients with knee osteoarthritis. Early-phase clinical studies concur on safety and provide some promising insight into efficacy, but the mechanism of action remains unclear. Recent studies implicate extracellular vesicles as important mediators of MSC action, offering exciting therapeutic prospects. Our increasing understanding of the mechanisms underlying intrinsic articular cartilage maintenance and repair fosters hope that novel/repurposed therapeutics could elicit repair through activation of endogenous stem/progenitor cells to maintain healthy joints and prevent osteoarthritis.