Purpose (the aim of the study): During previous experiments, in which we studied the capacity of Agrin to regenerate cartilage and bone in mice and sheep, we noticed that the animals in which Agrin was applied within an osteochondral defect spent more time being active compared to animals which received vehicle control - immediately after being administered Agrin. We hypothesised that this difference in behaviour was unlikely to be dependent on cartilage regeneration - which takes several weeks - and raised the possibility that Agrin may have a direct analgesic effect.

Chondral defects are common and disabling. The development of pharmacological approaches for cartilage repair requires the availability of in vivo models which are amenable for gain and loss of function and ideally to genetic modification. In this chapter, we describe a method to induce full-thickness cartilage defects which, in young DBA/1 mice, heal spontaneously, but fail to heal in C57BL/6 mice of the same age or in aged DBA/1 mice. This model (or variants) has been used for genetic screenings to identify genes associated to repair capacity, to study stem cells involved in cartilage repair, and to study the function of molecules involved in repair mechanisms.

We showed that the chemokine receptor C-X-C Motif Chemokine Receptor 2 (CXCR2) is essential for cartilage homeostasis.Here we reveal that the CXCR2 ligand granulocyte chemotactic protein 2 (GCP-2) was expressed, during embryonic development, within the prospective permanent articular cartilage, but not in the epiphyseal cartilage destined to be replaced by bone.GCP-2 expression was retained in adult articular cartilage.GCP-2 loss-of-function inhibited extracellular matrix production.GCP-2 treatment promoted chondrogenesis in vitro and in human cartilage organoids implanted in nude mice in vivo.To exploit the chondrogenic activity of GCP-2, we disrupted its chemotactic activity, by mutagenizing a glycosaminoglycan binding sequence, which we hypothesized to be required for the formation of a GCP-2 haptotactic gradient on endothelia.This mutated version (GCP-2-T) had reduced capacity to induce transendothelial migration in vitro and in vivo, without affecting downstream receptor signaling through AKT, and chondrogenic activity.Intra-articular adenoviral overexpression of GCP-2-T, but not wild type GCP-2, reduced pain and cartilage loss in instability-induced osteoarthritis in mice.We suggest that GCP-2-T may be used for disease modification in osteoarthritis.

Cartilage regeneration is a priority in medicine for the treatment of osteoarthritis and isolated cartilage defects. Several molecules with potential for cartilage regeneration are under investigation. Unfortunately, in vitro chondrogenesis assays do not always predict the stability of the newly formed cartilage in vivo. Therefore, there is a need for a stringent, quantifiable assay to assess in vivo the capacity of molecules to promote the stable formation of cartilage that is resistant to calcification and endochondral bone formation. We developed an ectopic cartilage formation assay (ECFA) that enables one to assess the capacity of bioactive molecules to support cartilage formation in vivo using cartilage organoids. The ECFA predicted good clinical outcomes when used as a quality control for efficacy of chondrocyte preparations before implantation in patients with cartilage defects. In this assay, articular chondrocytes from human donors or animals are injected either intramuscularly or subcutaneously in nude mice. As early as 2 weeks later, cartilage organoids can be retrieved. The size of the implants and their degree of differentiation can be assessed by histomorphometry, immunostainings of molecular markers and real-time PCR. Mineralization can be assessed by micro-computed tomography or by staining. The effects of molecules on cartilage formation can be tested following the systemic administration of the molecule in mice previously injected with chondrocytes, or after co-injection of chondrocytes with cell lines overexpressing and secreting the protein of interest. Here we describe the ECFA procedure, including steps for harvesting human and bovine articular cartilage, isolating primary chondrocytes, preparing overexpression cell lines, injecting the cells intramuscularly and retrieving the implants. This assay can be performed by technicians and researchers with appropriate animal training within 3 weeks.

WNT ligands can activate several signalling cascades of pivotal importance during development and regenerative processes. Their de-regulation has been associated with the onset of different diseases. Here we investigated the role of the WNT/Calcium Calmodulin Kinase II (CaMKII) pathway in osteoarthritis. We identified Heme Oxygenase I (HMOX1) and Sox-9 as specific markers of the WNT/CaMKII signalling in articular chondrocytes through a microarray analysis. We showed that the expression of the activated form of CaMKII, phospho-CaMKII, was increased in human and murine osteoarthritis and the expression of HMOX1 was accordingly reduced, demonstrating the activation of the pathway during disease progression. To elucidate its function, we administered the CaMKII inhibitor KN93 to mice in which osteoarthritis was induced by resection of the anterior horn of the medial meniscus and of the medial collateral ligament in the knee joint. Pharmacological blockade of CaMKII exacerbated cartilage damage and bone remodelling. Finally, we showed that CaMKII inhibition in articular chondrocytes upregulated the expression of matrix remodelling enzymes alone and in combination with Interleukin 1. These results suggest an important homeostatic role of the WNT/CaMKII signalling in osteoarthritis which could be exploited in the future for therapeutic purposes.

AbstractCartilage defects repair poorly. Recent genetic studies suggest that WNT3a may contribute to cartilage regeneration, however the dense, avascular cartilage extracellular matrix limits its penetration and signalling to chondrocytes. Extracellular vesicles actively penetrate intact cartilage. This study investigates the effect of delivering WNT3a into large cartilage defects in vivo using exosomes as a delivery vehicle. Exosomes were purified by ultracentrifugation from conditioned medium of either L‐cells overexpressing WNT3a or control un‐transduced L‐cells, and characterized by electron microscopy, nanoparticle tracking analysis and marker profiling. WNT3a loaded on exosomes was quantified by western blotting and functionally characterized in vitro using the SUPER8TOPFlash reporter assay and other established readouts including proliferation and proteoglycan content. In vivo pathway activation was assessed using TCF/Lef:H2B‐GFP reporter mice. Wnt3a loaded exosomes were injected into the knees of mice, in which large osteochondral defects were surgically generated. The degree of repair was histologically scored after 8 weeks. WNT3a was successfully loaded on exosomes and resulted in activation of WNT signalling in vitro. In vivo, recombinant WNT3a failed to activate WNT signalling in cartilage, whereas a single administration of WNT3a loaded exosomes activated canonical WNT signalling for at least one week, and eight weeks later, improved the repair of osteochondral defects. WNT3a assembled on exosomes, is efficiently delivered into cartilage and contributes to the healing of osteochondral defects.

More than 250 years ago, William Hunter stated that when cartilage is destroyed it never recovers. In the last 20 years, the understanding of the mechanisms that lead to joint formation and the knowledge that some of these mechanisms are reactivated in the homeostatic responses of cartilage to injury has offered an unprecedented therapeutic opportunity to achieve cartilage regeneration. Very large investments in ambitious clinical trials are finally revealing that, although we do not have perfect medicines yet, disease modification is a feasible possibility for human osteoarthritis.

Osteoarthritis is characterized by the loss of the articular cartilage, bone remodeling, pain, and disability. No pharmacological intervention can currently halt progression of osteoarthritis. Here, we show that blocking receptor tyrosine kinase-like orphan receptor 2 (ROR2) improves cartilage integrity and pain in osteoarthritis models by inhibiting yes-associated protein (YAP) signaling. ROR2 was up-regulated in the cartilage in response to inflammatory cytokines and mechanical stress. The main ligand for ROR2, WNT5A, and the targets YAP and connective tissue growth factor were up-regulated in osteoarthritis in humans. In vitro, ROR2 overexpression inhibited chondrocytic differentiation. Conversely, ROR2 blockade triggered chondrogenic differentiation of C3H10T1/2 cells and suppressed the expression of the cartilage-degrading enzymes a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-4 and ADAMTS-5. The chondrogenic effect of ROR2 blockade in the cartilage was independent of WNT signaling and was mediated by down-regulation of YAP signaling. ROR2 signaling induced G protein and Rho-dependent nuclear accumulation of YAP, and YAP inhibition was required but not sufficient for ROR2 blockade-induced chondrogenesis. ROR2 silencing protected mice from instability-induced osteoarthritis with improved structural outcomes, sustained pain relief, and without apparent side effects or organ toxicity. Last, ROR2 silencing in human articular chondrocytes transplanted in nude mice led to the formation of cartilage organoids with more and better differentiated extracellular matrix, suggesting that the anabolic effect of ROR2 blockade is conserved in humans. Thus, ROR2 blockade is efficacious and well tolerated in preclinical animal models of osteoarthritis.

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 β-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.

To explore potential biomarkers in a meniscectomy-induced knee osteoarthritis model, at forty years after meniscectomy.

Purpose: WNT16 is an injury response gene, absent in adult joints and briefly up-regulated in the initial phases following injury. Yet, its absence results in a worse outcome of disease. WNT16 has a unique ability to buffer the level of canonical WNT pathway activation and prevent excessive activity. In addition, WNT16 up-regulated Lubricin, a joint lubricant essential for healthy joints. In spite of the only transient expression of WNT16 shortly after injury, its functions are essential for the long term outcomes of osteoarthritis (OA). It is yet not understood what triggers WNT16 expression after injury. The expression of WNT16 coincides with a transient inflammatory response in animal models of OA. The objective of this study is to investigate whether such inflammatory response plays a role in the activation/regulation of WNT16. Methods: Human articular chondrocytes (HAC), were treated with inflammatory stimuli, various pathway inhibitors, as well as recombinant WNT proteins and the outcomes assessed by RT-QPCR and western blot. Results: In the destabilisation of the medial meniscus (DMM) model of instability induced OA, WNT16 was up-regulated in both DMM and sham operated knees as compared to non operated controls, although it persisted longer in DMM operated knees compared to sham operated knees. The period of WNT16 expression coincided with the presence of transient synovial inflammation. Suggesting that inflammatory cytokines may play a role in its regulation. In vitro, recombinant IL1-β treatment was sufficient to induce a robust WNT16 up-regulation in human articular chondrocytes. This up-regulation was dependent on activation of both JNK signalling and CamKII signalling as inhibition of these pathways abolished WNT16 expression. Interestingly, WNT16 expression further activated JNK, thereby suggesting a positive feedback loop. Importantly, exogenous WNT16 protected chondrocytes from IL-1 induced proteoglycan loss. Conclusions: WNT16 is up-regulated following IL1 stimulation and inflammation and is involved in modulating the effects of inflammation in the joint.

Purpose: Osteoarthritis (OA) is a leading cause of disability for which there is no cure. The identification of molecules supporting cartilage homeostasis and regeneration is therefore a major pursuit in musculoskeletal medicine. Agrin is a heparan sulfate proteoglycan which, through binding to low-density lipoprotein receptor-related protein 4 (LRP4), is required for neuromuscular synapse formation. We recently identified Agrin as an essential molecule in articular cartilage homeostasis, uniquely requiring both α-dystroglycan and LRP4 to support SOX9 expression and chondrocyte differentiation in vitro and in vivo. Despite identifying α-dystroglycan and LRP4 as necessary for the chondrogenic effects of Agrin in chondrocytes, the signalling pathway(s) by which this occurs remains unclear. LRP4 signalling is known to negatively regulate WNT signalling. Therefore, we tested whether Agrin is required for the WNT-inhibitory properties of LRP4 and whether this is the mechanism by which Agrin signals in chondrocytes. Methods: Mammalian Agrin or GFP was overexpressed in bovine primary chondrocytes and cultured in micromass for 5 days in the presence or absence of 100ng/ml WNT3a. Chondrogenic potential was assessed by qPCR for SOX9 mRNA expression. Agrin or GFP stably expressing COS7 cells were transfected with the SUPER8TOPFLASH reporter plasmid in combination with Vehicle or LRP4 plasmids. COS7 monolayer cultures were treated with vehicle or increasing doses of WNT3a (50, 100, 200ng/ml). WNT signalling activation was determined by luciferase expression. Results: LRP4 overexpression was sufficient to induce SOX9 upregulation in chondrocytes, however this effect was abolished following Agrin knockdown demonstrating the requirement of Agrin for the chondrogenic effect of LRP4 signalling. In addition, downregulation of SOX9 mRNA expression induced by WNT3a treatment was rescued by Agrin overexpression in micromass cultures. Using the WNT reporter assay, activation of canonical WNT signalling was inhibited in COS7 cells transduced with human Agrin compared to GFP-transduced control COS7 cells; and this effect was exacerbated by overexpression of LRP4. Conclusions: Agrin inhibits canonical WNT signalling through LRP4 and enhance chondrocyte differentiation. However, it remains unclear how Agrin-LRP4 signalling achieves its specificity in promoting chondrogenesis compared to other LRP4 ligands.

Purpose: There are many assays that assess the capacity of molecules to induce chondrogenic differentiation. However, most of these assays are in vitro and do not address the following important issues: 1. Does the induced differentiation result in stable articular cartilage or in an epiphyseal-like cartilage phenotype, which will undergo chondrocyte hypertrophy, calcification, vascular invasion and endochondral bone formation? 2. Does the bioactive molecule engage with the target molecule in human cartilage in vivo and result in biologically relevant outcomes? 3. PK/PD in human tissue in vivo. We have developed and validated an assay in which human articular chondrocytes form a human cartilage organoid in nude mice and methods to efficiently deliver bioactive molecules or RNA interference for gain or loss-of function respectively. Methods: As few as 1 million articular chondrocytes are suspended in 100μl of type 1 collagen gel (5mg/ml) and injected subcutaneously in immune-deficient mice. The gel sets spontaneously within seconds allowing formation of well-defined stable human cartilage pellets resistant to hypertrophy, vascularisation and endochondral bone formation within two weeks. Up to 6 pellets can be implanted in each mouse. The influence of secreted molecules/bioactive proteins on cartilage formation can be tested by local or systemic injection of compounds, or co-implanting the chondrocytes with stably or transiently transfected, growth-arrested cell lines overexpressing and secreting the molecule of interest. Loss of function can be achieved by transducing chondrocytes directly with a lentivirus containing shRNA prior to implantation. Outcomes can be assessed by in-vivo imaging, histomorphometry, immunohistochemistry and gene expression analysis using human-specific primers. Results: Newly-formed cartilage without evidence of vascular invasion and endochondral bone formation expressing markers and histological features of hyaline-like articular cartilage is retrievable after as little as 2 weeks. Conclusions: This is a time and cost-effective in vivo cartilage formation assay. The results of the ectopic cartilage formation assay are easily analyzed in a quantitative and qualitative manner. It allows assessment of the translational potential and validity of targets/ bioactive molecules in human cells in vivo. This assay is a clinically relevant tool to investigate the effect of bioactive molecules on the regenerative capacity of chondrocytes in vivo.

Purpose: We have previously demonstrated that Wnt3a-mediated activation of calcium calmodulin kinase II (CaMKII) in articular chondrocytes downregulates Sox9 and Col2A1 mRNA in vitro. We and others also showed that activated CaMKII is upregulated in human cartilage from osteoarthritis patients and in animal models of osteoarthritis. The aim of this study was to evaluate whether pharmacological blockade of CaMKII in an animal model of osteoarthritis could halt the progression of cartilage breakdown in vivo. Methods: Osteoarthritis was induced in C57BL/6 mice by surgical transection of the medial collateral ligament and removal of the anterior horn of the medial meniscus (MLI model). After 4 weeks from the surgery the mice started being systemically administered with either the CaMKII inhibitor KN93 or PBS via implantation of an osmotic minipump. The mice were sacrificed 8 weeks after surgery (4 weeks from the beginning of the treatment). The degree of cartilage damage was determined by two independent investigators by using the OARSI score and a novel histomorphometrical quantification by using ImageJ software. Bovine articular chondrocytes were removed from the metatarsal joints of young bovine obtained soon after death from a local abattoir. The cells were stimulated either in micromass or monolayer with IL-1 in presence or absence of two CaMKII inhibitors, KN93 and AIP. Metalloproteinases expression was evaluated by qPCR. Glycosaminoglycan content was assessed by alcian blue staining. Results: Mice treated with KN93 developed a more severe degree of cartilage damage in comparison to the control group. In vitro data revealed that CaMKII inhibition with both the inhibitors upregulated the expression of MMP-3 and ADAMTS-5, acting in a synergistic way to IL-1. CaMKII inhibition also induced decreased alcian blue staining of micromasses, suggesting increased proteoglycan remodelling. Conclusions: We showed that inhibition of CaMKII in a murine model of CaMKII is detrimental for the progression of cartilage damage in an in vivo model of osteoarthritis. In vitro inhibition of CaMKII showed uregulation of MMP-3 and ADAMTS-5 and decreased alcian blue staining of chondrocytes micromasses, suggesting CaMKII as a key molecule in modulating cartilage remodelling during OA progression.

Objectives Osteoarthritis (OA) is a leading cause of disability for which there is no cure. The identification of molecules supporting cartilage homeostasis and regeneration is therefore a major pursuit in musculoskeletal medicine. Agrin is a heparan sulfate proteoglycan which, through binding to low-density lipoprotein receptor-related protein 4 (LRP4), is required for neuromuscular synapse formation. In other tissues, it connects the cytoskeleton to the basement membrane through binding to α-dystroglycan. Prompted by an unexpected expression pattern, we investigated the role and receptor usage of agrin in cartilage. Methods Agrin expression pattern was investigated in human osteoarthritic cartilage and following destabilisation of the medial meniscus in mice. Extracellular matrix (ECM) formation and chondrocyte differentiation was studied in gain and loss of function experiments in vitro in three-dimensional cultures and gain of function in vivo, using an ectopic cartilage formation assay in nude mice. Receptor usage was investigated by disrupting LRP4 and α-dystroglycan by siRNA and blocking antibodies respectively. Results Agrin was detected in normal cartilage but was progressively lost in OA. In vitro, agrin knockdown resulted in reduced glycosaminoglycan content, downregulation of the cartilage transcription factor SOX9 and other cartilage-specific ECM molecules. Conversely, exogenous agrin supported cartilage differentiation in vitro and ectopic cartilage formation in vivo. In the context of cartilage differentiation, agrin used an unusual receptor repertoire requiring both LRP4 and α-dystroglycan. Conclusions We have discovered that agrin strongly promotes chondrocyte differentiation and cartilage formation in vivo. Our results identify agrin as a novel potent anabolic growth factor with strong therapeutic potential in cartilage regeneration.

Purpose: Osteoarthritis is a chronic disabling disease characterized by cartilage breakdown for which there is no cure. We recently discovered that the heparan sulphate proteoglycan Agrin is expressed in cartilage, where it is essential for the maintenance of the chondrocytic phenotype and for the production of cartilage extracellular matrix whilst exogenous Agrin enhances chondrocyte differentiation and cartilage formation in vitro and in vivo. Here we study the mechanism that leads to loss of Agrin in OA and the functional consequences of Agrin cleavage by metalloproteinase 3 (MMP3). Methods: Agrin expression was determined by immunohistochemistry. Osteoarthritis was induced in 8 week old 129sv mice by destabilisation of the medial meniscus (DMM) and N-Agrin and C-Agrin expression was evaluated by immunofluorescence at 7 days and 8 weeks post-surgery. Ex vivo human cartilage samples were treated with MMP3 and the cleavage of C-Agrin was compared by immunofluorescence. The effect of MMP3 on Agrin transfected chondrocytes was analysed by qPCR. Results: We discovered that detection of Agrin in osteoarthritic samples is lost in earlier stages of disease progression when using an antibody detecting the C-terminal portion of Agrin compared to when using an antibody recognizing the N-terminal domain. This could be explained by the well-described MMP3 mediated Agrin cleavage which separates the N-terminal domain (predicted to adhere to the basement membrane through interaction with laminin) from the C-terminal domain. In keeping with this hypothesis, treatment of chondrocyte cultures or cartilage explants with recombinant MMP3 resulted in the loss of Agrin detection when using the C-terminal, but not the N-terminal antibody. Using antibodies specific to the N and C-terminal portions of Agrin, we found that C-Agrin is lost in the articular cartilage as early as 2days post surgical-induction of OA (destabilization of the medial meniscus), whereas N-Agrin remains highly detectable for at least 7days post surgical-induction of OA. In functional terms, treatment with recombinant MMP3 reduced the capacity of Agrin to upregulate SOX9 mRNA. Conversely, overexpression of Agrin in chondrocytes strongly downregulated MMP3 mRNA expression, thereby suggesting a negative feedback loop. Conclusions: The cleavage of Agrin by MMP-3 in chondrocytes may contribute to the progression of OA by reducing SOX9 transcription.

Purpose: Osteoarthritis is a chronic disabling disease characterized by cartilage breakdown for which there is no cure. Disruption of the gene encoding for the heparan sulphate proteoglycan Agrin results in embryonic skeletal dysplasia suggesting a role for Agrin in cartilage biology and prompting us to study its role in articular cartilage biology and osteoarthritis. The aims of this study are to establish the expression pattern of Agrin in healthy/normal cartilage and compare it to the expression pattern in osteoarthritic or injured cartilage; determine the effects of knockdown and over-expression of Agrin within cartilage in vitro and in vivo and to determine the epistasis of Agrin in articular chondrocytes. Methods: Agrin expression was determined by immunohistochemistry and qPCR. Osteoathritis was induced in 8 week old 129sv mice by destabilisation of the medial meniscus (DMM) and Agrin expression was evaluated by immunofluorescence 8 weeks post-surgery. Paired human samples of preserved cartilage vs severely osteoarthritic cartilage were compared by immunofluorescence. Gain and loss of function experiments were performed using an expression plasmid encoding mammalian Agrin and siRNAs in C28/I2 and bovine chondrocytes in micromass culture. In vivo cartilage formation was assessed using an ectopic implantation model in nude mice; growth-arrested COS7 cells overexpressing Agrin or GFP were combined with bovine chondrocytes (ratio 1:10) and implanted ectopically into nude mice for two weeks. Retrieved implants were characterised by histology and qPCR. Results: Agrin and its known receptors were expressed in healthy adult human articular cartilage and downregulated in human and experimental murine osteoarthritis. Silencing of Agrin by siRNA resulted in reduced GAG production in C28/I2 and in chondrocyte de-differentiation characterised by decreased expression of SOX9, COL2A1 and ACAN mRNA. Overexpression of Agrin in the human chondrocyte cell line C28/I2 and bovine primary articular chondrocytes resulted in enhanced GAG production and SOX9 upregulation. Importantly, in contrast to BMP-2, Agrin over-expression did not induce markers of cartilage hypertrophy including COL10A1 and MMP-13. Delivering Agrin to primary bovine chondrocytes transplanted in the muscle of nude mice resulted in enhanced formation of ectopic cartilage, which did not display signs of hypertrophy, vascular invasion, or endochondral bone formation. Conclusions: Our data show that Agrin is essential for the maintenance of the chondrocytic phenotype and extracellular matrix production whilst exogenous Agrin enhances chondrocyte differentiation and cartilage formation in vitro and in vivo; and therefore may be a valuable chondrogenic molecule in tissue engineering technologies.

Cartilage breakdown is the disabling outcome of rheumatic diseases, whether prevalently inflammatory such as rheumatoid arthritis or prevalently mechanical such as osteoarthritis (OA). Despite the differences between immune-mediated arthritides and OA, common mechanisms drive cartilage breakdown. Inflammation, chondrocyte phenotype and homeostatic mechanisms have recently been the focus of research and will be summarised in this review.

Purpose of the study: Disruption of Wnt signalling results in osteoarthritis both in humans and experimental models. However, the role of Wnt pathways in the pathogenesis of this disease has not been fully characterized yet. We have recently demonstrated that Wnt-dependent activation of the Calcium Calmodulin Kinase II (CaMKII) is detrimental for the maintenance of articular chondrocyte homeostasis in vitro (Nalesso et al., JCB 2011). The aim of this study was then to determine the role of the Wnt/CaMKII pathway in human articular cartilage and in the development of osteoarthritis. Methods: Cartilage explants were removed from the knees of patients undergone joint replacement for osteoarthritis. Osteoarthritis was induced in 8 weeks old 129/Sv mice by destabilization of the medial meniscus (DMM) and the mice were sacrificed 8 weeks post-surgery. The expression level of the different CaMKII isoforms in articular cartilage was evaluated by PCR and immunohistochemistry. CaMKII phosphorylation was assessed by immunofluorescence. Articular chondrocytes were isolated by enzymatic digestion from the metatarsal joint of a bovine hoof obtained from a local abattoir. The chondrocytes were stimulated with recombinant Wnt5a in presence/absence of KN93, a CaMKII inhibitor. Calcium mobilization was detected by cellular accumulation of FURA-2 dye and phosphorylation of CaMKII by Western blotting analysis. Gene expression analysis of chondrocyte markers including Col2A1, Aggrecan and Sox9, was performed by quantitative real time PCR. Results: Only CaMKIIγ and -δ were expressed in human and murine articular cartilage. Phospho-CaMKII (pCaMKII), the activated form of CaMKII, was expressed only in the cartilage of osteoarthritic patients and was totally absent in normal cartilage. pCaMKII was also significantly upregulated in mice upon DMM in comparison with the contralateral sham-operated joint. The expression of Wnt5a, known to modulate the CaMKII pathway in different biological systems, was up-regulated in OA samples. Wnt5a stimulation promoted calcium mobilization and CaMKII phosphorylation in articular chondrocytes. Wnt5a induced loss of chondrocyte phenotype which was rescued by CaMKII inhibition. Conclusions: These results showed that activation of the Wnt5a/CaMKII pathway correlates with the development of osteoarthritis both in human and in animal models. CaMKII blockade rescued the loss of chondrocyte phenotype induced by Wnt5a in articular chondrocytes, therefore suggesting that the inhibition of this signalling cascade might represent a new therapeutic strategy useful to tackle the development/progression of osteoarthritis.