Objectives. Osteoarthritis (OA) is characterised by articular cartilage degradation. MicroRNAs (miRNAs) have been identified in the development of OA. The purpose of our study was to explore the functional role and underlying mechanism of miR-138-5p in interleukin-1 beta (IL-1β)-induced extracellular matrix (ECM) degradation of OA cartilage. Materials and Methods. Human articular cartilage was obtained from patients with and without OA, and chondrocytes were isolated and stimulated by IL-1β. The expression levels of miR-138-5p in cartilage and chondrocytes were both determined. After transfection with miR-138-5p mimics, allele-specific oligonucleotide (ASO)-miR-138-5p, or their negative controls, the messenger RNA (mRNA) levels of aggrecan (ACAN), collagen type II and alpha 1 (COL2A1), the protein levels of glycosaminoglycans (GAGs), and both the mRNA and protein levels of matrix metalloproteinase (MMP)-13 were evaluated. Luciferase reporter assay, quantitative real-time polymerase chain reaction (qRT-PCR), and Western blot were performed to explore whether Forkhead Box C1 (FOCX1) was a target of miR-138-5p. Further, we co-transfected OA chondrocytes with miR-138-5p mimics and pcDNA3.1 (+)-FOXC1 and then stimulated with IL-1β to determine whether miR-138-5p-mediated IL-1β-induced cartilage matrix degradation resulted from targeting FOXC1. Results. MiR-138-5p was significantly increased in OA cartilage and in chondrocytes in response to IL-1β-stimulation. Overexpression of miR-138-5p significantly increased the IL-1β-induced downregulation of COL2A1, ACAN, and GAGs, and increased the IL-1β-induced over expression of MMP-13.We found that FOXC1 is directly regulated by miR-138-5p. Additionally, co-transfection with miR-138-5p mimics and pcDNA3.1 (+)-FOXC1 resulted in higher levels of COL2A1, ACAN, and GAGs, but lower levels of MMP-13. Conclusion. miR-138-5p promotes IL-1β-induced cartilage degradation in human chondrocytes, possibly by targeting FOXC1. Cite this article: Y. Yuan, G. Q. Zhang, W. Chai,M. Ni, C. Xu, J. Y. Chen. Silencing of microRNA-138-5p promotes IL-1β-induced cartilage degradation in human chondrocytes by targeting FOXC1: miR-138 promotes cartilage degradation. Bone Joint Res 2016;5:523–530. DOI: 10.1302/2046-3758.510.BJR-2016-0074.R2

Introduction. Osteoarthritis (OA) causes pain, stiffness, and loss of function due to degenerative changes in joint cartilage and bone. In some forms of OA, exercise can alleviate symptoms by improving joint mobility and stability. However, excessive training after joint injury may have negative consequences for OA development. Sensory nerve fibers in joints release neuropeptides like alpha-calcitonin gene-related peptide (alpha-CGRP), potentially affecting OA progression. This study investigates the role of alpha-CGRP in OA pathogenesis under different exercise regimen in mice. Method. OA was induced in C57Bl/6J WT mice and alpha-CGRP KO mice via surgical destabilization of the medial meniscus (DMM) at 12 weeks of age (N=6). Treadmill exercise began 2 weeks post-surgery and was performed for 30 minutes, 5 days a week, for 2 or 6 weeks at intense (16 m/min, 15° incline) or moderate (10 m/min, 5° incline) levels. Histomorphometric assessment of cartilage degradation (OARSI scoring), serum cytokine analysis, immunohistochemistry, and nanoCT analysis were conducted. Result. OARSI scoring confirmed OA induction 4 weeks post-DMM surgery, with forced exercise exacerbating cartilage degradation regardless of intensity. No significant genotype-dependent differences were observed. Serum analysis revealed elevated cytokine levels associated with OA and inflammation in KO mice compared to WT mice 4 and 8 weeks post-surgery (VEGF-A, MCP-1, CXCL10, RANTES, MIP1-alpha, MIP1-beta, and RANKL). The observed effects were often exacerbated by intense exercise but rarely by DMM surgery. NanoCT analysis demonstrated increased sclerotic bone changes after 6 weeks of forced exercise in KO mice compared to WT mice. Conclusion. Our results suggest an OA promoting effect of exercise in early disease stages of posttraumatic OA. Intense exercise induced inflammatory processes correlated to increased cytokine levels in the serum that might exacerbate OA pathogenesis in later stages. The neuropeptide alpha-CGRP might play a role in protecting against these adverse effects

Development of osteoarthritis (OA) correlates with epigenetic alteration in chondrocytes. H3K27me3 demethylase UTX is known to regulate tissue homeostasis, but its role in the homeostasis of articulating joint tissue is poorly understood. Forced UTX expression upregulated H3K27me3 enrichment at the Sox9 promoter region to inhibit key extracellular matrix (ECM) molecules, like e.g. type II collagen, aggrecan, and glycosaminoglycans in articular chondrocytes. Utx loss in vitro altered the H3K27me3-binding epigenomic landscape, which contributes to mitochondrial activity, cellular senescence, and cartilage development. Functional target genes of Utx comprise insulin-like growth factor 2 (Igf2) and polycomb repressive complex 2 (PRC2) core components Eed and Suz12. Specifically, Utx deletion promoted Tfam transcription, mitochondrial respiration, ATP production and Igf2 transcription, but inhibited Eed and Suz12 expression. Igf2 inhibition or forced Eed or Suz12 expression increased H3K27 trimethylation and H3K27me3 enrichment at the Sox9 promoter, compromising Utx loss-induced ECM overproduction. Overexpression of Utx in murine knee joints aggravated OA development, including articular cartilage damage, synovitis, osteophyte formation, and subchondral bone loss. Transgenic mice with a chondrocytespecific Utx knockout develop thicker articular cartilage as compared to wild-type controls and show fewer gonarthrotic symptoms during destabilized medial meniscus- and collagenase-induced joint injury. In summary, UTX represses chondrocytic activity and accelerates cartilage degradation during OA, while Utx loss promotes cartilage integrity through epigenetic stimulation of mitochondrial biogenesis and Igf2 transcription. This highlights a novel noncanonical role of Utx that regulates articular chondrocyte anabolism and OA development

Osteoarthritis (OA) is a disease that affects both bone and cartilage. Typically, this disease leads to cartilage degradation and subchondral bone sclerosis but the link between the two is unknown. Also, while OA was traditionally thought of as non-inflammatory condition, it now seems that low levels of inflammation may be involved in the link between these responses. This is particularly relevant in the case of Post-Traumatic OA (PTOA), where an initial phase of synovial inflammation occurs after injury. The inflammatory mediator interleukin 1 beta (IL-1B) is central to this response and contributes to cartilage degradation. However, whether there is a secondary effect of this mediator on subchondral bone, via bone-cartilage crosstalk, is not known. To address this question, we developed a novel patellar explant model, to study bone cartilage crosstalk which may be more suitable than commonly used femoral head explants. The specific aim of this study was to validate this novel patellar explant model by using IL-1B to stimulate the inflammatory response after joint injury and the subsequent development of PTOA. Female Sprague Dawley rats (n=48) were used to obtain patellar explants, under an institutional ethical approval license. Patellae were maintained in high glucose media, under sterile culture conditions, with or without IL-1B (10ng/ml), for 7 days. Contralateral patellae served as controls. One group (n= 12) of patellae were assessed for active metabolism, using two both Live and Dead (L/D) staining and an Alamar Blue assay (AB). A second group (n=12) was used for tissue specific biochemical assays for both bone (Alkaline Phosphatase) and cartilage (sulfated proteoglycan and glycosaminoglycan (sGaG)). Finally, a third group (n=28) of explants were used for histologically analysis. Samples were decalcified, embedded in paraffin and sectioned to 7µm thickness, and then stained using H&E; and Safranin O with fast green. Additionally, toluidine blue and alkaline phosphatase staining were also performed. Our results demonstrate that our system can maintain good explant viability for at least 7 days, but that IL-1B reduces cell viability in patellar cartilage, as measured by both L/D and AB assays after 0, 2, 4 and 7 days in culture. In contrast, sGaG content in cartilage were increased by this treatment. Additionally, ALP, a marker of osteoblastic activity, was increased in IL-1B treated group 4 and 7 days, but was also showed some increase in control groups. Histological analyses showed that IL-1B treatment resulted in reduced proteoglycan staining, demonstrating the powerful effect of this factor in injury response over time. Thus, we conclude that IL-1B affects both bone and cartilage tissues independently in this system, which may have relevance in understanding bone-cartilage crosstalk after injury and how this is involved in PTOA development

Early changes within articular cartilage during human idiopathic osteoarthritis are poorly understood. However alterations to chondrocyte morphology occur with the development of fine cytoplasmic processes and cell clusters, potentially playing a role in cartilage degeneration. The aggrecanase ADAMTS-4 (A disintegrin and metalloproteinase with thrombospondin motifs-4) has been implicated as an important factor in cartilage degradation, so we investigated the relationship between chondrocyte morphology and levels of ADAMTS-4 in both non-degenerate and mildly osteoarthritic human cartilage. Human femoral heads were obtained following consent from patients undergoing hip arthroplasty following femoral neck fracture. Cartilage explants of normal (grade 0; G0) and mildly osteoarthritic (grade 1; G1) cartilage were labelled with the cytoplasmic dye CMFDA (5-chloromethylfluorescein-diacetate). Explants were cryosectioned (30μm sections), and labelled for ADAMTS-4 by fluorescence immunohistochemistry. Sections were imaged with confocal microscopy, allowing the semi-quantitative analysis of ADAMTS-4 and 3D visualisation of in situ cell morphology. With cartilage degeneration from G0 to G1, there was a decrease in the proportion of chondrocytes with normal rounded morphology (P<0.001) but an increase in the proportion of cells with processes (P<0.01) and those in clusters (P<0.001;[4(1653)]; femoral heads:cells). Although average levels of ADAMTS-4 for all cells was the same between G0 and G1 (P>0.05), a change was evident in the distribution curves for cell-specific ADAMTS-4 labelling. Cell-by-cell analysis showed that ADAMTS-4 levels were higher in chondrocytes with cytoplasmic processes compared to normal cells (P=0.044) however cells in clusters had lower levels than normal cells (P=0.003;[3(436)]). Preliminary data suggested that ADAMTS-4 levels increased with larger chondrocyte clusters. These results suggest complex heterogeneous changes to levels of cell-associated ADAMTS-4 with early cartilage degeneration – increasing in cells with processes and initially decreasing in clusters. Increased levels of ADAMTS-4 are likely to produce focal areas of matrix weakness potentially leading to early cartilage degeneration

Summary Statement. The deletion of gangliosides enhanced OA development by elevating MMP-13 and ADAMTS-5 expression and accelerating chondrocyte apoptosis. Gangliosides possibly play suppressive roles in IL-1α-induced inflammatory signaling cascades. Introduction. We have previously reported that glycosphingolipids (GSLs) play chondroprotective roles in the cartilage degradation process [1]. Gangliosides, one of the series of GSLs, are known to be important in intercellular signal transduction and cell-to-cell recognition [2]. Therefore, we hypothesised that gangliosides are important in cartilage metabolisms among the GSLs species. The purpose of this study was to determine the functional role of gangliosides in the development of OA in murine models. Materials & Methods. We adopted systemic GM3 synthase deficient mice (GM3S. −/−. ) which lack most of the gangliosides [3], and wild-type C57BL/6 mice as controls (WT). We applied instability-induced OA model (transecting the medial collateral ligament and removing the cranial half of the medial meniscus [4]) and age-associated OA model (following up to 15 months) with these mice. We also applied IL-1α-induced OA model with femoral head cartilage explants ex vivo. Histological evaluation and quantification of released proteoglycan (PG), secreted MMP-13, and NO in the cultured media were performed. In vitro experiments with chondrocytes extracted from articular cartilages of both genotypes (GM3S. −/−. , WT) were also performed to check the mRNA expression of cartilage degrading enzymes (MMP-13, ADAMTS-5). To test the functional roles of gangliosides, transient GM3S transfection was applied to WT chondrocytes and quantification of MMP-13 and ADAMTS-5 expression was performed. Results. Both age-associated and instability-induced OA models showed cartilage degradation in GM3S. −/−. mice were significantly more severe than WT mice. The results of IL-1α-induced OA model showed gangliosides deletion enhanced chondrocyte apoptosis and accelerated cartilage degradation. Femoral heads from GM3S. −/−. showed significantly higher concentration of MMP-13 and NO in the cultured media than those from WT. In vitro experiments revealed that ganglioside deletion enhanced MMP-13 and ADAMTS-5 expression in the chondrocytes stimulated with IL-1α. The expression of these enzymes was significantly suppressed by overexpressed GM3S in WT chondrocytes. Discussion/Conclusion. The deletion of gangliosides enhanced OA development by elevating MMP-13 and ADAMTS-5 expression and accelerating chondrocyte apoptosis. The results of this study raised the possibility that gangliosides, synthesised mainly from GM3, would play crucial roles in maintaining cartilage homeostasis among the GSLs species. Moreover, the result of overexpression experiment indicated that gangliosides play suppressive roles in IL-1α-triggered inflammatory signaling cascades. Although further studies are required to confirm our speculation, gangliosides may be the target molecules of a novel and effective strategy for the treatment of OA

When joints sustain injury, the release of inflammation cytokines can cleavage matrix proteins and result in cartilage degradation and the subsequent osteoarthritis. RNA therapeutics emerging recently is a very promising approach to efficiently and specifically inhibit disease gene expression. However, the major challenge is how to deliver therapeutic RNA into joint and cartilage. Janus base nanotubes are self-assembled from synthetic Janus bases inspired from DNA base pairs. Based on the charge interaction, we are able to “sandwich” small RNAs among Janus base nanotubes to form tiny, nano-rod shaped delivery vehicles. Such vehicles can be engineered into different sizes and shapes. We have found that short and slim morphologies can greatly increase their penetration to extracellular matrix and delivery into “difficult-to-reach” tissues, such as cartilage and brain. Moreover, by delivering therapeutic siRNA, we have demonstrated its high-efficacy in inhibiting expression of an inflammatory regulator, Interleukin-1 receptor (IL-1R) in articular cartilage. Moreover, the inhibition effect is long-lasting so that joint inflammation and cartilage degradation caused by meniscus injury are greatly inhibited in a mouse model. Therefore, the Janus base nanotubes present a great potential in engineering into nano-structures for RNA delivery. Such approach may become an effective therapeutic against joint inflammation and arthritis

Summary Statement. We observed that severe muscle weakness leads to OA, whereas a transient inflammatory stimulus did not have a significant effect on cartilage degradation. This arises the thought that a severe but transient inflammation may not be an independent risk factor for OA. Introduction. Biomechanical disturbances and joint inflammation are known risk factors, which may provoke or advance osteoarthritis (OA). However, the effect of interactions of such risk factors on the onset and progression of OA are still poorly understood. Therefore, the goal of this study was to investigate the in vivo effects of muscle weakness, joint inflammation, and the combination of these two risk factors, on the onset and progression of OA in the rabbit knee. Patients & Methods. Thirty 1-year-old skeletally mature female New Zealand White rabbits (weight: average 5.7kg, range 4.8–6.6kg) were used in this study. The animals were divided into four experimental groups: (i) surgical transection of the nerve branch of the common femoral nerve leading to the vastus lateralis muscle; (ii) muscle weakness of the quadriceps muscle induced by a chronic intramuscular injection of Botulinum toxin A (BTX-A) (3); (iii) intraarticular injection in the experimental knee joint with commercially available sterile Carrageenan solution to induce a transient severe inflammatory reaction (4); (iv) administration of both intraarticular injection of Carrageenan and intramuscular injection of BTX-A. In each animal, one hind limb was randomly assigned to the experimental intervention, while the contralateral side acted as its own control. Ninety days following intervention, muscle mass, joint diameter and cartilage histology of the femur, femoral groove, tibia and patella were assessed and microscopically analyzed using the OARSI histology score. Results. Transection of the femoral branch leading to the vastus lateralis as well as the administration of BTX-A led to a significant muscle mass loss for the vastus lateralis and the total quadriceps group, respectively. Similar results were seen in the combined Carrageenan/BTX-A group. There were no changes in total quadriceps muscle mass in the Carrageenan group. Knee joint diameters of the experimental limb were significantly increased in the Carrageenan and Carrageenan/BTX groups. VL transection and BTX-A injection did not cause significant increases in joint diameter. Histologic assessment of the cartilage showed that weakness of the vastus lateralis resulted in significantly higher OARSI scores in the patella and femoral groove, but not the tibiofemoral articulation. The administration of BTX-A caused significant cartilage damage in all 4 compartments (patella, femur, tibia, femoral groove). Intraarticular injection of Carrageenan did not cause significant cartilage damage in any compartment compared to the contralateral side. The combination of BTX-A and Carrageenan resulted in severe cartilage damage in the patella in all four compartments of the knee. The most severe damage was found on the medial side of the tibiofemoral joint and the lateral side of the patellofemoral joint. Conclusion. Severe muscle weakness over a three months period leads to the onset and progression of OA in the rabbit knee. A transient local inflammatory stimulus did not promote cartilage degradation, nor did it enhance cartilage degradation when it was combined with muscle weakness. This result is surprising and adds to the literature the idea that a severe but transient inflammation may not be an independent risk factor for OA

TRIM32 is a candidate gene at the 9q33.1 genetic susceptibility locus for hip osteoarthritis (OA). Increased cartilage degradation typical of OA has previously been demonstrated in Trim32 knockout mice. Our aim is to investigate the role of TRIM32 in human and murine articular tissue. TRIM32 expression in human articular cartilage was examined by immunostaining. TRIM32 expression was compared in femoral head chondrocytes from patients with and without primary hip OA (n=6/group) and examined by Western blotting. Aggrecanolysis by femoral head explants from Trim32 knockout (T32KO) and wild-type (WT) mice was compared following stimulation with IL1α or retinoic acid (RA) and was assessed by DMMB assay (n=4/group). Expression of chondrocyte phenotype markers was measured by qPCR and compared between articular chondrocytes from WT and T32KO mice following catabolic (IL1α/TNFα) or anabolic (Oncostatin-M (OSM)/IGF1) stimulation. TRIM32 expression was demonstrated in human articular cartilage; TRIM32 expression by chondrocytes was reduced in patients with hip OA (p=0.03). Greater aggrecanolysis occurred in cartilage explants from T32KO mice after treatment with no stimulation (p=0.03), IL1α (p=0.02), and RA (p=0.001). Unstimulated T32KO chondrocytes expressed reduced Col2a1 (p=8.53×10. −5. ), and Sox9 (p=2.35×10. −6. ). Upon IL1α treatment, T32KO chondrocytes expressed increased Col10a1 (p=0.0003). Upon anabolic stimulation, T32KO chondrocytes expressed increased Col2a1 (OSM: p=0.001; IGF: p=0.001), and reduced Sox9 (OSM: p=0.0002; IGF: p=0.0006). These results indicate that altered TRIM32 expression in human articular tissue is associated with OA, and that Trim32 knockout results in increased cartilage degradation in murine femoral head explants. Predisposition to cartilage degeneration with reduced Trim32 expression may involve increased chondrocyte hypertrophy upon catabolic cytokine stimulation and dysregulation of Col2a1 and Sox9 expression upon anabolic stimulation

Objectives. This study aimed to explore the role of miR-320a in the pathogenesis of osteoarthritis (OA). Methods. Human cartilage cells (C28/I2) were transfected with miR-320a or antisense oligonucleotides (ASO)-miR-320a, and treated with IL-1β. Subsequently the expression of collagen type II alpha 1 (Col2α1) and aggrecan (ACAN), and the concentrations of sulfated glycosaminoglycans (sGAG) and matrix metallopeptidase 13 (MMP-13), were assessed. Luciferase reporter assay, qRT-PCR, and Western blot were performed to explore whether pre-B-cell leukemia Homeobox 3 (PBX3) was a target of miR-320a. Furthermore, cells were co-transfected with miR-320a and PBX3 expressing vector, or cells were transfected with miR-320a and treated with a nuclear factor kappa B (NF-κB) antagonist MG132. The changes in Col2α1 and ACAN expression, and in sGAG and MMP-13 concentrations, were measured again. Statistical comparisons were made between two groups by using the two-tailed paired t-test. Results. Expression of miR-320a was elevated in OA cartilage tissues and chondrocytes, and in IL-1β-stimulated C28/I2 cells (p < 0.05 or p < 0.01). MiR-320a overexpression enhanced IL-1β-induced down-regulation of Col2α1 and ACAN and sGAG, and increased the IL-1β-induced overexpression of MMP-13 (p < 0.01). PBX3 was a direct target of miR-320a. PBX3 and MG132 co-transfection attenuated the effects of miR-320a on the expression of Col2α1, ACAN, sGAG and MMP-13(p < 0.01). Conclusion. Overexpression of miR-320a might enhance IL-1β-induced cartilage degradation factors. These effects might be via targeting PBX3 and regulating NF-κB. Cite this article: Y. Jin, X. Chen, Z. Y. Gao, K. Liu, Y. Hou, J. Zheng. The role of miR-320a and IL-1β in human chondrocyte degradation. Bone Joint Res 2017;6:–203. DOI: 10.1302/2046-3758.64.BJR-2016-0224.R1

Cartilage neoangiogenesis holds a key role in the development of osteoarthritis (OA) by promoting cartilage degradation with proteoglycan loss, subchondral bone sclerosis, osteophyte formation and synovial hyperplasia. This study aimed to assess the in vivo efficacy of bevacizumab, an antibody against vascular endothelial growth factor (VEGF) in an OA animal model. 24 New Zealand white rabbits underwent anterior cruciate ligament transection in order to spontaneously develop knee OA. Animals were divided into four groups: one receiving a sham intraarticular knee injection (saline) and three groups treated with 5, 10, and 20 mg intraarticular bevacizumab injections. The biological effect of the antibody on cartilage and synovium was evaluated through histology and quantified with the Osteoarthritis Research Society International (OARSI) scores. Immunohistochemical analysis was conducted to investigate type 2 collagen, aggrecan, and matrix metalloproteinase 13 (MMP-13) expression in both cartilage and synovium. Intraarticular bevacizumab led to a significant reduction of cartilage degeneration and synovial OA alterations. Immunohistochemistry showed a significantly reduced MMP-13 expression in all experimental groups, with the one receiving 20 mg bevacizumab showing the lowest. Furthermore, the antibody showed to increment the production of aggrecan and type 2 collagen after administration of 5, 10, and 20 mg. The group treated with 20 mg showed the highest levels of type 2 collagen expression, while aggrecan content was even higher than in the healthy cartilage. Intraarticular bevacizumab has demonstrated to effectively arrest OA progression in our model, with 20 mg being the most efficacious dose. By inhibiting cartilage and synovial neoangiogenesis, bevacizumab may serve as a possible disease-modifying osteoarthritis drug (DMOAD) in the next future

Introduction and Objective. Traditionally, osteoarthritis (OA) has been associated mostly with degradation of cartilage only. More recently, it has been established that other joint tissues, in particular bone, are also centrally involved. However, the link between these two tissues remains unclear. This relationship is particularly evident in post-traumatic OA (PTOA), where bone marrow lesions (BMLs), as well as fluctuating levels of inflammation, are present long before cartilage degradation begins. The process of bone-cartilage crosstalk has been challenging to study due to its multi-tissue complexity. Thus, the use of explant model systems have been crucial in advancing our knowledge. Thus, we developed a novel patellar explant model, to study bone cartilage crosstalk, in particular related to subchondral bone damage, as an alternative to traditional femoral head explants or cylindrical core specimens. The commonly used osteochondral explant models are limited, for our application, since they involve bone damage during harvest. The specifics aim of this study was to validate this novel patellar explant model by using IL-1B to stimulate the inflammatory response and mechanical stimulation to determine the subsequent developments of PTOA. Materials and Methods. Lewis rats (n=48) were used to obtain patellar and femoral head explants which were harvested under an institutional ethical approval license. Explants were maintained in high glucose media (containing supplements), under sterile culture conditions. Initially, we characterised undamaged patellar explants and compared them with the commonly used femoral head. First, tissue viability was assessed using an assay of metabolic activity and cell damage. Second, we created chemical and mechanical damage in the form of IL-1B treatment, and mechanical stimulation, to replicate damage. Standard biochemical assays, histological assays and microstructural assays were used to evaluate responses. For chemical damage, explants were exposed to 10ng/ml of IL-1B for 24 hours at 0, 1, 3 and 7 days after harvesting. For mechanical damage, tissues were exposed to mechanical compression at 0.5 Hz, 10 % strain for 10 cycles, for 7 days. Contralateral patellae served as controls. In both groups, sGAG, ADAMTS4, and MMP-13 were measured as an assessment of representative cartilage responses while ALP, TRAP and CTSK were assessed as a representative of bone responses. In addition to this, histomorphometric, and immunohistochemical, evaluations of each explant system were also carried out. Results. Our results confirm that the patellar explant system is an excellent ex vivo model system to study bone-cartilage crosstalk, and one which does not induce any bone damage at the time of tissue harvest. We successfully established culture conditions to maintain viability in these explants for up to 28 days. Rat IL-1B treatment resulted in increased both proteoglycan content and bone metabolism markers after 7 days when compared with the controls. To confirm this finding, qualitative immunohistochemical staining showed chondrocytes increased expression of MMP13 after treatment with IL-1B. Furthermore, we observed that the levels of ADAMTS4 decreased in 48 hours after IL-1B exposure. Contrastingly IL-1B treatment had the opposite effect on CTSK markers when compared with the control. Mechanically compressed patellae showed a decrease in compressive moduli from day 3 to day 7, suggesting that tissue remodelling may have taken place as a compensatory mechanism in response to damage. In addition, MMP13 release decreased over 48 hours after mechanical compression, while TRAP levels were increased compared with the control. Conclusions. Thus, we successfully demonstrated that IL-1B and mechanical stimulation affects both bone and cartilage tissues independently in this system, which may have relevance in the understanding of bone-cartilage crosstalk after injury and how this is involved in PTOA development

Post-traumatic osteoarthritis (PTOA) is a subset of osteoarthritis, which occurs secondary to traumatic joint injury which is known to cause pathological changes to the osteochondral unit. Articular cartilage degradation is a primary hallmark of OA, and is normally associated with end-stage disease. However, subchondral bone marrow lesions are associated with joint injury, and may represent localized bone microdamage. Changes in the osteochondral unit have been traditionally studied using explant models, of which the femoral-head model is the most common. However, the bone damage caused during harvest can confound studies of microdamage. Thus, we used a novel patellar explant model to study osteochondral tissue dynamics and mechanistic changes in bone-cartilage crosstalk. Firstly, we characterized explants by comparing patella with femoral head models. Then, the patellar explants (n=269) were subjected to either mechanical or inflammatory stimulus. For mechanical stimulus 10% strain was applied at 0.5 and 1 Hz for 10 cycles. We also studied the responses of osteochondral tissues to 10ng/ml of TNF-α or IL-1β for 24hrs. In general the findings showed that patellar explant viability compared extremely well to the femoral head explant. Following IL-1β or TNF-α treatment, MMP13, significantly increased three days post exposure, furthermore we observed a decrease in sulfate glycoaminoglycan (sGAG) content. Bone morphometric analysis showed no significant changes. Contrastingly, mechanical stimulation resulted in a significant decrease sGAG particularly at 0.5Hz, where an increase in MMP13 release 24hrs post stimulation and an upregulation of bone and cartilage matrix degradation markers was observed. Furthermore, mechanical stimulus caused increases in TNF-α, MMP-8, VEGF expression. In summary, this study demonstrates that our novel patella explant model is an excellent system for studying bone-cartilage crosstalk, which responds well to both mechanical and inflammatory stimulus and is thus of great utility in the study of PTOA

Osteoarthritis (OA) is a joint degenerative disease leading to chronic pain and disability, thus resulting in a major socioeconomic health burden. OA, which has long been believed to be a cartilage disease, is now considered a whole-joint disorder affecting various anatomical structures, including subchondral bone. Hyaluronic Acid (HA) is commonly used as intra-articular viscosupplementation therapy for its mechanical features and biological effects. Bisphosphonates (BPs) are antiresorptive agents inhibiting recruitment and maturation of osteoclast precursors and activity of mature osteoclasts in the bone. Pre-clinical evidences in the literature, show that intra-articular BPs could impact on OA progression, slowing down or reversing it. The combination of HA biological and mechanical role and Alendronate (ALD) antiresorptive effect could be an interesting strategy for OA treatment. This study describes the synthesis and characterization of FID-134, a new chemical derivative of HA conjugated with ALD by means of a covalent bond, cleavable in physiological condition. FID-134 was synthesized starting from 500 kDa HA: chemical structure and functionalization degree with ALD were investigated by NMR and ICP-OES. Kinetics of ALD release from FID-134 was determined in TRIS buffer at 37°C and compared to a simple mixture of HA+ALD. 20mg/mL formulations of FID-134 and HA+ALD were investigated for viscoelastic properties, in absence and presence of Ca. 2+. ions. The cytotoxicity of FID-134 and free ALD were tested on Saos-2 osteoblasts (ATCC HTB-85) and on primary bovine chondrocytes (PBC) at day 1, 3 and 7. The efficacy of FID-134 was assessed in an inflammatory arthritis in vitro model, where bovine cartilage biopsies were exposed to IL-1β/OSM (10ng/mL) for 3 weeks; at the same time, cartilage explants were treated with FID-134. Collagen release in the surnatants was quantified and compared to controls. FID-134 structure was confirmed by NMR and the 20% mol/mol functionalization degree was determined by ICP-OES. Only about 50% of total bound ALD was released from FID-134 within 7 days, resulting slower compared to HA+ALD mixture. In presence of Ca. 2+. ions, viscoelastic properties of FID-134 dramatically improved, while HA+ALD formulation remained unaffected. The cytotoxicity of ALD was evident at 100 μM on Saos-2 and PBC after 3 days, while no cytotoxicity was observed at 7 days with FID-134. In the cartilage explant model, a strong collagen release was detected in inflammatory conditions after 3 weeks; this tendency was reversed, and collagen release halved when FID-134 was added to the biopsies. The synthesized HA-ALD adduct, FID-134, opens the door for a new approach for OA treatment. The results suggest that FID-134 could be beneficial in cartilage degradation and in restoration of subchondral bone function. Finally, local administration and controlled BP release would likely overcome the drawbacks of ALD oral administration, such as unspecific features and long-term toxic side effects

Summary Statement. IL-1β stimulation of human OA chondrocytes induces NFκB, ERK1/2, c-JUN, IκB and P38 signalling pathways. Pre-treatment with cannabinoid WIN-55 for 48 hours inhibits certain pathways, providing mechanisms for cannabinoids inhibitory actions on IL-1β induced cartilage degradation. Matrix metalloproteinases (MMPs) are involved in extracellular matrix (ECM) breakdown in osteoarthritis (OA) and their expression is regulated by nuclear factor kappa B (NFκB). In addition signalling pathways ERK1/2, c-JUN, IκB and P38 are activated in OA and are induced by inflammatory cytokine interleukin 1 (IL-1). Cannabinoids have been shown to reduce joint damage in animal models of arthritis. Synthetic cannabinoid WIN-55, 212-2 mesylate (WIN-55) significantly reduces IL-1β induced expression of MMP-3 and -13 in human OA chondrocytes, indicating a possible mechanism via which cannabinoids may act to prevent ECM breakdown. Here the effects of WIN-55 on IL-1β induced NFκB, ERK1/2, c-JUN, IκB and P38 phosphorylation in human OA chondrocytes has been investigated. Primary human chondrocytes were obtained from articular cartilage removed from patients with symptomatic OA during total knee replacement (Ethic approval:SMB002). Cartilage tissue was graded macroscopically 0–4 using the Outerbridge Classification method. Chondrocytes isolated from grade 2 cartilage and cultured in monolayer were pre-treated with 10 μM WIN-55 for 1 hour prior to stimulation with 10 ng/ml IL-1β for 30 minutes for investigation of NFκB, c-JUN, IκB and P38 phosphorylation. In addition chondrocytes were pre-treated with 10 μM WIN-55 for 30 minutes, 1, 3, 6, 24 and 48 hours prior to 10 ng/ml IL-1β stimulation for 30 minutes to investigate ERK1/2 phosphorylation. Dimethyl sulfoxide (DMSO) was used as a vehicle control at 0.1%. Immunocytochemistry was used to investigate the phosphorylation and translocation of NFκB. ERK1/2, c-JUN, IκB, and P38 activation was investigated using cell based ELISA. Immunocytochemical analysis showed chondrocytes stimulated with IL-1β induced NFκB phosphorylation and translocation to the nucleus. Chondrocytes treated with IL-1β with WIN-55 for 1 hour pre-treatment showed no inhibition of the IL-1β induced NFκB phosphorylation and translocation to the nucleus. WIN-55 treatment alone for 1 hour stimulated NFκB phosphorylation in the cytoplasm but not the nucleus. ELISA showed that phosphorylation of ERK1/2, c-JUN, IκB, and P38 was significantly induced by IL-1β following 30 minutes stimulation (p<0.05). Pre-treatment with WIN-55 for 1 hour had no significant effect on this IL-1β induced phosphorylation. However WIN-55 pre-treatment for 48 hours prior to IL-1β stimulation for 30 minutes, resulted in a significant decrease in ERK1/2 phosphorylation compared to IL-1β stimulation alone (p<0.05). WIN-55 treatment alone for 1 hour significantly induced c-JUN phosphorylation (p<0.05), but had no effect on IκB and P38 phosphorylation compared to DMSO control. IL-1β stimulation of ERK1/2 phosphorylation was not significantly affected by WIN-55 pre-treatment of 30 minutes, 1, 3, 6 and 24 hours. WIN-55 treatment alone for 48 hours significantly reduced ERK1/2 phosphorylation compared to DMSO control (p<0.05). WIN-55 treatment alone for 30 min, 1, 3, 6 and 24 hours had no significant effect on ERK1/2 phosphorylation compared to DMSO control. The results show that following 48 hours pre-treatment WIN-55 inhibits IL-1β induced ERK1/2 phosphorylation in human OA chondrocytes. Thus inhibitory effects of cannabinoids on IL-1β induced cartilage degradation may be mediated via modulation of ERK1/2 signalling

Inflammation has been associated with early degradative changes in articular cartilage and immune responses are key factor influencing normal tissue regeneration and repair. With synovitis a prominent feature in osteoarthritis (OA) and associated with the progressive degradation of articular cartilage, immune factors need to be factored into efforts to achieve efficient cartilage repair/regeneration. Recent efforts have focused on the use of autologous or allogeneic mesenchymal stem/stromal cells (MSCs) to modulate the inflammatory environment in the injured or osteoarthritic joint. Intraarticular injection of MSCS has modulated cartilage degradation in a variety of pre-clinical OA models. Results from early clinical trials have also shown effects on pain and function-associated outcome measures. Other cell types may also have some capacity for use as a therapy for OA. For example, primary allogeneic chondrocytes also seem to have some immune-privilege in the synovial joint and are immunomodulatory in a rat model. Although MSCs isolated from bone marrow that are induced to undergo chondrogenic differentiation do not retain these properties, MSCs isolated from the synovium or chondroprogenitors generated from cartilage itself may represent the future of cell therapy for OA

Osteoarthritis (OA) is the most common cause of joint disease and associated disability. Despite this, its pathogenesis remains incompletely understood and no specific drug exists to prevent or reverse the structural changes in OA. Basic calcium phosphate (BCP) crystals are extremely common in OA. BCP crystals consist primarily of hydroxyapatite, with smaller amounts of octacalcium phosphate, tricalcium phosphate and magnesium whitlockite. They are present in 100% of joints at the time of knee and hip joint replacement surgery. Their presence strongly correlates with radiographic severity of osteoarthitis. In mice, intra-articular BCP crystals elicit synovial inflammation and cartilage degradation. The potential mechanisms by which calcium-containing crystals may promote articular damage have been studied in the laboratory setting and in vitro properties of BCP crystals have been observed that emphasise their pathogenic potential. BCP crystals interact with articular cells such as fibroblasts and chondrocytes to induce mitogenesis with resultant cellular proliferation likely leading to synovial lining hypertrophy. BCP crystals also upregulate production of cytokines such as tumour necrosis factor alpha (TNF-α), interleukin 1 (IL-1), increase prostaglandin E2 via the cyclooxygenase pathway, stimulate matrix metalloproteinases production and increase nitrous oxide production. Therefore, BCP crystals have potent biologic effects and represent a potential therapeutic target in OA

Osteoarthritis (OA) is a leading cause of joint pain, deformity and functional limitation. An imbalance of anabolic and catabolic activity results in destruction of the extracellular matrix of articular cartilage. While there is evidence to support the role of DNA methylation in the pathogenesis of OA, the effect of other epigenetic modifications is yet to be described. This study looks at the effect of two novel epigenetic modifiers, PFI-1, a bromodomain inhibitor, and SGC707, a histone methytransferase inhibitor, on gene expression in the pathogenesis of OA. Chondrocytes were extracted from OA femoral heads (n=6), cultured and incubated with increasing concentrations of the compounds. Cells were treated with media alone (control), interleukin 1-beta (IL-1β) plus oncostatin M (OSM) alone, or in combination with PFI-1 or SGC707. Levels of expression of iNOS, COX2, IL8, IL1B, matrix metalloproteinase-13 (MMP13), RUNX2 and COL9A1 were measured using qRT-PCR. PFI-1 (0.5 and 5µM) suppressed expression of catabolic genes in OA chondrocytes, at basal levels and when co-stimulated with IL-1β+OSM. While there was a decrease in catabolic gene expression (iNOS, COX2, IL8, IL1B and MMP13), RUNX2 expression was also supressed. There was no effect on expression of COL9A1, an anabolic chondrocytic gene. SGC707 (0.1 and 1µM) did not induce a reduction in expression of all the catabolic genes, with a less predictable effect on gene expression than PFI-1. This study has demonstrated that the BET inhibitor PFI-1 has a potent protective effect against cartilage degradation, through its action as an epigenetic modifier in modulating the expression of catabolic genes in OA chondrocytes. This further validates the role of epigenetics in OA, with potential implications for therapeutic interventions

Osteoarthritis (OA) is a leading cause of joint deformity and functional limitation. An imbalance of anabolic and catabolic activity results in destruction of the extracellular matrix of articular cartilage. There is evidence to support the role of DNA methylation in the pathogenesis of OA, but the effect of other epigenetic modifiers is yet to be described. This study looks at the effect of novel epigenetic modulators, PFI-1, a bromodomain inhibitor, and SGC707, a histone methytransferase inhibitor, and their effects on gene expression in the pathogenesis of OA. Chondrocytes were extracted from OA femoral heads (n=6), cultured and incubated. Samples were treated with media alone (control), interleukin 1-beta (IL-1β) plus oncostatin M (OSM) alone, or in combination with increasing concentrations of PFI-1 or SGC707. Levels of expression of iNOS, COX2, IL8, IL1B, matrix metalloproteinase-13 (MMP13), RUNX2 and COL9A1 were measured using qRT-PCR, and expressed relative to GAPDH. PFI-1 (0.5 and 5µM) suppressed expression of catabolic genes in OA chondrocytes, at basal levels and when co-stimulated with IL-1β+OSM. Catabolic gene expression decreased (iNOS, COX2, IL-8, IL-1β and MMP), and RUNX2 expression was also supressed. There was no effect on expression of the anabolic gene COL9A1. SGC707 (0.1 and 1µM) did not induce a reduction in expression of all the catabolic genes. This study has demonstrated that PFI-1 has a potent protective effect against cartilage degradation, by modulating the expression of catabolic genes in OA chondrocytes. This further validates the role of epigenetics in OA, with implications for therapeutic interventions in the future

Osteoarthritis (OA) is traditionally believed to affect the osteochondral unit by wear-and-tear from the superficial zone to the deep zone of cartilage and extended to subchondral plate. Obesity is commonly considered as a risk of OA development and hence total knee replacement (TKR), but the mechanism remains unclear. We hypothesized that obesity accelerated OA development by deteriorating tidemarks and increasing bone remodelling. 616,495 cases of TKR for OA from Australia and British joint replacement registries were collected, and data indicated that patients with higher BMI had TKR at earlier age. Specifically, patients with BMI ≤25kg/m. 2. showed 8 years younger than patients with BMI ≥40kg/m. 2. (P<0.0001) when they received TKR. We next examined tibia plateaus of 88 knee OA patients by micro-CT and histomorphometry. Linear regression showed that less cartilage degradation was associated with increased BMI in the load-bear compartment (p<0.05), while 58.3% of patients with BMI≥40kg/m. 2. demonstrated a clear anatomical separation close to tidemarks filled with fibrosis, erythrocytes and bone fragments (compared to BMI ≤25kg/m. 2. group: 7.7%, p<0.01). In subchondral bone, elevated bone formation was associated with increased BMI, as higher thickness of osteoid (p<0.01), percent osteoid volume (p<0.01), percent osteoid surface (p<0.01) were found in obese patients. However, no alteration of bone resorption and microstructural parameters was found to be associated with BMI. We suspected that the abnormal loading in knee joint due to high BMI led to the direct deterioration of binding site of osteochondral unit, which might be the mechanism of the rapid progression in obesity-related OA