Objectives. Metabolic syndrome and low-grade systemic inflammation are associated with knee osteoarthritis (OA), but the relationships between these factors and OA in other synovial joints are unclear. The aim of this study was to determine if a high-fat/high-sucrose (HFS) diet results in OA-like joint damage in the shoulders, knees, and hips of rats after induction of obesity, and to identify potential joint-specific risks for OA-like changes. Methods. A total of 16 male Sprague-Dawley rats were allocated to either the diet-induced obesity group (DIO, 40% fat, 45% sucrose, n = 9) or a chow control diet (n = 7) for 12 weeks. At sacrifice, histological assessments of the shoulder, hip, and knee joints were performed. Serum inflammatory mediators and body composition were also evaluated. The total Mankin score for each animal was assessed by adding together the individual Modified Mankin scores across all three joints. Linear regression modelling was conducted to evaluate predictive relationships between serum mediators and total joint damage. Results. The HFS diet, in the absence of trauma, resulted in increased joint damage in the shoulder and knee joints of rats. Hip joint damage, however, was not significantly affected by DIO, consistent with findings in human studies. The total Mankin score was increased in DIO animals compared with the chow group, and was associated with percentage of body fat. Positive significant predictive relationships for total Mankin score were found between body fat and two serum mediators (interleukin 1 alpha (IL-1α) and vascular endothelial growth factor (VEGF)). Conclusion. Systemic inflammatory alterations from DIO in this model system may result in a higher risk for development of knee, shoulder, and multi-joint damage with a HFS diet. Cite this article: K. H. Collins, D. A. Hart, R. A. Seerattan, R. A. Reimer, W. Herzog. High-fat/high-sucrose diet-induced obesity results in joint-specific development of osteoarthritis-like degeneration in a rat model. Bone Joint Res 2018;7:274–281. DOI: 10.1302/2046-3758.74.BJR-2017-0201.R2

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

Introduction. Piezo1 is a mechanosensitive Ca. 2+. ion channel that has been shown to transduce hyper-physiologic mechanical loads in chondrocytes. In osteoarthritic cartilage, Piezo1 expression was shown to be upregulated by interleukin-1 alpha (IL-1α) and resulted in altered calcium dynamics and actin cytoskeleton rarefication. Together these studies highlight the importance of Piezo1 channels during joint injury. However, the mechanism by which Piezo1 regulates chondrocyte physiology and mechanotransduction during homeostasis is still largely unknown. In this study, we investigate the impact of Piezo1 activation on nuclear mechanics and chromatin methylation state. Methods. Porcine chondrocytes (n=3-5 pigs) were treated with Yoda1, a Piezo1-specific agonist, for either 2, 5, 15 or 180 minutes. To characterize chromatin state, we monitored the abundance of a chromatin methylation marker (H3K9Me3) using immunofluorescence (IF). Atomic force microscopy (AFM, 25 nm cantilever) was employed to quantify the nuclear elastic modulus (NEM) of individual cell nuclei. To explore the interplay between cytoskeletal dynamics and nuclear mechanics, chondrocytes were treated with Latrunculin A (LatA), an actin polymerization inhibitor. Result. IF experiments showed chromatin methylation was the lowest 2 minutes post Yoda1 activation of Piezo1 (p=0.027). Additionally, we found that 2 or 5 minutes post-Piezo1 activation resulted in a significantly lower NEM when compared to the control (p<0.00001). The observed decrease in NEM at 2 and 5 minutes post-Piezo1 activation was not observed after knocking down Piezo1 (p>0.99). In LatA treated cells, the elevated NEM persisted even after Piezo1 activation with Yoda1 (p>0.75). Conclusion. These findings illuminate the mechanism by which Piezo1 activation and actin remodeling regulate transient mechanotransduction during homeostasis. Further research into the transient decrease in nuclear stiffness and chromatin methylation observed during the initial 5 minutes of Piezo1-induced Ca2+ signaling, may contribute to a better understanding of the role of Piezo1 channels in joint injury and development of therapeutic interventions for osteoarthritis

Senescent chondrocyte and subchondral osteoclast overburden aggravate inflammatory cytokine and pro-catabolic proteinase overproduction, accelerating extracellular matrix degradation and pain during osteoarthritis (OA). Fibronectin type III domain containing 5 (FNDC5) is found to promote tissue homeostasis and alleviate inflammation. This study aimed to characterize what role Fndc5 may play in chondrocyte aging and OA development. Serum and macroscopically healthy and osteoarthritic cartilage were biopsied from patients with knee OA who received total knee replacement. Murine chondrocytes were transfected with Fndc5 RNAi or cDNA. Mice overexpressing Fndc5 (Fndc5Tg) were operated to have destabilized medial meniscus mediated (DMM) joint injury as an experimental OA model. Cellular senescence was characterized using RT-PCR analysis of p16INK4A, p21CIP1, and p53 expression together with ß-galactosidase activity staining. Articular cartilage damage and synovitis were graded using OARSI scores. Osteophyte formation and mechanical allodynia were quantified using microCT imaging and von Frey filament, respectively. Osteoclast formation was examined using tartrate-resistant acid phosphatase staining. Senescent chondrocyte and subchondral osteoclast overburden together with decreased serum FNDC5 levels were present in human osteoarthritic cartilage. Fndc5 knockdown upregulated senescence program together with increased IL-6, MMP9 and Adamts5 expression, whereas Alcian blue-stained glycosaminoglycan production were inhibited. Forced Fndc5 expression repressed senescence, apoptosis and IL-6 expression, reversing proliferation and extracellular matrix production in inflamed chondrocytes. Fndc5Tg mice showed few OA signs, including articular cartilage erosion, synovitis, osteophyte formation, subchondral plate sclerosis and mechanical allodynia together with decreased IL-6 production and few senescent chondrocytes and subchondral osteoclast formation during DMM-induced joint injury. Mechanistically, Fndc5 reversed histone H3K27me3-mediated IL-6 transcription repression to reduce reactive oxygen species production. Fndc5 loss correlated with OA development. It was indispensable in chondrocyte growth and anabolism. This study sheds light onto the anti-ageing and anti-inflammatory actions of Fndc5 to chondrocytes; and highlights the chondroprotective function of Fndc5 to compromise OA

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

Glenohumeral joint injuries frequently result in shoulder instability. However, the biomechanical effect of cartilage loss on shoulder stability remains unknown. The aim of the current study was to investigate biomechanically the effect of two severity stages of cartilage loss in different dislocation directions on shoulder stability. Joint dislocation was provoked for 11 human cadaveric glenoids in seven different dislocation directions between 3 o'clock (anterior) to 9 o'clock (posterior) dislocation. Shoulder stability ratio (SSR) and concavity gradient were assessed in intact condition, and after 3 mm and 6 mm simulated cartilage loss. The influence of cartilage loss on SSR and concavity gradient was statistically evaluated. Between intact state and 6 mm cartilage loss, both SSR and concavity gradient decreased significantly in every dislocation direction (p≤0.038), except the concavity gradient in 4 o'clock dislocation direction (p=0.088). Thereby, anterior-inferior dislocation directions were associated with the highest loss of SSR and concavity gradient of up to 59.0% and 49.4%, respectively, being significantly higher for SSR compared to all other dislocation directions (p≤0.04). The correlations between concavity gradient and SSR for pooled dislocation directions were significant for all three conditions of cartilage loss (p<0.001). From a biomechanical perspective, articular cartilage of the glenoid contributes significantly to the concavity gradient, correlating strongly with the associated loss in glenohumeral joint stability. The highest effect of cartilage loss was observed in anterior-inferior dislocation directions, suggesting that surgical intervention should be considered for recurrent shoulder dislocations in the presence of cartilage loss

Introduction. Anterior shoulder instability results in labral and osseous glenoid injuries. With a large osseous defect, there is a risk of recurrent dislocation of the joint, and therefore the patient must undergo surgical correction. An MRI evaluation of the patient helps to assess the soft tissue injury. Currently, the volumetric three-dimensional (3D) reconstructed CT image is the standard for measuring glenoid bone loss and the glenoid index. However, it has the disadvantage of exposing the patient to radiation and additional expenses. This study aims to compare the values of the glenoid index using MRI and CT. Method. The present study was a two-year cross-sectional study of patients with shoulder pain, trauma, and dislocation in a tertiary hospital in Karnataka. The sagittal proton density (PD) section of the glenoid and enface 3D reconstructed images of the scapula were used to calculate glenoid bone loss and the glenoid index. The baseline data were analyzed using descriptive statistics, and the Chi-square test was used to test the association of various complications with selected variables of interest. Result. The glenoid index calculated in the current study using 3D volumetric CT images and MR sagittal PD images was 0.95±0.01 and 0.95±0.01, respectively. The CT and MRI glenoid bone loss was 5.41±0.65% and 5.38±0.65%, respectively. When compared, the glenoid index and bone loss calculated by MRI and CT revealed a high correlation and significance with a p-value of <0.001. Conclusions. The study concluded that MRI is a reliable method for glenoid measurement. The sagittal PD sequence combined with an enface glenoid makes it possible to identify osseous defects linked to glenohumeral joint damage and dislocation. The values derived from 3D CT are identical to the glenoid index and bone loss determined using the sagittal PD sequence in MRI

Osteoarthritis (OA) is the leading cause of pain and disability worldwide and is characterized by the degenerative changes of articular cartilage. Joint loading is required for cartilage maintenance; however, hyper-physiologic loading is a risk factor for OA. Mechanosensitive ion channels Piezo1 and Piezo2 synergistically transduce hyper-physiologic compression of chondrocytes, leading to chondrocyte death and onset of OA. This injury response is inhibited by Piezo channel loss of function, however the mechanistic role of Piezo channels in vivo is unknown. We examined the hypothesis that deletion of Piezo in chondrocytes will protect mice from joint damage and pain-related behaviors following a surgical destabilization of the medial meniscus (DMM), investigating a key mechanistic and mechanobiological role of these channels in the pathogenesis of OA. Aggrecan-Cre Piezo1 and Piezo1/2 knockout mice ((Agc)1-CRE. ERT2. ;Piezo1. fl/fl. Piezo2. fl/fl. ) were generated and given a 5-day Tamoxifen regimen at 12-weeks of age (n=6–12/group/sex). Cre-negative mice served as controls. At 16-weeks, mice received DMM surgery on the left knee. 12-weeks following DMM prior to sacrifice, activity and hyperalgesia were measured using spontaneous running wheels and a small animal algometer. Structural changes in bone, cartilage, and synovium were characterized using microCT, histology, and Modified Mankin Score criteria. Knockout of Piezo1/2 channels was chondroprotective in both sexes following DMM surgery as demonstrated by reduced Modified Mankin Score compared to control animals. Piezo1 KO was chondroprotective in only female mice, indicating a sexually dimorphic response. Piezo1 and Piezo1/2 KO was protective against pain in male mice, while females displayed no differences compared to controls. No changes were observed in bone morphology. Chondrocyte-specific Piezo1/2 knockout protects the knee joint from structural damage, hyperalgesia and functional deficits in a surgical model of PTOA in male and female mice, illustrating the importance of Piezo channels in response to injury in vivo. Future work aims to interrogate potential sexually dimorphic responses to cartilage damage and investigating Piezo2 KO mice

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

Abstract. Objectives. The syndesmosis joint, located between the tibia and fibula, is critical to maintaining the stability and function of the ankle joint. Damage to the ligaments that support this joint can lead to ankle instability, chronic pain, and a range of other debilitating conditions. Understanding the kinematics of a healthy joint is critical to better quantify the effects of instability and pathology. However, measuring this movement is challenging due to the anatomical structure of the syndesmosis joint. Biplane Video Xray (BVX) combined with Magnetic Resonance Imaging (MRI) allows direct measurement of the bones but the accuracy of this technique is unknown. The primary objective is to quantify this accuracy for measuring tibia and fibula bone poses by comparing with a gold standard implanted bead method. Methods. Written informed consent was given by one participant who had five tantalum beads implanted into their distal tibia and three into their distal fibula from a previous study. Three-dimensional (3D) models of the tibia and fibula were segmented (Simpleware Scan IP, Synopsis) from an MRI scan (Magnetom 3T Prisma, Siemens). The beads were segmented from a previous CT and co-registered with the MRI bone models to calculate their positions. BVX (125 FPS, 1.25ms pulse width) was recorded whilst the participant performed level gait across a raised platform. The beads were tracked, and the bone position of the tibia and fibula were calculated at each frame (DSX Suite, C-Motion Inc.). The beads were digitally removed from the X-rays (MATLAB, MathWorks) allowing for blinded image-registration of the MRI models to the radiographs. The mean difference and standard deviation (STD) between bead-generated and image-registered bone poses were calculated for all degrees of freedom (DOF) for both bones. Results. The absolute mean tibia and fibula bone position differences (Table 1) between the bead and BVX poses were found to be less than 0.5 mm for both bones. The bone rotation differences were found to be less than 1° for all axes except for the fibula Z axis rotation which was found to be 1.46°. One study. 1. has reported the kinematics of the syndesmosis joint and reported maximum ranges of motion of 9.3°and translations of 3.3mm for the fibula. The results show that the accuracy of the methodology is sufficient to quantify these small movements. Conclusions. BVX combined with MRI can be used to accurately measure the syndesmosis joint. Future work will look at quantifying the accuracy of the talus to provide further understanding of normal ankle kinematics and to quantify the kinematics across a healthy population to act as a comparator for future patient studies. Declaration of Interest. (b) declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported:I declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research project

Hip Osteoarthritis (HOA) is the most common joint disorder and a major cause of disability in the adult population, leading to total hip replacement (THR). Recently, evidence has mounted for a prominent etiologic role of femoroacetabular impingement (FAI) in the development of early OA in the non-dysplastic hip. FAI is a pathological mechanical process, caused by abnormalities of the acetabulum and/or femur leading to damage the soft tissue structures. FAI can determine chondro-labral damage and groin pain in young adults and can accelerate HOA progression in middle-aged adults. The aim of the study was to determine if the presence of calcium crystal in synovial fluid (SF) at the time of FAI surgery affects the clinical outcomes to be used as diagnostic and predictive biomarker. 49 patients with FAI undergoing arthroscopy were enrolled after providing informed consent; 37 SFs were collected by arthrocentesis at the time of surgery and 35 analyzed (66% males), median age 35 years with standard deviation (SD) 9.7 and body mass index (BMI) 23.4 kg/m. 2. ; e SD 3. At the time of surgery, chondral pathology using the Outerbridge score, labral pathology and macroscopic synovial pathology based on direct arthroscopic visualization were evaluated. Physical examination and clinical assessment using the Hip disability & Osteoarthritis Outcome Score (HOOS) were performed at the time of surgery and at 6 months of follow up. As positive controls of OA signs, SF samples were also collected from cohort of 15 patients with HOA undergoing THR and 12 were analysed. 45% FAI patients showed CAM deformity; 88% presented labral lesion or instability and 68% radiographic labral calcification. 4 patients out of 35 showed moderate radiographic signs of OA (Kellegren-Lawrence score = 3). Pre-operative HOOS median value was 61.3% (68.10-40.03) with interquartile range (IQR) of 75-25% and post-operative HOOS median value 90% with IQR 93.8-80.60. In both FAI and OA patients the calcium crystal level in SFs negatively correlated with glycosaminoglycan (component of the extracellular matrix) released, which is a marker of cartilage damage (Spearman rho=-0.601, p<0.001). In FAI patients a worst articular function after surgery, measured with the HOOS questionnaire, was associated with both acetabular and femoral chondropathy and degenerative labral lesion. Moreover, radiographic labral calcification was also significantly associated with pain, worst articular function and labral lesion. Calcium crystal level in SFs was associated with labral lesions and OA signs. We concluded that the levels of calcium crystals in FAI patients are correlated with joint damage, OA signs and worst post-operative outcome. The presence of calcium crystals in SF of FAI patients might be a potential new biomarker that might help clinicians to make an early diagnosis, evaluate disease progression and monitor treatment response

Although remnant-preserved ACL reconstruction (ACLR) restores knee joint stability and dampens the problem of acute ACL rupture-induced knee pain, an increasing number of patients still develop post-traumatic osteoarthritis (PTOA) after 10 to 15 years of ACLR. We previously found that remnant-preserved ACLR with concomitant medial and lateral meniscus repair may not prevent cartilage degeneration and weaken muscle strength, while the clinical features of PTOA are not clear. We hypothesized that remnant-preserved ACLR with concomitant medial and lateral meniscus tears is related to early cartilage damage, worse function recovery, patient-reported outcomes (PROs) and delayed duration to return to sports. The aim is to evaluate the remnant-preserved ACLR with complicated meniscal injuries in predicting which patients are at higher risk of osteoarthritic changes, worse function and limited activities after ACLR for 12 months. Human ethical issue was approved by a committee from Xi'an Jiaotong University. 26 young and active patients (24 male, 2 female) with ACL injuries (Sherman type I and II) with concomitant medial and lateral meniscus within 2 months were included from January 2014 to March 2022. The average age of the ACLR+ meniscus repair was 26.77±1.52 (8 right, 5 left) and isolated ACLR control was 31.92±2.61 years old (7 left, 6 right). Remnant-preserved ACLR with a 5- to 6-strand hamstring tendon graft was operated on by the same sports medicine specialists. MRI CUBE-T. 2. scanning with 48 channels was conducted by a professional radiologist. The volume of the ACL graft was created through 3 dimensional MRI model (Mimics 19, Ann Arbor). Anterior Cruciate Ligament OsteoArthritis Score (ACLOAS) was applied to score visible cartilage damage. IKDC 2000 score and VAS were assessed by two blinded researchers. Results were presented as mean± SEM of each group. The cross-sectional area and 3D volume of the ACL graft were greater in the remnant-preserved ACLR+meniscus group compared with isolated ACLR (p=0.01). It showed that ACLR+ meniscus group had early signs of joint damage and delayed meniscus healing regarding ACLOAS compared to control group (p=0.045). MRI CUBE-T. 2. prediction of radiographic cartilage degeneration was not obvious in both groups post remnant-preserved ACLR over 12 months (p>0.05). However, higher VAS scores, lower IKDC scores, and long-last joint swelling were reported in the ACLR+ meniscus repair group at the end of 12 months follow-up. Although remnant-preserved ACLR+ meniscus was able to maintain the restore the knee function, it showed delayed timing (>12 months) to return to play at the pre-injury stage, while no difference between the timing of returning to the normal daily routine of their ACLR knee compared to control (p=0.30). The cost of ACLR+ meniscus (average 10,520.76$) was higher than the control group (6,452.92$, p=0.018). Remnants-preserved ACLR with concomitant injured medial and lateral meniscus repair shows a higher risk of cartilage damage, greater cost, worse functional performance, and longer time for young male patients to return to sports after 12-month follow-up compared to isolated ACLR. Further evidence and long-term follow-up are needed to better understand the association between these results and the risk of development of PTOA in this patient cohort

Abstract. INTRODUCTION. The mechanisms underlying abnormal joint mechanics are poorly understood despite it being a major risk factor for developing osteoarthritis. Glutamate signalling has been implicated in osteoarthritic bone changes and AMPA/kainate glutamate receptor (GluR) antagonists alleviate degeneration in rodent models of osteoarthritis. We investigated whether glutamate signalling molecules are mechanically regulated in a human, cell-based 3D model of bone. METHODS. Human Y201 MSC cells embedded in 3D type I collagen gels (0.05 × 106 cell/gel) differentiated to osteocytes were mechanically loaded in silicone plates (5000 µstrain, 10Hz, 3000 cycles) or not loaded (n=5/group). RNA extracted 1-hr post load was quantified by RTqPCR and RNAseq whole transcriptome analysis (NovaSeq S1 flow cell 2 × 100bp PE reads). Differentially expressed GluRs and glutamate transporters (GluTs) were identified using DEseq2 analysis on normalised count data. Genes were considered differentially expressed if >2 fold change and FDR p<0.05. RESULTS. Cells expressed mature osteocyte markers (E11, sclerostin, DMP-1). DEseq2 analysis, revealed 981 mechanically regulated genes. Mechanical loading upregulated kainate GluRs, GRIK2 (1.6 fold, p=0.024) and GRIK5 (4.2 fold, p=0.045); the NMDA GluR GRIN3B (3.25 fold, p=0.047) and the GluT SLC1A1 (3 fold, p=0.037). Conversely, AMPA GRIA3, NMDA GluRs GRIN2A&C, and the GluT SLC1A2 were down regulated by 50–60%, although not significant. Kainate GRIK3&4; AMPA GRIA2, NMDA GRIN1, and GluTs SLC1A6&A7 were not expressed in control or loaded osteocytes, whereas GluRs (GRIK1, GRIA1&4, GRIN2B&2D&3A) and GluT SLC1A3 were expressed but not regulated by mechanical loading. DISCUSSION. Mechanical loading of human osteocytes in 3D revealed that they regulated expression of glutamate receptors and transporters. This is consistent with our observation that mechanical perturbation after joint injury in rodent models of OA regulates glutamatergic signalling in the bone thus linking mechanical stimuli to inflammatory and nociceptive pathways mediated by glutamate receptors. Declaration of Interest. (a) fully declare any financial or other potential conflict of interest

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

Post-traumatic arthritis is a frequent consequence of articular fracture. The mechanisms leading to its development after such injuries have not been clearly delineated. A potential contributing factor is decreased viability of the articular chondrocytes. The object of this study was to characterise the regional variation in the viability of chondrocytes following joint trauma. A total of 29 osteochondral fragments from traumatic injuries to joints that could not be used in articular reconstruction were analysed for cell viability using the fluorescence live/dead assay and for apoptosis employing the TUNEL assay, and compared with cadaver control fragments. Chondrocyte death and apoptosis were significantly greater along the edge of the fracture and in the superficial zone of the osteochondral fragments. The middle and deep zones demonstrated significantly higher viability of the chondrocytes. These findings indicate the presence of both necrotic and apoptotic chondrocytes after joint injury and may provide further insight into the role of chondrocyte death in post-traumatic arthritis

The paramount importance of synovial fluid in lubrication and protection of articular joints has long been recognized. Synovial fluid, a dialysate of plasma, forms an interface with both the synovium and cartilage and plays a crucial role in joint lubrication and bearing functions. In an osteoarthritic joint, damage to the articular cartilage causes modifications in the rheological properties of synovial fluid and, reducing the viscoelasticity and increasing the friction between articular surfaces. Viscosupplementation is a treatment for osteoarthritis that uses hyaluronic acid as a (visco)supplement to the diseased joint. The aim of this treatment is to restore the rheological properties of synovial fluid. Osteoarthritis is the most common disease affecting the joints in human population and among the most important causes of pain, disability and economic loss. Therefore, innovative methods are needed to more effectively treat osteoarthritis, directly addressing the disease process. Among various locomotor mechanisms that could serve to illustrate the integrated nature of functional morphology, perhaps none is more complex than the equine locomotor system. Confronting the need for evaluating the current methods to control joint disease, the horse provides an excellent animal model. As it suffers similar clinical manifestations to those seen in human, it may provide tentative biomedical extrapolations

Osteoarthritis (OA) is the most common musculoskeletal disease in the EU and is characterized by cartilage degeneration, pain and restricted movement. Post-Traumatic OA (PTOA) is a specific disease subset that occurs subsequent to traumatic injury, such as ACL rupture and makes up 12% of the overall disease burden. Our current understanding PTOA is that initial injury affects multiple tissues, and many/all contribute to overall ‘joint failure.’ MRI scans show that subchondral bone marrow lesions (BMLs) are present in 80% of ACL rupture cases in the immediate aftermath of joint injury. Their presence indicates an acute consequence in subchondral bone. It has also been suggested that BMLs overlap with, or directly represent, bone microdamage. Microdamage is known to induce osteoclast-mediated remodelling in bone. Therefore, the inhibition of subchondral bone remodelling, particularly in the early phase post-injury, may be a candidate therapeutic approach for preventing PTOA. Finally, the contiguous link between subchondral bone and articular cartilage, can allow transport of small molecules across this boundary, this suggests that bone/cartilage crosstalk is likely to be a key factor in PTOA development after injury. This presentation will summarize recent advances in our understanding these phenomena in both animal and human studies

In least 12% of patients with symptomatic OA, the cause is joint injury that progressed over time to post-traumatic OA. Human adult articular cartilage has a limited innate ability to regenerate. Available treatment options are unable to restore native structure and function of hyaline cartilage. Agili-C (CartiHeal, Israel) is a first-in-class acellular scaffold consisted of two layers corresponding to cartilage and bone that is capable of attracting stem cells and guide a regenerative process in both tissues. Agili-C has been extensively tested in vitro in our laboratory using human normal cartilage and in vivo in preclinical and currently clinical studies. This scaffold consists of a natural crystalline aragonite, derived from corals, to which hyaluronic acid is added. It showed a great ability to induce regeneration of chondral and osteochondral lesions and attract chondrocytes and stems cells to fill the defect area. Cells remained viable over the course of the study (up to 2 months). Signs of the extracellular matrix formation were evident inside 3D structure of the scaffold. PG synthesis and gene expression of collagen type II and aggrecan were elevated by more than 2.5-fold in cartilage with the scaffold vs corresponding controls. Agili-C scaffold displays a potential in the treatment of focal chondral and osteochondral defects

Joint injuries often result in inflammation and cartilage defects. When inflamed, the synovium secretes factors that prevent successful cartilage repair by inhibiting chondrogenic differentiation of progenitor cells. In particular the pro-inflammatory macrophages in the synovium are indicated to contribute to this anti-chondrogenic effect. Thus, we aimed to counteract the anti-chondrogenic effect of inflamed synovium by modulating synovial inflammation and its macrophages. Synovium tissue obtained from osteoarthritic patients undergoing a total knee replacement was cut into explants and cultured for 72 hours +/− 1 µM of the anti-inflammatory drug triamcinolone acetonide (TAA) (Sigma Aldrich). TAA significantly decreased gene expression of TNFA, IL1β and IL6, and increased expression of CCL18, IL1RA in synovial explants (all with p < 0.001). On the other hand, TAA significantly decreased the percentages of pro-inflammatory CD14+/CD80+ and CD14+/CD86+ macrophages in the synovium (both p < 0.001) as assessed by flow cytometry analyses. The percentages of anti-inflammatory CD14+/CD163+ macrophages, is significantly increased (p < 0.001) in TAA treated synovium. Conditioned medium (CM) from synovium explants downregulated the gene expression of cartilage matrix components collagen type-2 and aggrecan expression in chondrogenic MSCs. This chondrogenesis inhibiting effect was reduced by treating synovium with TAA during the production of the CM. Our findings indicate that reducing synovial inflammation might improve the joint environment for better cartilage repair, possibly by modulation of macrophage phenotypes

Onset and progression of osteoarthritis (OA) is affected by a plethora of factors, including joint injury, obesity, aging, and heredity. This multi-factorial etiology obstructs our understanding of driving molecular mechanisms, which likely comprise an interplay between systemic and local factors. Next to biomechanical factors and cytokines, the course of OA appears to be altered by microenvironmental oxidative stress: cumulative evidence now suggests a prominent participation of cell signalling mediated by nuclear factor (erythroid-derived 2)-like 2 (Nrf2), a master regulator of cellular protective processes, in this process. Nrf2 activation through phosphorylation of mitogen-activated protein kinases (MAPKs) regulates Nrf2 target genes, like hemeoxygenase-1 (HO-1), superoxide dismutase 2 (SOD2), or NAD(P)H Quinone Dehydrogenase 1 (NQO1) in OA chondrocytes. Maintaining high levels of HO-1 appears to be beneficial against OA development. Experimental manipulation of putative antioxidant response element (ARE) binding sites alters the in vitro expression of key transcription factors of chondrocyte markers in promoter-reporter assays. Potentially, Nrf2 is involved in autophagy, intermediary metabolism and unfolded protein response. RNAi-mediated depletion of Nrf2 further significantly abrogated anti-inflammatory and chondroprotective effects and epigenetics link transcriptional pathways of ‘N-factors’, Nrf2 and NFATs, to micro-RNA signalling. Current findings thus reveal novel mechanisms regulating extracellular matrix synthesis by chondrocytes. A further understanding of these pathways and their regulation will lead to important novel targets to slow OA progression