Cranial cruciate ligament (CrCL) disease/rupture is a highly prevalent orthopaedic disease in dogs and common cause of pain, lameness, and secondary joint osteoarthritis (OA). Previous experiments investigating the role of glutamate receptors (GluR) in arthritic degeneration and pain revealed that OA biomarkers assessing early bone turnover and inflammation, including osteoprotegerin (OPG) and the receptor activator of nuclear factor kappa-B ligand (RANKL) are more likely to be influenced by glutamate signalling. Moreover, interleukin-6 (IL-6) has a complex and potentially bi directional (beneficial and detrimental) effect, and it is a critical mediator of arthritic pain, OA progression and joint destruction. 1) to recruit dogs undergoing CrCL disease/rupture surgery and obtain discarded synovial fluid (SF) and serum/plasma (ethics approval, RCVS:2017/14/Alves); 2) to quantify the biomarkers listed above in the SF and serum/plasma by enzyme linked immunosorbent assay (ELISA); 3) to assess radiographic OA at the time of surgery and correlate it with the biomarkers and clinical findings. Abnova, Abcam and AMSBIO ELISA kits were tested using a validation protocol relating the standard curve to a dilution series of SF and serum/plasma (1× to 1/50×), with and without SF hyaluronidase treatment to evaluate linearity, specificity and optimal dilutions. Validated ELISA kits were used to measure [IL-6], glutamate [glu], [RANKL] and [OPG] in SF and serum/plasma. For each dog, CrCL disease pre-operative lameness scores were graded as: (1) mild, (2) moderate (easily visible), (3) marked (encumbered), (4) non-weightbearing lameness. Blinded OA scoring was performed on radiographs [15–60, normal-severe OA].Abstract
Objectives
Methods
Knee varus malalignment increases medial knee compartment loading and is associated with knee osteoarthritis (OA) progression and severity1. Altered biomechanical loading and dysregulation of joint tissue biology drive OA progression, but mechanistic links between these factors are lacking. Subchondral bone structural changes are biomechanically driven, involve bone resorption, immune cell influx, angiogenesis, and sensory nerve invasion, and contribute to joint destruction and pain2. We have investigated mechanisms underlying this involving RANKL and alkaline phosphatase (ALP), which reflect bone resorption and mineralisation respectively3 and the axonal guidance factor Sema3A. Sema3A is osteotropic, expressed by mechanically sensitive osteocytes, and an inhibitor of sensory nerve, blood vessel and immune cell invasion4. Sema3A is also differentially expressed in human OA bone5.HYPOTHESIS: Medial knee compartment overloading in varus knee malalignment patients causes dysregulation of bone derived Sema3A signalling directly linking joint biomechanics to pathology and pain. Synovial fluid obtained from 30 subjects with medial knee OA (KL grade II-IV) undergoing high tibial osteotomy surgery (HTO) was analysed by mesoscale discovery and ELISA analysis for inflammatory, neural and bone turnover markers. 11 of these patients had been previously analysed in a published patient-specific musculoskeletal model6 of gait estimating joint contact location, pressure, forces, and medial-lateral condyle load distribution in a published data set included in analyses. Data analysis was performed using Pearson's correlation matrices and principal component analyses. Principal Components (PCs) with eigenvalues greater than 1 were analysed.Abstract
OBJECTIVE
METHODS
Changes in subchondral bone are one of few disease characteristics to correlate with pain in OA1. Profound neuroplasticity and nociceptor sprouting is displayed within osteoarthritic (OA) subchondral bone and is associated with pain and pathology2. The cause of these neural changes remains unestablished. Correct innervation patterns are indispensable for bone growth, homeostasis, and repair. Axon guidance signalling factor, Sema3A is essential for the correct innervation patterning of bony tissues3, expressed in osteocytes4 and known to be downregulated in bone OA mechanical loading5. Bioinformatic analysis has also shown Sema3a as a differentially expressed pathway by bone in human OA patients6.HYPOTHESIS: Pathological mechanical load and inflammation of bone causes dysregulation of Sema3A signalling leading to perturbed sensory nerve plasticity and pain. Human KOLF2-C1 iPSC derived nociceptors were generated by TALEN-mediated insertion of transcription factors NGN2+Brn3A and modified chambers differentiation protocol to produce nociceptor-like cells. Nociceptor phenotype was confirmed by immunocytochemistry. Human Y201-MSC cells were embedded in 3D type-I collagen gels (0.05 × 106 cell/gel), in 48-well plates and silicone plates, were differentiated to osteocytes for 7 days before stimulation with IL-6 (5ng/ml) and soluble IL-6 receptor (sIL-6r (40ng/ml), IL6/sIL6r and mechanical load mimetic Yoda1 (5μM) or unstimulated (n=5/group) (48-well plates) or were mechanically loaded in silicone plates (5000μstrain, 10Hz, 3000 cycles) or not loaded (n=5/group). Conditioned media transfer was performed from osteocyte to nociceptor cultures assessed by continuous 24-hour phase contrast confocal microscopy. 24-hours after stimulation RNA was quantified by RT-qPCR (IL6) or RNAseq whole transcriptome analysis/DEseq2 analysis (Load). Protein release was quantified by ELISA. Normally distributed data with homogenous variances was analysed by two-tailed t test.Abstract
OBJECTIVE
METHODS
Stimulation of the mechanosensitive ion channel, Piezo1 promotes bone anabolism and SNPs in the Piezo1 locus are associated with changes in fracture risk. Osteocytes function as critical regulators of bone homeostasis by sensing mechanical signals. The current study used a human, cell-based physiological, 3D in vitro model of bone to determine whether loading of osteocytes in vitro results in upregulation of the Piezo1 pathway. Human Y201 MSCs, embedded in type I collagen gels and differentiated to osteocytes for 7-days, were subjected to pathophysiological load (5000 µstrain, 10Hz, 5 mins; n=6) with unloaded cells as controls (n=4). RNA was extracted 1-hr post load and assessed by RNAseq analysis. To mimic mechanical load and activate Piezo1, cells were differentiated to osteocytes for 13 days and treated ± Yoda1 (5µM, 2- and 24-hs, n=4); vehicle treated cells served as controls (n=4). RNA was subjected to RT-qPCR and data normalised to the housekeeping gene, YWHAZ. Media was analysed for IL6 release by ELISA. Mechanical load upregulated Piezo1 gene expression (16.5-fold, p<0.001) and expression of the transcription factor NFATc1, and matricellular protein CYR61, known regulators of Piezo1 mechanotransduction (3-fold; p= 5.0E-5 and 6.8-fold; p= 6.0E-5, respectively). After 2-hrs, Yoda1 increased the expression of the early mechanical response gene, cFOS (11-fold; p=0.021), mean Piezo1 expression (2.3-fold) and IL-6 expression (103-fold, p<0.001). Yoda1 increased the release of IL6 protein after 24 hours (7.5-fold, p=0.001). This study confirms Piezo1 as an important mechanosensor in osteocytes. Piezo1 activation mediated an increase in IL6, a cytokine that drives inflammation and bone resorption providing a direct link between mechanical activation of Piezo1, bone remodeling and inflammation, which may contribute to mechanically induced joint degeneration in diseases such as osteoarthritis. Mechanistically, we hypothesize this may occur through promoting Ca2+ influx and activation of the NFATc1 signaling pathway.
Osteoarthritis (OA) is a common cause of chronic pain. Subchondral bone is highly innervated, and bone structural changes directly correlate with pain in OA. Mechanisms underlying skeletal–neural interactions are under-investigated. Bone derived axon guidance molecules are known to regulate bone remodelling. Such signals in the nervous system regulate neural plasticity, branching and neural inflammation. Perturbation of these signals during OA disease progression may disrupt sensory afferents activity, affecting tissue integrity, nociception, and proprioception. Osteocyte mechanical loading and IL-6 stimulation alters axon guidance signalling influencing innervation, proprioception, and nociception. Human Y201 MSC cells, embedded in 3D type I collagen gels (0.05 × 106 cell/gel) in 48 well plastic or silicone (load) plates, were differentiated to osteocytes for 7 days before stimulation with IL-6 (5ng/ml) with soluble IL-6 receptor (sIL-6r (40ng/ml) or unstimulated (n=5/group), or mechanically loaded (5000 μstrain, 10Hz, 3000 cycles) or not loaded (n=5/group). RNA extracted 1hr and 24hrs post load was quantified by RNAseq whole transcriptome analysis (NovaSeq S1 flow cell 2 × 100bp PE reads and differentially expressed neurotransmitters identified (>2-fold change in DEseq2 analysis on normalised count data with FDR p<0.05). After 24 hours, extracted IL-6 stimulated RNA was quantified by RT-qPCR for neurotrophic factors using 2–∆∆Ct method (efficiency=94-106%) normalised to reference gene GAPDH (stability = 1.12 REfinder). Normally distributed data with homogenous variances was analysed by two-tailed t test. All detected axonal guidance genes were regulated by mechanical load. Axonal guidance genes were both down-regulated (Netrin1 0.16-fold, p=0.001; Sema3A 0.4-fold, p<0.001; SEMA3C (0.4-fold, p<0.001), and up-regulated (SLIT2 2.3-fold, p<0.001; CXCL12 5-fold, p<0.001; SEMA3B 13-fold, p<0.001; SEMA4F 2-fold, p<0.001) by mechanical load. IL6 and IL6sR stimulation upregulated SEMA3A (7-fold, p=0.01), its receptor Plexin1 (3-fold, p=0.03). Neutrophins analysed in IL6 stimulated RNA did not show regulation. Here we show osteocytes regulate multiple factors which may influence innervation, nociception, and proprioception upon inflammatory or mechanical insult. Future studies will establish how these factors may combine and affect nerve activity during OA disease progression.
Mechanical loading of joints with osteoarthritis (OA) results in pain-related functional impairment, altered joint mechanics and physiological nociceptor interactions leading to an experience of pain. However, the current tools to measure this are largely patient reported subjective impressions of a nociceptive impact. A direct measure of nociception may offer a more objective indicator. Specifically, movement-induced physiological responses to nociception may offer a useful way to monitor knee OA. In this study, we gathered preliminary data on healthy volunteers to analyse whether integrated biomechanical and physiological sensor datasets could display linked and quantifiable information to a nociceptive stimulus. Following ethical approval, 15 healthy volunteers completed 5 movement and stationary activities in 2 conditions; a control setting and then repeated with an applied quantified thermal pain stimulus to their right knee. An inertial measurement unit (IMU) and an electromyography (EMG) lower body marker set were tested and integrated with ground reaction force (GRF) data collection. Galvanic skin response electrodes for skin temperature and conductivity and photoplethysmography (PPG) sensors were manually timestamped to the integrated system. Pilot data showed EMG, GRF and IMU fluctuations within 0.5 seconds of each other in response to a thermal trigger. Preliminary analysis on the 15 participants tested has shown skin conductance, PPG, EMG, GRFs, joint angles and kinematics with varying increases and fluctuations during the thermal condition in comparison to the control condition. Preliminary results suggest physiological and biomechanical data outputs can be linked and identified in response to a defined nociceptive stimulus. Study data is currently founded on healthy volunteers as a proof-of-concept. Further exploratory statistical and sensor readout pattern analysis, alongside early and late-stage OA patient data collection, can provide the information for potential development of wearable nociceptive sensors to measure disease progression and treatment effectiveness.
Involving research users in setting priorities for research is essential to ensure research outcomes are patient-centred and to maximise research value and impact. The Musculoskeletal (MSK) Disorders Research Advisory Group Versus Arthritis led a research priority setting exercise across MSK disorders. The Child Health and Nutrition Research Initiative (CHRNI) method of setting research priorities with a range of stakeholders were utilised. The MSKD RAG identified, through consensus, four research Domains: Mechanisms of Disease; Diagnosis and Impact; Living Well with MSK disorders and Successful Translation. Following ethical approval, the research priority exercise involved four stages and two surveys, to: 1) gather research uncertainties; 2) consolidate these; 3) score uncertainties using agreed criteria of importance and impact on a score of 1–10; and 4) analyse scoring, for prioritisation.Background
Methods
Cranial cruciate ligament (CrCL) disease/rupture causes pain and osteoarthritis (OA) in dogs. α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA)-2 and kainate (KA)-1 glutamate receptors (GluR) and the excitatory amino acid transporter-1 (EAAT-1) and EAAT-3 are expressed in joint tissues from OA patients and rodent arthritis models and represent potential therapeutic targets. To evaluate glutamate signalling in canine diseased and normal CrCL and meniscus by immunohistochemistry (IHC). Surgical waste (CrCL, n=5 and medial meniscus, n=3) were obtained from canines with CrCL disease (RCVS ethics approval:2017/14/Alves) and normal analogous tissues (n=2). IHC optimization was performed for rabbit polyclonal (AMPA-2:ab52176, KA-1:ab67402, EAAT-1:ab416) and monoclonal (EAAT-3:ab124802) antibodies from Abcam. IHC was optimised over antibody dilutions from 1:100 to 1:5000 alongside equivalent IgG isotype controls (ab37415 and ab172730) and negative controls (TBS/Tween buffer without primary antibodies). IHC staining was compared in diseased and normal tissues and disclosed with 3,3’-Diaminobenzidine (DAB).Abstract
Objectives
Methods
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. 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.Abstract
INTRODUCTION
METHODS
Knee tactile afferents act as synovial joint limit detectors, eliciting signalling upon excessive fibrous tissue strain but play little role in joint function as disruption of their activity does not induce impairments in movement or sensation. In contrast, knee nociceptive afferents gain activity upon inflammation producing painful sensation in pathology such as osteoarthritis. We hypothesize that similar in origin, fast-conducting tactile afferents become sensitized by inflammatory mediators and gain activity causing proprioceptive sensation impairment in patients with knee pathology, driving gait abnormalities and osteoarthritis progression. To investigate the activity of these neurons, we will produce a co-culture model using our existing 3D bone mimetic and iPSC derived tactile sensory neurons by utilizing the NGN2-BRN3A plasmid produced by Nickolls et al producing a model of these tactile neurons at their position within the joint at the fibrous/bony interface. Human Y201 MSC cells embedded in type I collagen gels (0.05 × 106 cell/gel) were differentiated to osteocytes andmechanically loaded in silicone plates (5000 µstrain, 10Hz, 3000 cycles) (n=5). RNA quantified by RNAseq analysis (NovaSeq S1) and neuronal communication pathways identified using DEseq2 analysis.Abstract
INTRODUCTION
METHODS
Current tools to measure pain are broadly subjective impressions of the impact of the nociceptive impulse felt by the patient. A direct measure of nociception may offer a more objective indicator. Specifically, movement-induced physiological responses to nociception may offer a useful way to monitor knee OA. In this proof-of-concept study, we evaluated whether integrated biomechanical and physiological sensor datasets could display linked and quantifiable information to a nociceptive stimulus. Following ethical approval, we applied a quantified thermal pain stimulus to a volunteer during stationary standing in a gait lab setting. An inertial measurement unit (IMU) and an electromyography (EMG) lower body marker set were tested and integrated with ground reaction force (GRF) data collection. Galvanic skin response electrodes and skin thermal sensors were manually timestamp linked to the integrated system.Abstract
Objectives
Method
Osteocytes function as critical regulators of bone homeostasis by sensing mechanical signals. Stimulation of the mechanosensitive ion channel, Piezo1 promotes bone anabolism and deletion of Piezo1 in osteoblasts and osteocytes decreases bone mass and bone strength in mice. This study determined whether loading of osteocytes in vitro results in upregulation of the Piezo1 pathway. Human MSC cells (Y201), embedded in type I collagen gels and differentiated to osteocytes in osteogenic media for 7-days, were subjected to pathophysiological load (5000 µstrain, 10Hz, 5 mins; n=6) with unloaded cells as controls (n=4). RNA was extracted 1-hr post load and Piezo1 activation assessed by RNAseq analysis (NovaSeq S1 flow cell 2 × 100bp PE reads). To mimic mechanical load and activate Piezo1, Y201s were differentiated to osteocytes in 3D gels for 13 days and treated, with Yoda1 (5µM, 2 hours, n=4); vehicle treated cells served as controls (n=4). Extracted RNA was subjected to RT-qPCR and data analysed by Minitab.Abstract
Objectives
Methods
Cranial cruciate ligament (CrCL) disease in dogs causes pain and osteoarthritis (OA) and surgical treatment does not prevent OA progression. Glutamate receptor (GluR) antagonists alleviate pain and degeneration in rodent models of OA, but it is unknown whether they are a suitable treatment for dogs. Understanding GluR signalling in CrCL disease may lead to novel therapeutics in both veterinary and human medicine. To determine whether age, breed, sex, weight, and therapeutic(s) influence lameness and pre-operative radiographic OA scoring in dogs with CrCL disease and whether GluRs are expressed, in this disease. Surgical waste (CrCL and medial meniscus), clinical data, stifle radiographs, lameness scores (1–4, mild-unloading limb) were obtained with full informed consent (RCVS ethics approval, ref: 2017/14/Alves). OA scoring was performed on radiographs [VCOT, 2017, 30(6):377–384, 15–60, normal-severe OA], and α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA)-2 and kainate (KA)-1 GluR expression compared in diseased Abstract
Objectives
Methods
Osteoarthritis therapies are limited to symptom management and joint replacement. AMPA/kainate glutamate receptor (GluR) antagonists (NBQX/DNQX, 2.5–20mM) alleviate symptoms and disease in rodent models of osteoarthritis. We hypothesised that poly(lactic-co-glycolic) acid (PLGA) nanoparticles and thermoresponsive hydrogels sustain GluR antagonist release to improve their efficacy in an humanised 3D bone model of inflammation. Drug release in PBS (37 °C) was measured by HPLC of samples taken from 2.5mM NBQX/DNQX loaded PLGA nanoparticles (double emulsion) and thermosetting hydrogels (homogenised Pluronic-F127 (22%/25% w/v) and Carbopol 934 (0.5% w/v) with 2.5mM NBQX/DNQX in dH2O)(n=3). Y201 MSCs were cultured in 3D in rat tail collagen type I gels and exposed to IL-6/sIL-6r (5/40ng/ml), free NBQX (200μM) or NBQX loaded PLGA nanoparticles for 24 and 72hrs. Bone turnover, inflammatory and glutamate signalling markers were quantified by immunoassay and RTqPCR. Data analysed using t-test/ANOVA with Tukeys and principal component analysis (PCA)(SPSS).Abstract
OBJECTIVES
METHODS
Valgus high tibial osteotomy (HTO) represents an effective treatment for patients with medial compartment osteoarthritis (OA) in a varus knee. However, the mechanisms which cause this clinical improvement are unclear. Previous studies suggest a wider stance gait can reduce medial compartment loading via reduction in the external knee adduction moment (KAM); a measure implicated in progression of medial compartment OA. This study aimed to measure whether valgus HTO is associated with a postoperative increase in static stance width. 32 patients, recruited in the Biomechanics and Bioengineering Centre Versus Arthritis HTO study, underwent valgus (medial opening wedge) HTO. Weightbearing pre- and post- operative radiographs were taken showing both lower limbs. The horizontal distance, measured from a fixed point on the right talus to the corresponding point on the left, was divided by the talus width to give a standardised “stance width” for each radiograph. The difference between pre- and post- operative stance width was compared for each patient using a paired sample t-test.Abstract
OBJECTIVES
METHODS
The mechanisms underlying abnormal joint mechanics are poorly understood despite it being a major risk factor for developing osteoarthritis. This study investigated the response of a 3D in vitro bone cell model to mechanical load. Human MSC cells (Y201) embedded in 3D type I collagen gels were differentiated in osteogenic media for 7-days in deformable, silicone plates. Gels were loaded once (5000 µstrain, 10Hz, 3000 cycles), RNA extracted 1-hr post load and assessed by RT-qPCR and RNAseq analysis (n=5/treatment). Cell shape and phenotype were assessed by immunocytochemistry and phalloidin staining. Data was analysed by Minitab.Abstract
Objectives
Methods
The AMPA/kainate glutamate receptor (GluR) antagonist NBQX reduced bone destruction when injected intra-articularly, in rat antigen induced arthritis (AIA) and is similarly protective in rodent models of osteoarthritis. NBQX reduced bone turnover
Osteoarthritis (OA), characterised by pain, disability and joint degeneration, is common and has no cure. Prevalence of severe radiographic knee OA is 19% in over 45's and 50% in over 75's in the US and Europe. Abnormal joint loading, or injury, increase risk of OA. We have discovered that glutamatergic signalling is mechanically regulated and glutamate receptors (GluR) drive inflammation, degeneration and pain representing potential drug targets in osteoarthritic joints. Joints from OA and knee injured patients, and rodent models of arthritis, show increased synovial fluid glutamate concentrations and abundant GluR expression. Since AMPA/kainate GluRs regulate IL-6, a critical mediator of arthritic degeneration, we tested protective effects of the AMPA/KA GluR antagonist, NBQX in animal models of arthritis. In rodent antigen induced arthritis, and osteoarthritis (meniscal transection and anterior cruciate ligament rupture), NBQX reduced joint swelling, degeneration and pain, exceeding anti-degenerative effects of other drugs tested similarly. 3D osteocyte/osteoblast co-cultures and human bone samples taken from patients undergoing high tibial osteotomy joint realignment surgery, revealed underlying cellular mechanisms mediated by bone cells. Related drugs, already used in humans for epilepsy and migraine, represent a repurposing opportunity and are effective in our models of arthritis.
Total Knee Arthroplasty (TKA) aims to deliver relief from pain and restore normal function. Unfortunately, a significant cohort of patients report poor outcomes. Synovial fluid metabolite concentrations at surgery predict outcome of TKA, assessed by a validated measure.Introduction
Hypothesis
High tibial Osteotomy (HTO) realigns the forces in the knee to slow the progression of osteoarthritis. This study relates the changes in knee joint biomechanics during level gait to glutamate signalling in the subchondral bone of patients pre and post HTO. Glutamate transmits mechanical signals in bone and activates glutamate receptors to influence inflammation, degeneration and nociception in arthritic joints. Thus glutamate signalling is a mechanism whereby mechanical load can directly modulate joint pathology and pain. 3D motion analysis was used to assess level gait prior to HTO (n=5) and postoperatively (n=2). A biomechanical model of each subject was created in Visual3D (C-motion. Inc) and used for biomechanical analysis. Gene expression was analysed by RT-PCR from bone cores from anterior and posterior drill holes, subdivided according to medial or lateral proximal tibia from HTO patients (n=5).BACKGROUND
METHODS
To determine whether cycles of pivot shift testing prior to anterior cruciate ligament (ACL) reconstruction alters metabolite levels in synovial fluid. Testing for pivot shift is a standard aspect of the EUA prior to an ACL reconstruction. Teaching 2 trainees to perform the pivot test will result in the knee being pivoted 5 times. All cases were isolated ACL deficiency, without meniscal or chondral damage (n=3). Each knee had synovial fluid extracted under aseptic conditions following anaesthesia. The pivot shift test was then performed and demonstrated 5 times. After preparation of the knee for surgery, a second synovial fluid sample was extracted. The time between samples was 5 minutes. Synovial fluids were analysed using 500 MHz 1H NMR spectroscopy. Chemical shifts were referenced to known concentration NMR internal standard (TSP), peaks identified and peak integrals measured using the Bruker software Topspin 2.0.Purpose of study
Method
Mechanical loading is a potent stimulator of bone formation. A screen for genes associated with mechanically-induced osteogenesis implicated the glutamate transporter GLAST-1 (1), in the mechanoresponse. We are investigating whether modulation of glutamate transporters represents a potential anabolic therapy in bone. Bone cells express functional components from each stage of the glutamate signalling pathway and activation of ionotropic glutamate receptors on osteoblasts can increase bone forming activity (2). Five high affinity Na+-dependant excitatory amino acid transporters (EAATs 1-5) regulate glutamatergic signalling. EAAT1 (GLAST-1) is expressed by osteocytes and bone-forming osteoblasts in vivo. We quantified transcripts for EAATs 1-3 and two splice variants (EAAT1a and EAAT1ex9skip) in human osteoblasts (MG63, SaOS-2 and primary) using real time-PCR. EAAT1a expression was very low whilst levels of the dominant negative EAAT1ex9skip were much higher in all cell types. EAAT1 and EAAT3 proteins were detected by immunofluorescence. We also demonstrated that glutamate transporters function in human osteoblasts. Sodium-dependent 14C-labelled glutamate uptake, sensitive to pharmacological EAAT inhibitors (t-PDC, TBOA) and extracellular glutamate concentration (10-500μM) was detected in MG63 and SaOS-2 cells. To determine whether modulation of EAATs can influence bone formation, we used pharmacological inhibitors of EAATs 1-5 (t-PDC and TBOA) and also over-expressed EAAT1exon9skip using antisense oligonucleotides (AONs) targeted to splice donor sequence of exon 9. Experiments were performed in 0-500μM glutamate. Pharmacological inhibition of EAATs over 5-21 days increased alkaline phosphatase activity and mineralisation of SaOS-2 cells and human primary osteoblasts. Over-expression of EAAT1ex9skip significantly increased cell number and decreased cell death as well as significantly increasing PCNA, Osteonectin and Type I collagen mRNAs in MG63 cells. Furthermore, over-expression of EAAT1ex9skip increased mean alkaline phosphatase activity over 48hrs in SaOS-2 cells. These data show that EAATs are expressed and functional in osteoblasts and that pharmaceutical and genetic inhibition of their activity increases bone formation. These mechanically regulated glutamate transporters are important in regulating bone homeostasis and their manipulation may represent a new anabolic therapy for the treatment of disorders such as osteoporosis or non-union fractures.
is the most common arthritic condition. OA causes joint pain, loss of mobility and significantly affects the quality of life for the affected individual. The major burden to patients with arthritis is pain. However, often radiological joint destruction and the extent of pain do not correlate. This causes a dilemma for clinicians in advising timing for joint replacement surgery. In arthritis, concentrations of the neurotransmitter, glutamate is increased within the synovial fluid activating both peripheral pain mechanisms and pathological processes (1). Other pathological/pain related metabolites are also released into synovial fluid, which provides a real time snap shot of the joint pathology. We have tested the hypothesis that ‘The increased levels of pain and disease-related metabolites within human synovial fluids from arthritic joints can be detected and quantified ex vivo using high resolution 1H-NMR.’ OA synovial fluid samples were obtained during arthroscopy or total knee replacements from patients with varying degrees of pain and pathology (cartilage graded 0-4; n=21). Pain perception was determined using the Oxford knee score and samples sub-classified as mild, moderate and severe pain. All samples were analysed using 500 MHz 1H NMR spectroscopy. Chemical shifts were referenced to a known concentration NMR internal standard (TSP), peaks identified by reference to published synovial fluid NMR spectra (2) and peak integrals measured using the Bruker software Topspin 2.0. Results: Using NMR we were able to detect around 26 metabolite-specific peaks in synovial fluid spectra (such as glutamate/glutamine, isoleucine, acetyl glucoproteins, beta-hydroxbutyrate, CH2 lipids, lactate, glucose). Some specific metabolites varied significantly with pain or pathological score. For example, we found significantly more glutamate/glutamine, isoleucine and beta-hydroxybutyrate (p<0.05, T test) in OA samples reporting mild to moderate levels of pain (n=14) compared to severe pain (n=7). Significantly more CH2 lipids (p<0.05, T-test) were also present in samples indicating severe pain compared to mild/moderate pain.Osteoarthritis (OA)
Method
Meniscal tears commonly occur after a traumatic twisting injury to the knee (acute) or can form over time (degenerate). Symptoms include pain, swelling, and ‘locking’ of the knee. These symptoms are also commonly associated with osteoarthritis (OA). In some cases of OA, degenerative meniscal tears can also be present making it difficult to determine the cause of symptoms. Furthermore, acute meniscal lesions may be associated with early stage OA but often no radiological signs are evident. Many metabolites associated with joint disorders are released into the synovial fluid providing a real-time snap shot of joint pathology. The ability to examine concentrations of specific metabolites within synovial fluid could provide invaluable clinical information about the cause and stage of joint pathology. We have tested the hypothesis that ‘high resolution 1H-NMR can discriminate between osteoarthritic and meniscal tear-related metabolites within human synovial fluids and aid in clinical diagnosis.’ Synovial fluid samples have been obtained during arthroscopy or knee replacement from patients with varying degrees of joint pathology (cartilage graded 0-4; meniscal tears classified as acute or degenerative). Samples were also taken from patients undergoing Anterior Cruciate Ligament (ACL) reconstruction with no additional pathology. Samples were analysed using 500 MHz 1H NMR spectroscopy. Chemical shifts were referenced to known concentration NMR internal standard (TSP), peaks identified by reference to published synovial fluid NMR spectra (1) and peak integrals measured using the Bruker software Topspin 2.0. Spectroscopy revealed a number of differences in metabolites between OA, meniscal tear and ACL pathologies. These included significantly increased concentrations of glutamate, n-acetyl glycoprotein and β-hydroxybutyrate in OA (n=10) and acute meniscal tears (n=6) compared to ACL samples (p<0.05, T-test, n=6). Specific metabolites were also able to discriminate between OA with no meniscal tear and OA with meniscal tear synovial fluids. For example, concentrations of n-acetyl glycoproteins, glutamate and CH3 lipids were significantly increased in OA without tears (n=10) compared to OA plus meniscal tears (n=12); conversely ceramide concentrations were significantly increased in OA plus tears compared to OA only samples (p<0.05, T-test).Method
Results
Rheumatoid arthritis (RA) is a systemic autoimmune disease affecting 350,000 people in the UK. Within synovial joints, synoviocytes form a destructive pannus that degrades articular cartilage and bone. Synovial fluid glutamate levels increase 54 fold in RA patients and are also elevated in animal models of inflammatory and osteoarthritis. To determine whether elevated glutamate levels contribute to RA pathology we investigated which synovial joint tissues express glutamate receptors and whether glutamate stimulation influences synovio-cyte phenotype. Various glutamate receptor mRNAs (NMDAR1, KA1, AMPAGluR2, AMPA GluR3, mGluR4) were expressed in tissues of the rat knee. All receptors were expressed in the patella. The fibrocartilagenous menis-cus and articular cartilage chondrocytes expressed mGluR4 and both AMPA receptor subunits. Human synoviocytes expressed NMDAR1 and KA1 mRNA. To determine whether glutamate receptors were functional in human synoviocytes, cells were preloaded with a fluorescent indicator of intracellular calcium (iCa 2+) and stimulated with glutamate or specific agonists (NMDA or kainate, 500mM). Glutamate stimulated release of iCa2+ in 25% of synoviocytes whereas NMDA and Kainate each stimulated 15% of cells. NMDA responses increased to 57% in the absence of Mg2+ consistent with the inhibitory effect of Mg2+ on this receptor. To determine whether activation of glutamate receptors can influence human synoviocyte phenotype, we cultured synoviocytes in various glutamate concentrations (50mM to 2mM) and measured effects of glutamate receptor antagonists on release of a proinflammatory cytokine (IL-6) and degradative enzymes (MMP2 and 9). In some RA patients, glutamate stimulation increased synoviocyte pro MMP-2 release. TIMP1 and TIMP2 release were not affected by glutamate stimulation or co-treatment with receptor antagonists. IL-6 expression varied greatly in human synoviocytes derived from different RA patients (0–120pg/ml media). However, the AMPA/KA receptor antagonist NBQX significantly reduced IL-6 release at all glutamate concentrations. This inhibition was greater than that by CFM2 (AMPAR antagonist), indicating that activation of kainate receptors in human synoviocytes may induce IL-6 release. We conclude that glutamate receptors are functional in human synoviocytes and regulate release of MMP-2 and IL-6 Thus glutamatergic signalling may contribute to RA pathology and represent a new therapeutic target.
