Aseptic loosening is a major cause of revision surgeries and occurs when osteolysis is stimulated around the implant by pro-inflammatory cytokines including IL-1β. Production of active IL-1β in response to orthopedic wear particles depends on processing by the NLRP3 inflammasome which requires priming followed by activation. We found that pathogen associated molecular patterns (PAMPs) adherent to wear particles are necessary to prime the NLRP3 inflammasome. In contrast, in pre-primed macrophages, particles themselves are sufficient to activate the NLRP3 inflammasome and induce secretion of active IL-1β. Particles themselves also induce cell death, kinetically preceding the release of active IL-1β. Phagocytosis of particles is required to initiate both responses as the phagocytosis inhibitor cytochalasin blocks cell death and IL-1β release. Lysosome membrane destabilization is also critical as inhibition of lysosomal function with bafilomycin or chloroquine significantly abrogated the release of active IL-1β and cell death in response to wear particles. The pan-cathepsin inhibitors Ca-074-Me or K777 also inhibit cell death and IL-1β release indicating that cathepsin release from lysosomes is also a necessary step in the particle-induced response. Our results open the possibility of clinical intervention with lysosomal or cathepsin inhibitors to treat aseptic loosening as these drugs have better specificity and less in vivo toxicity than the phagocytosis inhibitors. Testing of these inhibitors in vivo in models of particle induced osteolysis is a key future direction.

Unresolved inflammatory processes in tendon healing have been related to the progression of tendinopathies. Thus, the management of tendon injuries may rely on cell-based strategies to identify and modulate tendon inflammatory cues. Pulsed electromagnetic field (PEMF) has been approved by FDA for orthopedics therapies and has been related to a reduction in pain and to improve healing. However, the influence of PEMF in tendon healing remains largely unknown. Human tendon resident cells (hTDCs) were cultured in an inflammatory environment induced by exogenous supplementation of IL-1β and their response assessed after exposure to different PEMF treatments. This study demonstrates that IL-1β induced up-regulation of pro-inflammatory factors (IL-6 and TNFα) and extracellular matrix components (MMP−1, −2, −3) whereas reduces the expression of TIMP-1, suggesting IL-1β as a candidate inflammation model to study hTDCs response to inflammation cues. Moreover, in both homeostatic and inflammatory environments, hTDCs respond differently to PEMF treatment suggesting that cells are sensitive to magnetic field parameters such as strength (1.5 – 5mT), frequency (5–17Hz) and duration (10–50% duty cycle, dc). Among the conditions studied, PEMF treatment with 4mT/5Hz/50%dc suppresses the inflammatory response of hTDCs to the IL-1β stimulation, as evidenced by the decreases amount of IL-6, TNFα and downregulation of MMP-1, −2, −3 and COX-2, IL-8, IL-6, TNFα genes. These results demonstrate the potential of PEMF, in particular 4mT/5Hz/50%dc PEMF in treating tendon inflammation suppressing the inflammatory stimulation induced by IL-1β, which may be beneficial for tendon healing strategies.

The biological understanding for the disease progression osteoarthritis (OA) has uncovered specific biomarkers from either synovial fluid, articular chondrocytes or synoviocytes that can be used to diagnose the disease. Examples of these biomarkers include interleukin-1β (IL-1β) or collagen II fragments (1, 2). In parallel, isolation of chondrocytes or bone marrow derived mesenchymal stromal cells (MSCs) has yielded cell-based strategies that have shown long- term beneficial effects in a specific cohort of patients, specifically in traumatic cartilage lesions (2). This latter finding shows that patient stratification of OA is an important tool to both match patients for a specific treatment and to develop novel therapies, especially disease modifying drugs. In order to create disease stage specific therapies, the use of next generation analysis tools such as RNAseq and metabolomics, has the potential to decipher specific cellular and molecular endotypes. Alongside greater understanding of the clinical phenotype (e.g. imaging, pain, co- morbidities), therapies can be designed to alleviate the symptoms of OA at specific points of the disease in patients. This talk will outline the current biological understanding of OA and discuss how patient stratification could assist in the design of innovative therapies for the disease. Acknowledgements: This presentation was supported by the COST action, CA21110 – Building an open European Network on Osteoarthritis Research (NetwOArk)

Introduction:. Exercise has showed to reduce pain and improve function in patients with discogenic low back pain (LBP). Although there is currently no biologic evidence that the intervertebral disc (IVD) can respond to physical exercise in humans, a recent study has shown that chronic running exercise is associated with increased IVD hydration and hypertrophy1. Irisin, a myokine released upon muscle contraction, has demonstrated to yield anabolic effects on different cell types, including chondrocytes2. This study aimed to investigate the effect of irisin on human nucleus pulposus cells (hNPCs). Our hypothesis is that irisin may improve hNPCs metabolism and proliferation. METHODS:. The hNPCs, isolated from discectomy surgical waste material (n = 5), were expanded and encapsulated in alginate beads. The hNPCs were treated with: i) only growth medium (control); ii) medium with recombinant irisin (r-IR) at different concentrations (5, 10 and 25 ng / mL); iii) medium with Interleukin-1β (IL1β); iv) medium with IL1β for 24 h and then with IL1β and r-IR; v) medium with r-IR for 24 h and then with r-IR and IL1 β. We evaluated proliferation (trypan blue and PicoGreen), metabolic activity (MTT), nitrite concentration (Griess), and expression levels of catabolic and anabolic genes via real-time polymerase chain reaction (qPCR). Each analysis was performed in triplicate for each donor and each experiment was performed three times. Data were expressed as mean ± S.D. One-way ANOVA was used for the groups under exam. RESULTS:. Irisin increased hNPCs proliferation (p < 0.001), metabolic activity at 10 ng/mL (p < 0.05), and GAG content at concentration of 10 ng/mL and 25 ng/mL (p < 0.01; p < 0.001, respectively). The production of nitrites, used as an indicator of cellular oxidative stress, was significantly decreased (p < 0.01). Gene expression levels compared to the control group increased for COL2A1 (p < 0.01), ACAN (p < 0.05), TIMP-1 and −3 (p < 0.01), while a decrease in mRNA levels of MMP-13 (p < 0.05) and IL1β (p < 0.001) was noticed. r-IR pretreatment of hNPCs cultured in pro-inflammatory conditions resulted in a rescue of metabolic activity (p < 0.001), as well as a decrease of IL-1β (p < 0.05) levels. Similarly, incubation of hNPCs with IL-1β and subsequent exposure to r-IR led to an increment of hNPC metabolic activity (p < 0.001), COL2A1 gene expression (p < 0.05) and a reduction of IL-1β (p < 0.05) and ADAMTS-5 gene levels (p < 0.01). CONCLUSIONS:. The present study suggested that irisin may stimulate hNPCs proliferation, metabolic activity, and anabolism by reducing the expression of IL-1β and catabolic enzymes while promoting the synthesis of extracellular matrix components. Furthermore, this myokine was able to blunt the catabolic effect of in vitro inflammation. Our results indicate that irisin may be one of the mediators by which physical exercise and muscle tissues modulate IVD metabolism, thus suggesting the existence of a biological cross-talk mechanism between the muscle and the IVD

Objectives. Recently, high failure rates of metal-on-metal (MOM) hip implants have raised concerns of cobalt toxicity. Adverse reactions occur to cobalt nanoparticles (CoNPs) and cobalt ions (Co. 2+. ) during wear of MOM hip implants, but the toxic mechanism is not clear. Methods. To evaluate the protective effect of zinc ions (Zn. 2+. ), Balb/3T3 mouse fibroblast cells were pretreated with 50 μM Zn. 2+. for four hours. The cells were then exposed to different concentrations of CoNPs and Co. 2+. for four hours, 24 hours and 48 hours. The cell viabilities, reactive oxygen species (ROS) levels, and inflammatory cytokines were measured. Results. CoNPs and Co. 2+. can induce the increase of ROS and inflammatory cytokines, such as tumour necrosis factor α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6). However, Zn pretreatment can significantly prevent cytotoxicity induced by CoNPs and Co. 2+. , decrease ROS production, and decrease levels of inflammatory cytokines in Balb/3T3 mouse fibroblast cells. Conclusion. These results suggest that Zn pretreatment can provide protection against inflammation and cytotoxicity induced by CoNPs and Co. 2+. in Balb/3T3 cells. Cite this article: Y. Liu, H. Zhu, H. Hong, W. Wang, F. Liu. Can zinc protect cells from the cytotoxic effects of cobalt ions and nanoparticles derived from metal-on-metal joint arthroplasties? Bone Joint Res 2017;6:649–655. DOI: 10.1302/2046-3758.612.BJR-2016-0137.R2

Although 80% of fractures typically heal without any problems, there is a small proportion (<20%) that suffer complications such as delayed healing and potential progression to non-union. In patients with healing complications, the coordinated regulation between pro- and anti-inflammatory cytokines, such as interleukin-1β (IL-1β) and interleukin-1 receptor antagonist (IL-1Ra) respectively, is often dysregulated. The aim of this study is to develop a therapeutic strategy based on the local delivery of genes to reparative mesenchymal stromal cells (MSCs) migrating into the local fracture microenvironment, thereby promoting a more favourable healing environment to enhance fracture repair. Our approach involves the local delivery of nanoparticles complexing the non-viral vector polyethyleneimine (PEI) with therapeutic plasmid DNA (pDNA) encoding for IL-1Ra. pDNA encoding green fluorescent protein and Gaussia luciferase were used as reporter genes to determine the transfection efficiency of both rat and human MSCs using flow cytometry and to assess the transgene expression profile using a luciferase expression assay. The effect of transfection with PEI on the viability of MSCs was assessed using the metabolic assay Cell Titer Blue and dsDNA quantification. Levels of IL-1Ra produced by cells following transfection with nanoparticles encoding IL-1Ra was assessed using enzyme-linked immunosorbent assays (ELISA). HEK-Blue IL-1β reporter cells, which secrete alkaline phosphatase in response to IL-1β stimulation, were used to confirm that the IL-1Ra produced by transfected cells is functionally active, i.e. the successful antagonism of IL-1β bioactivity. We have determined that using PEI-based nanoparticles we can achieve a transfection efficiency of 14.8 + 1.8% in rat MSCs. Transgene expression was found to be transient, with a peak in expression at 7 days post-transfection and a gradual decrease over time, which was maintained for up to 4 weeks. Using an optimized concentration of PEI, the impact of the nanoparticles on MSC viability was limited, with no significant difference in cellular metabolic activity compared to non-transfected cells at 10 days post-transfection. We have additionally demonstrated the capacity to successfully transfect both rat and human MSCs with pDNA encoding for IL-1Ra, resulting in enhanced levels of IL-1Ra, which is functionally active. The use of non-viral gene therapy to locally deliver immunomodulatory genes, such as IL-1Ra, to MSCs presents a promising strategy to enhance bone healing. Specifically, the transgene expression levels achieved with such an approach can remain therapeutically effective and are transient in nature, presenting an advantage over other methods such as recombinant protein delivery and viral-based gene delivery methodologies

Objective. Excessive mechanical stress on synovial joints causes osteoarthritis (OA) and results in the production of prostaglandin E2 (PGE2), a key molecule in arthritis, by synovial fibroblasts. However, the relationship between arthritis-related molecules and mechanical stress is still unclear. The purpose of this study was to examine the synovial fibroblast response to cyclic mechanical stress using an in vitro osteoarthritis model. Method. Human synovial fibroblasts were cultured on collagen scaffolds to produce three-dimensional constructs. A cyclic compressive loading of 40 kPa at 0.5 Hz was applied to the constructs, with or without the administration of a cyclooxygenase-2 (COX-2) selective inhibitor or dexamethasone, and then the concentrations of PGE2, interleukin-1β (IL-1β), tumour necrosis factor-α (TNF-α), IL-6, IL-8 and COX-2 were measured. Results. The concentrations of PGE2, IL-6 and IL-8 in the loaded samples were significantly higher than those of unloaded samples; however, the concentrations of IL-1β and TNF-α were the same as the unloaded samples. After the administration of a COX-2 selective inhibitor, the increased concentration of PGE2 by cyclic compressive loading was impeded, but the concentrations of IL-6 and IL-8 remained high. With dexamethasone, upregulation of PGE2, IL-6 and IL-8 was suppressed. Conclusion. These results could be useful in revealing the molecular mechanism of mechanical stress in vivo for a better understanding of the pathology and therapy of OA. Cite this article: Bone Joint Res 2014;3:280–8

Osteoarthritis is a degenerative disease mainly caused by aging, although in younger patients (aged 25 – 50) it can be a consequence of sports-related injuries or trauma. This results in early osteoarthritis with subsequent changes in cartilage extracellular matrix. Cell-based tissue engineering approaches using mesenchymal stem cells (MSCs) are an ideal cell type for the treatment of early osteoarthritc defects. Our group has demonstrated in a clinical study, that interleukin-1β (IL-1β) was expressed in cartilage plugs from patients with early osteoarthritis. In vitro studies have shown that IL-1β inhibits cartilage formation in chondrocytes or MSCs undergoing chondrogenesis. However, these studies show complete inhibition of tissue formation, whereas in the context of early osteoarthritis, cartilage extracellular matrix remains around the defect site. Thus, the present study sought to develop a model mimicking early osteoarthritis using MSCs. Method. Human MSCs (Male donors; aged 18–60 years, n = 6) were isolated from bone marrow and expanded in culture for one passage. 2 × 10. 5. MSCs were aliquoted into wells of a 96-well cell culture plate in the presence of 10ng/ml TGF-β. 1. or in combination with IL-1β administered at a range of concentrations (0.1, 0.5, 1 and 10ng/ml) and centrifuged to form pellets. Pellets were removed from culture on days 7, 14 and 21. Pellets were evaluated for wet weight, pellet area, histological (DMMB staining, collagen type I, II, MMP-13 and TGF-β receptor II) and collagen type II ELISA analysis. Results. Chondrogenic pellets in the presence of IL-1β demonstrated a dose-dependant inhibition in chondrogenesis. Concentrations equal or greater than 0.5ng/ml IL-1β showed significant reduction (p < 0.05) in pellet area and wet weight, with no positive staining for collagen type I, II (including ELISA analysis) and DMMB. However, at 0.1ng/ml IL-1β, despite a slight reduction in pellet area, positive staining for collagen type I, II and DMMB was observed. Furthermore, MMP-13 matrix staining was increased and TGF-b receptor II staining was decreased in pellets at IL-1β concentrations above 0.5ng/ml. Discussion. A dose dependant catabolic response in cartilage extracellular matrix formation was demonstrated for IL-1β treated MSCs undergoing chondrogenesis. At concentrations equal or greater than 0.5ng/ml IL-1β, MMP13 expression was observed in the matrix, indicative of osteoarthritis. Furthermore, there was reduced expression of TGF-β receptor II under these conditions that is required for TGF-b induced chondrogenesis. However, at 0.1ng/ml IL-1β, a reduced catabolic response in extracellular matrix components was observed, whilst showing a moderate expression in MMP-13 and the presence of cellular TGF-β receptor II expression. Therefore, this latter model may be used to develop pro-chondrogenic strategies for the treatment of early osteoarthritic defects

Sterilisation by gamma irradiation in the presence of air causes free radicals generated in polyethylene (PE) to react with oxygen, which could lead to loss of physical properties and reduction in fatigue strength. Tissue retrieved from failed total hip replacements often has large quantities of particulate PE and most particles associated with peri-implant osteolysis are oxidised. Consequently, an understanding of the cellular responses of oxidised PE particles may lead to clarification of the pathogenesis of osteolysis and aseptic loosening. We have used the agarose system to demonstrate the differential effects of oxidised and non-oxidised PE particles on the release of proinflammatory products such as interleukin-1β (IL-1β), IL-6, and tumour necrosis factor-α (TNF-α) from monocytes/ macrophages (M/M). Oxidised PE particles were shown to stimulate human M/M to phagocytose and to release cytokines. Oxidation may alter the surface chemistry of the particles and enhance the response to specific membrane receptors on macrophages, such as scavenger-type receptors

Aseptic loosening and osteolysis around prosthetic joints are the principal causes of failure and consequent revision. During this process activated macrophages produce cytokines which are thought to promote osteolysis by osteoclasts. Changes in pressure within the space around implants have been proposed as a cause of loosening and osteolysis. We therefore studied the effect of two different regimes of cyclic pressure on the production of interleukin-1β (IL-1β), IL-6 and tumour necrosis factor-α (TNF-α) by cultured human monocyte-derived (M-D) macrophages. There was a wide variation in the expression of cytokines in non-stimulated M-D macrophages from different donors and therefore cells from the same donor were compared under control and pressurised conditions. Both regimes of cyclic pressure were found to increase expression of IL-6 and TNF-α. Expression of IL-1β was increased by a higher-frequency regime only. Our findings suggest that M-D macrophages are activated by cyclic pressure. Further work will be required to understand the relative roles of frequency, amplitude and duration of applied pressure in the cellular effects of cyclic pressure in this system

Aims

This study intended to investigate the effect of vericiguat (VIT) on titanium rod osseointegration in aged rats with iron overload, and also explore the role of VIT in osteoblast and osteoclast differentiation.

Introduction. Osteoarthritis (OA) involves pathological change in all joint tissues, including cartilage degradation and synovitis. Synovial inflammation is significantly associated with pain severity and incidence in knee OA. It is becoming evident that synovitis also plays an active role in the initiation and progression of cartilage erosion in OA, through direct secretion of catabolic enzymes as well as factors that stimulate chondrocyte catabolic activity. Therapeutic agents that target both synovitis and cartilage pathology are likely to be maximally beneficial in treating pain and slowing cartilage breakdown in OA. We have previously shown that an amide-derivative of HA (HYMOVIS™) was superior to native HA of the same MW in improving gait, and reducing synovial hyperplasia in a sheep OA model. In the present study the mechanisms whereby the chemically modified HA may be beneficial were examined using chondrocytes and synovial fibroblasts from knees of OA patients. Patients & Methods. Chondrocytes (HAC, n=6) and synovial fibroblasts (HSF, n=6) were isolated from OA patients at the time of knee replacement. HYMOVIS™ (0, 0.5, 1.0 or 1.5mg/mL) was added to simultaneously or 1 hour before interleukin-1β (IL1, 2ng/mL). Cultures were terminated 30 minutes later for Bioplex. ®. quantitation of p-JNK, p-NFκB and p-p38; or 24 hours later for RNA isolation and analysis of gene expression by real time RT-PCR, and measurement of MMP13 activity in the media. Only statistically significant results are reported. Results. In HAC in the absence of IL1, HYMOVIS™ decreased MMP13, ADAMTS5, PTGS2 and IL6 and increased COL2A1 mRNA (2–10fold). In HSF in absence of IL1, HYMOVIS™ decreased TIMP1, TIMP3, CD44, IL6 and increased PTGS2 (2–3fold). In HAC and HSF, IL1 increased expression of MMP1, MMP13, PTGS2, IL6 (>100fold), ADAMTS4 (∼10 fold), all phosphoproteins (3–10fold), and APMA-activated MMP13 activity in media. IL1 increased expression of ADAMTS5 (∼10fold) only in HSF. As expected, IL1 reduced expression of the key matrix proteins in HAC (2–3 fold decrease in COL2A1 and ACAN) and HSF (2 fold decrease in COL1A1). When added simultaneously with IL1, HYMOVIS™ decreased expression of MMP13, ADAMTS5, PTGS2, IL6 expression, and normalised matrix protein expression in both HAC and HAS. Pre-incubation with HYMOVIS™ for 1 hour inhibited IL1-stimulated p-JNK, p-NFκB and p-p38 in both cell types (excluding p-JNK in HSF). In HAC, HYMOVIS™ pre-incubation was superior to simultaneous addition in reducing expression of MMP1, MMP13, ADAMTS4, PTGS2, and IL6 expression. There was a less dramatic effect of HYMOVIS™ pre-incubation on gene expression in HSF compared with HAC. The inhibitory effects of HYMOVIS™ on IL1 stimulated gene expression in HAC and HSF was partially ameliorated by pre-incubation with a CD-44 blocking antibody. Discussion/Conclusions. The present studies have demonstrated several potential key mechanisms whereby the intra-articular injection of a hexadecylamide-derivative of HA (HYMOVIS™) may have both symptom and disease-modifying effects in OA. The previously described increased joint retention of the hexadecylamide-derivative, might act in a similar manner to the pre-incubation studies in our cell culture studies, to reduce the initiation of degradative events with recurrent/cyclic inflammatory episodes that typify OA

Objectives

This study was conducted to evaluate the cytokine-release kinetics of platelet-rich plasma (PRP) according to different activation protocols.

Osteoarthritis (OA) is an important cause of pain, disability and economic loss in humans, and is similarly important in the horse. Recent knowledge on post-traumatic OA has suggested opportunities for early intervention, but it is difficult to identify the appropriate time of these interventions. The horse provides two useful mechanisms to answer these questions: 1) extensive experience with clinical OA in horses; and 2) use of a consistently predictable model of OA that can help study early pathobiological events, define targets for therapeutic intervention and then test these putative therapies. This paper summarises the syndromes of clinical OA in horses including pathogenesis, diagnosis and treatment, and details controlled studies of various treatment options using an equine model of clinical OA.

Several experimental models have been used to produce intravascular fat embolism. We have developed a simple technique to induce fat embolism using corn oil emulsified with distilled water to form fatty micelles. Fat embolism was produced by intravenous administration of these fatty micelles in anaesthetised rats, causing alveolar oedema, haemorrhage and increased lung weight.

Histopathological examination revealed fatty droplets and fibrin thrombi in the lung, kidney and brain. The arteriolar lumen was filled with fatty deposits. Following fat embolism, hypoxia and hypercapnia occurred. The plasma phospholipase A₂, nitrate/nitrite, methylguidanidine and proinflammatory cytokines were significantly increased. Mass spectrometry showed that the main ingredient of corn oil was oleic acid.

This simple technique may be applied as a new animal model for the investigation of the mechanisms involved in the fat embolism syndrome.