Aims. Osteoarthritis (OA) is the most common chronic pathema of human joints. The pathogenesis is complex, involving physiological and mechanical factors. In previous studies, we found that ferroptosis is intimately related to OA, while the role of Sat1 in chondrocyte ferroptosis and OA, as well as the underlying mechanism, remains unclear. Methods. In this study, interleukin-1β (IL-1β) was used to simulate inflammation and Erastin was used to simulate ferroptosis in vitro. We used small interfering RNA (siRNA) to knock down the spermidine/spermine N1-acetyltransferase 1 (Sat1) and arachidonate 15-lipoxygenase (Alox15), and examined damage-associated events including inflammation, ferroptosis, and oxidative stress of chondrocytes. In addition, a destabilization of the medial meniscus (DMM) mouse model of OA induced by surgery was established to investigate the role of Sat1 inhibition in OA progression. Results. The results showed that inhibition of Sat1 expression can reduce inflammation, ferroptosis changes, reactive oxygen species (ROS) level, and lipid-ROS accumulation induced by IL-1β and Erastin. Knockdown of Sat1 promotes nuclear factor-E2-related factor 2 (Nrf2) signalling. Additionally, knockdown Alox15 can alleviate the inflammation-related protein expression induced by IL-1β and ferroptosis-related protein expression induced by Erastin. Furthermore, knockdown Nrf2 can reverse these protein expression alterations. Finally, intra-articular injection of diminazene aceturate (DA), an inhibitor of Sat1, enhanced type II collagen (collagen II) and increased Sat1 and Alox15 expression. Conclusion. Our results demonstrate that inhibition of Sat1 could alleviate chondrocyte ferroptosis and inflammation by downregulating Alox15 activating the Nrf2 system, and delaying the progression of OA. These findings suggest that Sat1 provides a new approach for studying and treating OA. Cite this article: Bone Joint Res 2024;13(3):110–123

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.

Purpose: The inflammatory response around herniated tissue in the epidural space is believed to play a major role in the spontaneous regression of herniated lumbar disc. Numerous macrophages invade the herniated tissue along with newly formed blood vessels which influence oxygen gradient. Inflammatory cytokines such as interleukin-1 are produced by macrophages. These chemical mediators could stimulate disc cells to produce proteases such as MMPs which degrade the intervertebral disc matrix and could hence influence regression of the herniation. Here we have examined the influence of IL-1β and oxygen tension on proteoglycan turnover using a three-dimensional disc-cell culture system.

Methods: Cells were isolated from the nucleus pulposus of 18–24 month bovine caudal discs by enzyme digestion. They were initially cultured for 14 days in alginate beads in DMEM containing 6% FBS at 4.10⁶ cells/ml under 21% oxygen to accumulate matrix. They were then cultured for 6 days under 0% or 21% oxygen and with or without IL-1β. Glycosaminoglycan (GAG) accumulation (as a measure of proteoglycan content) was measured using a DMB assay. Lactate and glucose production were measured using a standard enzymatic method. Rates of sulfated GAG synthesis was measured from rates of ³⁵S-sulfate accumulation. MMP activity was measured using coumarin fluorescent assay.

Results: The results showed that IL-1β had a significant effect on GAG accumulation and production and that its effect was dependent on oxygen tension. GAG production and sulfate incorporation rates decreased in the presence of IL-1β at high oxygen but low oxygen inhibited the effects of this cytokine. MMP activity increased with IL-1β under 21% oxygen, but not at low oxygen.

Conclusion: Exogenous IL-1β can activate MMP activity and digest the extracellular matrix of the disc but only at high oxygen tensions. Angiogenesis as well as inflammation is thus required for resorption of herniations.

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

Aims

Osteoarthritis (OA) is a prevalent joint disorder with inflammatory response and cartilage deterioration as its main features. Dihydrocaffeic acid (DHCA), a bioactive component extracted from natural plant (gynura bicolor), has demonstrated anti-inflammatory properties in various diseases. We aimed to explore the chondroprotective effect of DHCA on OA and its potential mechanism.

Introduction. Within the intervertebral disc (IVD), nucleus pulposus (NP) cells reside within a unique microenvironment. Factors such as hypoxia, osmolality, pH and the presence of cytokines all dictate the function of NP cells and as such the cells must adapt to their environment to survive. Previously we have identified the expression of aquaporins (AQP) within human IVD tissue. AQPs allow the movement of water across the cell membrane and are important in cellular homeostasis. Here we investigated how AQP gene expression was regulated by the microenvironment of the IVD. Methods. Human NP cells were cultured in alginate beads prior to cytokine, osmolality, pH and hypoxia treatments and subsequent RT-qPCR to assess regulation of AQP gene expression. Results. Physiological conditions observed within the native IVD regulated AQP gene expression in human NP cells. Hyperosmotic treatment up-regulated the expression of AQP1 and 5 during hypoxic conditions, whereas AQP4 expression was down-regulated. During hypoxia and physiological pH treatments AQP5 expression was increased. Pro-inflammatory cytokines, increased during IVD degeneration, also altered AQP gene expression. Interleukin-1β (IL-1β) decreased expression of AQP1 and 3 yet up-regulated AQP9, interleukin-6 (IL-6) increased expression of AQP1, 3, and 9 and tumour necrosis factor α (TNFα) upregulated the gene expression of both AQP2 and 9. Conclusion. The microenvironment in which NP cells reside in vivo directly contributes to their correct function and survival. AQP gene expression was differentially regulated under healthy compared to degenerate conditions; this potentially highlights that during IVD degeneration NP cells differentially express AQPs. No conflicts of interest. Funded by BMRC, Sheffield Hallam University

Aims

Osteoarthritis (OA) is a common degenerative disease. PA28γ is a member of the 11S proteasome activator and is involved in the regulation of several important cellular processes, including cell proliferation, apoptosis, and inflammation. This study aimed to explore the role of PA28γ in the occurrence and development of OA and its potential mechanism.

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

Aims

cAMP response element binding protein (CREB1) is involved in the progression of osteoarthritis (OA). However, available findings about the role of CREB1 in OA are inconsistent. 666-15 is a potent and selective CREB1 inhibitor, but its role in OA is unclear. This study aimed to investigate the precise role of CREB1 in OA, and whether 666-15 exerts an anti-OA effect.

Aims: The purpose of this study is to examine the relationship between synovitis and shoulder pain in rotator cuff disease. Methods: Thirty-nine patients with rotator cuff disease were candidates. Subacromial synovium around the greater tuberosity and glnohu-meral synovium around the rotator interval were harvested for specimens during operation. The expression levels of inflammatory cytokine mRNA of interleukin-1β and its naturally occurring antagonists (secreted and intracellular interleukin-1 receptor antagonists) were measured by reverse transcription plolymerase chain reaction (RT-PCR). The cytokine-mRNAs producing cells were identified by RT- in situPCR. For control specimens, subacromial bursae were obtained from 10 patients with anterior instability of the shoulder that exhibited no signs of subacromial impingement. All specimens were obtained with patient’s informed consent. The level of shoulder pain was evaluated in each patients before the operation with a visual analogue scale: 0 as no pain, 5 as moderate, and 10 as severe. Results:The expression levels of the cytokine-mRNAs in the subacromial bursa well correlated to the level of shoulder pain, but not those in the glenohumeral joint. A variety of the cells (synovial and inflamatory cells) produced the cytokines at the protein and gene level in both subacromial and glenohuemeral synovium. Conclusions: Subacromial bursa is the site associated the shoulder pain caused by rotator cuff disease, and targeting the subacromial bursa for treatment leads to successful pain relief in patients with the disease

Bone regeneration and repair are crucial to ambulation and quality of life. Factors such as poor general health, serious medical comorbidities, chronic inflammation, and ageing can lead to delayed healing and nonunion of fractures, and persistent bone defects. Bioengineering strategies to heal bone often involve grafting of autologous bone marrow aspirate concentrate (BMAC) or mesenchymal stem cells (MSCs) with biocompatible scaffolds. While BMAC shows promise, variability in its efficacy exists due to discrepancies in MSC concentration and robustness, and immune cell composition. Understanding the mechanisms by which macrophages and lymphocytes – the main cellular components in BMAC – interact with MSCs could suggest novel strategies to enhance bone healing. Macrophages are polarized into pro-inflammatory (M1) or anti-inflammatory (M2) phenotypes, and influence cell metabolism and tissue regeneration via the secretion of cytokines and other factors. T cells, especially helper T1 (Th1) and Th17, promote inflammation and osteoclastogenesis, whereas Th2 and regulatory T (Treg) cells have anti-inflammatory pro-reconstructive effects, thereby supporting osteogenesis. Crosstalk among macrophages, T cells, and MSCs affects the bone microenvironment and regulates the local immune response. Manipulating the proportion and interactions of these cells presents an opportunity to alter the local regenerative capacity of bone, which potentially could enhance clinical outcomes.

Cite this article: Bone Joint Res 2024;13(9):462–473.

Aims

The present study aimed to investigate whether patients with inflammatory bowel disease (IBD) undergoing joint arthroplasty have a higher incidence of adverse outcomes than those without IBD.

Aims

The aim of this study was to determine the fracture haematoma (fxH) proteome after multiple trauma using label-free proteomics, comparing two different fracture treatment strategies.

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.

Aims

This study aimed, through bioinformatics analysis and in vitro experiment validation, to identify the key extracellular proteins of intervertebral disc degeneration (IDD).