Aims. This study aimed to explore whether serum combined with synovial interleukin-6 (IL-6) measurement can improve the accuracy of prosthetic joint infection (PJI) diagnosis, and to establish the cut-off values of IL-6 in serum and synovial fluid in detecting chronic PJI. Methods. Patients scheduled to have a revision surgery for indications of chronic infection of knee and hip arthroplasties or aseptic loosening of an implant were prospectively screened before being enrolled into this study. The Musculoskeletal Infection Society (MSIS) definition of PJI was used for the classification of cases as aseptic or infected. Serum CRP, ESR, IL-6, and percentage of polymorphonuclear neutrophils (PMN%) and IL-6 in synovial fluid were analyzed. Statistical tests were performed to compare these biomarkers in the two groups, and receiver operating characteristic (ROC) curves and area under the curve (AUC) were analyzed for each biomarker. Results. A total of 93 patients were enrolled. There was no difference in demographic data between both groups. Synovial fluid IL-6, with a threshold of 1,855.36 pg/ml, demonstrated a mean sensitivity of 94.59% (95% confidence interval (CI) 81.8% to 99.3%) and a mean specificity of 92.86% (95% CI 82.7 to 98.0) for detecting chronic PJI. Then 6.7 pg/ml was determined to be the optimal threshold value of serum IL-6 for the diagnosis of chronic PJI, with a mean sensitivity of 97.30% (95% CI 85.8% to 99.9%) and a mean specificity of 76.79% (95% CI 63.6% to 87.0%). The combination of synovial IL-6 and serum IL-6 led to improved accuracy of 96.77% in diagnosing chronic PJI. Conclusion. The present study identified that a combination of IL-6 in serum and synovial IL-6 has the potential for further improvement of the diagnosis of PJI. Cite this article: Bone Joint Res 2020;9(9):587–592

Aims. Insufficient treatment response in rheumatoid arthritis (RA) patients requires novel treatment strategies to halt disease progression. The potential benefit of combination of cytokine-inhibitors in RA is still unclear and needs further investigation. To explore the impact of combined deficiency of two major cytokines, namely interleukin (IL)-1 and IL-6, in this study double deficient mice for IL-1αβ and IL-6 were investigated in different tumour necrosis factor (TNF)-driven inflammatory bone disorders, namely peripheral arthritis and sacroiliitis, as well as systemic bone loss. Methods. Disease course, histopathological features of arthritis, and micro-CT (µCT) bone analysis of local and systemic bone loss were assessed in 15-week-old IL1-/-IL6-/-hTNFtg in comparison to IL1-/-hTNFtg, IL6-/-hTNFtg, and hTNFtg mice. µCT bone analysis of single deficient and wild-type mice was also performed. Results. Combined deficiency of IL-1/IL-6 markedly ameliorated TNF-mediated arthritis and bilateral sacroiliitis, but without additive benefits compared to single IL-1 deficiency. This finding confirms the important role of IL-1 and the marginal role of IL-6 in TNF-driven pathways of local joint damage, but questions the efficacy of potential combinatorial therapies of IL-1 and IL-6 in treatment of RA. In contrast, combined deficiency of IL-1/IL-6 led to an additive protective effect on TNF-driven systemic bone loss compared to single IL-1 and IL-6 deficiency. This finding clearly indicates a common contribution of both IL-1 and IL-6 in TNF-driven systemic bone loss, and points to a discrepancy of cytokine dependency in local and systemic TNF-driven mechanisms of inflammatory arthritis. Conclusion. Combinatorial treatments in RA might provide different benefits to inflammatory local arthritis and systemic comorbidities. Cite this article: Bone Joint Res 2022;11(7):484–493

Aims. In the repair of condylar cartilage injury, synovium-derived mesenchymal stem cells (SMSCs) migrate to an injured site and differentiate into cartilage. This study aimed to confirm that histone deacetylase (HDAC) inhibitors, which alleviate arthritis, can improve chondrogenesis inhibited by IL-1β, and to explore its mechanism. Methods. SMSCs were isolated from synovium specimens of patients undergoing temporomandibular joint (TMJ) surgery. Chondrogenic differentiation potential of SMSCs was evaluated in vitro in the control, IL-1β stimulation, and IL-1β stimulation with HDAC inhibitors groups. The effect of HDAC inhibitors on the synovium and condylar cartilage in a rat TMJ arthritis model was evaluated. Results. Interleukin (IL)-1β inhibited the chondrogenic differentiation potential of SMSCs, while the HDAC inhibitors, suberoylanilide hydroxamic acid (SAHA) and panobinostat (LBH589), attenuated inhibition of IL-1β-induced SMSC chondrogenesis. Additionally, SAHA attenuated the destruction of condylar cartilage in rat TMJ arthritis model. IL-6 (p < 0.001) and matrix metalloproteinase 13 (MMP13) (p = 0.006) were significantly upregulated after IL-1β stimulation, while SAHA and LBH589 attenuated IL-6 and MMP13 expression, which was upregulated by IL-1β in vitro. Silencing of IL-6 significantly downregulated MMP13 expression and attenuated IL-1β-induced chondrogenesis inhibition of SMSCs. Conclusion. HDAC inhibitors SAHA and LBH589 attenuated chondrogenesis inhibition of SMSC induced by IL-1β in TMJ, and inhibition of IL-6/MMP13 pathway activation contributes to this biological progress. This study provides a theoretical basis for the application of HDAC inhibitors in the treatment of TMJ arthritis. Cite this article: Bone Joint Res 2022;11(1):40–48

Purpose: The role of the pro-inflammatory cytokine HMGB1 (alarmins) has not been investigated in the clinical setting. This study aims to assess its relationship to IL-6 release, ISS, and to quantify the second hit phenomenon after femoral nailing. Methods: 22 (13 males, mean age 37.5y) consecutive patients were entered into this prospective randomised trial. All patients underwent stabilisation of the femoral shaft fracture with reamed (10 patients) or unreamed nailing. Patient demographics, ISS, and complications were recorded prospectively. Peripheral blood samples were collected on admission, induction of anaesthesia, entry into femoral canal, wound closure and on day 1, 3, and 6. Serum HMGB1 and IL-6 concentrations were measured using ELISAs. 6 healthy volunteers formed the control group. Results: The median ISS was 14.5 (9–29). Admission median HMGB1 and IL-6 concentrations were 7.2 ng/ml and 169 pg/ml respectively. A direct correlation was observed between ISS and IL-6 concentrations. HMGB1 concentrations reached to peak levels on day-6. On the contrary, the median concentration of IL-6 peaked around day 1 postoperatively (reamed: 780 vs. unreamed: 376 pg/ml) and then showed a downward trend. The median increase of HMGB1 by day 6 was 4.21ng/ml in the reamed and 2.98ng/ml in the unreamed population; the median increase of IL-6 by day 1 measured 462 pg/ml and 232 pg/ml in the respective groups. Conclusion: Femoral nailing and reaming induces a second hit response as supported by the post-operative increased levels of IL-6. There appears to be an inverse relationship in the concentrations of IL-6 and HMGB-1. Serum concentration of IL-6 unlike HMGB-1 strongly correlate with ISS. While IL-6 has been suggested as a marker of assessment of the early inflammatory response, alarmins can provide useful information at the later stage of an evolving immune-inflammatory process

Aims. Synovial fluid white blood cell (WBC) count and percentage of polymorphonuclear cells (%PMN) are elevated at periprosthetic joint infection (PJI). Leucocytes produce different interleukins (IL), including IL-6, so we hypothesized that synovial fluid IL-6 could be a more accurate predictor of PJI than synovial fluid WBC count and %PMN. The main aim of our study was to compare the predictive performance of all three diagnostic tests in the detection of PJI. Methods. Patients undergoing total hip or knee revision surgery were included. In the perioperative assessment phase, synovial fluid WBC count, %PMN, and IL-6 concentration were measured. Patients were labeled as positive or negative according to the predefined cut-off values for IL-6 and WBC count with %PMN. Intraoperative samples for microbiological and histopathological analysis were obtained. PJI was defined as the presence of sinus tract, inflammation in histopathological samples, and growth of the same microorganism in a minimum of two or more samples out of at least four taken. Results. In total, 49 joints in 48 patients (mean age 68 years (SD 10; 26 females (54%), 25 knees (51%)) were included. Of these 11 joints (22%) were infected. The synovial fluid WBC count and %PMN predicted PJI with sensitivity, specificity, accuracy, PPV, and NPV of 82%, 97%, 94%, 90%, and 95%, respectively. Synovial fluid IL-6 predicted PJI with sensitivity, specificity, accuracy, PPV, and NPV of 73%, 95%, 90%, 80%, and 92%, respectively. A comparison of predictive performance indicated a strong agreement between tests. Conclusions. Synovial fluid IL-6 is not superior to synovial fluid WBC count and %PMN in detecting PJI. Level of Evidence: Therapeutic Level II. Cite this article: Bone Jt Open 2020;1-12:737–742

The role of the pro-inflammatory cytokine HMGB1 (alarmins) has not been investigated in the clinical setting. This study aims to assess its relationship to IL-6 release, ISS, and to quantify the second hit phenomenon after femoral nailing. 22 (13 males, mean age 37.5y) consecutive patients entered in this prospective randomised trial. All patients underwent stabilisation of the femoral shaft fracture with reamed (10 patients) or unreamed nailing. Patient demographics, ISS, and complications were recorded prospectively. Peripheral blood samples were collected on admission, induction of anaesthesia, entry into femoral canal, wound closure and on day 1, 3, and 6. Serum HMGB1 and IL-6 concentrations were measured using ELISAs. 6 healthy volunteers formed the control group. The median ISS was 14.5 (9–29). Admission median HMGB1 and IL-6 concentrations were 7.2 ng/ml and 169 pg/ml respectively. A direct correlation was observed between ISS and IL-6 and HMGB1 concentrations. HMGB1 concentrations reached to peak levels on day-6. On the contrary, the median concentration of IL-6 peaked around day 1 postoperatively (reamed: 780 vs. unreamed: 376 pg/ml) and then showed a downward trend. The median increase of HMGB1 by day 6 was 4.21ng/ml in the reamed and 2.98ng/ml in the unreamed population; the median increase of IL-6 by day 1 measured 462 pg/ml and 232 pg/ml in the respective groups. Day 6 concentration of HMGB1 in patients with an ICU stay > 5 days (n=4), compared to the rest of the patients (n=16), was 11.04ng/ml (6.13 – 35.84) vs. 7.14ng/ml (4.06 – 12.8), (p=0.03). Femoral nailing and reaming induces a second hit as supported by the post-operative increased levels of both IL-6 and HMGB1. While IL-6 has been suggested as a marker of assessment of the early inflammatory response, alarmins can provide useful information at the later stage of an evolving immuno-inflammatory process

Introduction: Abnormalities of mesenchymal stem cells and osteoblastic cells (Obs) might play a role in producing bone collapse due to insufficient repair of the necrotic area in osteonecrosis of the femoral head (ON). Osteoblast and osteocyte apoptosis should be increased at the osteonecrotic site. Materials and Methods: We compared the TRAIL (TNF-related apoptosis-inducing ligand) cytotoxicity in primary Obs isolated from femoral heads from patients with ON or osteoarthritis (OA) and on two human osteosarcoma cell lines, MG-63 and SaOS. 2. . Results and Discussion: We showed that ON but also OA Obs were sensitive to TRAIL. We also observed TRAIL cytotoxicity on MG-63 but not SaOS. 2. cells. Moreover, we saw that TRAIL negatively regulated Akt and ERK survival pathways in MG-63 cells. We also investigated the IL-6 influences on apoptotic response of Obs to TRAIL. Even though decreased IL-6 and sIL-6R levels were observed at peripheral sites of the ON in regard to the levels produced in the iliac crest, IL-6 had no protective effects on TRAIL-induced apoptosis in ON Obs and only a weak protective effect in MG-63 cells. However, TRAIL stimulated IL-6 production in MG-63 cells and, cells and OA Obs, suggesting to a lesser extend, in SaOS. 2. other roles of TRAIL in the bone environment

Aims: As inflammation plays a key role in the etiology of intervertebral disc degeneration, we suggest a possible contribution of pro-inflammatory gene polymorphisms in the pathogenesis of adolescent idiopathic scoliosis (AIS). The nucleus pulposus of scoliotic discs responds to exogenous stimuli by secreting interleukin-6 (IL-6) and other inflammatory cytokines. The association between matrix metalloproteinases (MMPs) and disc degeneration has been reported by several investigators. A human MMP-3 promoter 5A/6A gene polymorphism regulates MMP-3 genes expression, while the G/C polymorphism of the promoter region of IL-6 gene influences levels and functional activity of the IL-6 protein. Methods: We conducted a case-control study to investigate whether the 5A/6A polymorphism of the MMP-3 gene and the G/C polymorphism of the promoter region of IL-6 gene were associated with susceptibility to AIS. Results: The frequency of the 5A/5A genotype of MMP-3 gene polymorphism in patients with scoliosis was almost 3 times higher than in controls (30.2 % vs 11.2 %, p 0.001) and the frequency of the G/G genotype of IL-6 gene polymorphism in patients with scoliosis was almost 2 times higher than in controls (52.8 % vs 26.2 %, p < 0.001). 5A/5A genotype of MMP-3 gene polymorphism and G/G genotype of IL-6 gene polymorphism are independently associated with an higher risk of scoliosis (odds ratio respectively 3.34 and 10.54). Conclusions: This is the first study that has evaluated the possibility that gene variants of IL-6 and MMPs might be associated with scoliosis and suggests that MMP-3 and IL-6 promoter polymorphisms constitute important factors for the genetic predisposition to scoliosis

Introduction: After a meniscus trauma, preservation of the meniscus is the most important surgical goal. The use of scaffolds colonized with meniscus cells (fibrochondrocytes) to reconstruct meniscal defects seem to be a promising way for the treatment of a meniscus trauma. The goal of our investigations was the analysis of expression of different anabolic and catabolic factors in human fibrochondrocytes after seeding these cells onto a collagen I scaffold to investigate the regenerative potential of such a construct for the treatment of meniscus tears. Material and Methods: Human meniscus tissue was digested in collagenase and dispase and cells were characterized by immunohistochemistry. To test scaffolds, we used a commercially available bovine collagen I matrix approved for surgical purposes. The scaffold was colonized with human fibrochondrocytes in a density of 106 cells per cm2. Cells expanded at the same ínoculation density w/o scaffold served as mock-controls. After 14 and 28 days in culture, the cells were extracted from the scaffold by aid of collagenase (Sigma, Deisenhofen, FGR) and analyzed for the expression of different factors, including IL-1β, IL-6, TGF-β, TIMP-1, TIMP-3, MMP-1, and MMP-3 using a quantitative RT-PCR-technology. Results: Bovine collagen I matrices could be colonized with human fibrochondrocytes. After 14 and 28 days of incubation on the scaffolds, the cells show the same mRNA expression levels of IL-1β, TIMP-1, TIMP-3, and TGF-β when compared to controls. In contrast, after 14 days IL-6 (12.7-fold ± 4.4, p< 0.001), MMP-1 (11.3-fold ± 2.4, p< 0.001), and MMP-3 (13.7-fold ± 6.8, p< 0.031) were upregulated on transcription levels in the scaffold when compared to controls after the same period of culture. After 28 days of culture in scaffold the expression of MMP-3 was upregulated 78.2-fold (± 7.4, p< 0.0001), MMP-1 (71.3-fold ± 5.9, p< 0.0001) and IL-6 was elevated 98.9-fold (± 9.1, p< 0.0001) compared to controls. Discussion/Conclusion: We were able to cultivate and characterize human fibrochondrocytes from menisci of the knee joint colonized onto a bovine collagen I matrix. We could show that meniscus cells revealed a significantly increased expression of MMP-1 and MMP-3, and also a significant elevation of IL-6 mRNA after 14 and 28 days of culture. No changes were found in the expression levels of IL-1β, TGF-β, and the TIMPs. This suggests that the meniscus cells colonized onto a bovine collagen I scaffold produce a considerable amount of catabolic or inflammatory factors. This may lead to a destruction of the scaffold-matrix itself and the extracellular matrix of the meniscus. Secondly, IL-6 could induce a global inflammation around the scaffold by activating the IL-6 inflammation cascade

Osteosarcoma (OS) is an aggressive bone malignancy with a high relapse rate despite combined treatment with surgery and multiagent chemotherapy. As for other cancers, OS-associated microenvironment may contribute to tumor initiation, growth, and metastasis. We consider mesenchymal stromal cells (MSC) as a relevant cellular component of OS microenvironment, and have previously found that the interaction between MSC and tumor cells is bidirectional: tumor cells can modulate their peripheral environment that in turn becomes more favourable to tumor growth through metabolic reprogramming (1). Stem-like cells were derived from HOS osteosarcoma cell line by using the spherogenic system (2). CSC isolated from HOS (HOS-CSC) were co-coltured with MSC isolated from bone marrow. Cell lysates and supernatants were collected for the analysis of RNA expression and of secreted cytokines, by Q-RT-PCR and specific ELISA assays, respectively. Here, we determined the effects of MSC on OS stemness and migration, two major features associated with recurrence and chemoresistance. Recruitment of MSC to the tumor environment leads to enhanced proliferation of OS stem cells, which increase the expression levels of TGFβ1. The latter, in turn, could be responsible for the activation of NF-kB genes and IL-6 secretion by MSC. Pro-tumorigenic effects of MSC, via IL-6, including induction of HOS-CSC migration and sphere growth, can be counteracted by IL-6 neutralizing antibody. The presence of MSC is also responsible for increased expression of adhesion molecules involved in intra- or extra-vasation. Stromal cells in combination with OS spheres exploit a vicious cycle where the presence of CSC stimulates mesenchymal cytokine secretion, which in turn increases stemness, proliferation, migration, and metastatic potential of CSC. Furthermore, for the first time we identified a novel OS stem cell marker, the Met proto-oncogene, that is frequently overexpressed and is pathogenetically relevant in OS (2 and 3). Altogether, our data corroborates the concept that a comprehensive knowledge of the interplay between tumor and stroma that also includes the stem-like fraction of tumor cells is needed to develop novel and effective anti-cancer therapies

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

Introduction: The development of periprosthetic osteolysis (OL) in total hip arthroplasty (THA) depends on activation of distinct pathways by wear particles eventually leading to predominance of osteoclasts over osteoblasts at the bone-implant interface. These processes are orchestrated by many cytokines and chemokines. However, interindividual variability in OL was observed even in cases of comparable wear rates and identical prosthesis. To explain it, we hypothesize genetic susceptibility to OL underlined by single nucleotide polymorphisms (SNP) for genes for key signal molecules. Patients and Methods: In this case-control association study we investigated patients with severe OL around THA (n=116). The control group included patients with the same THA and mild OL (n=89). All were Caucasian, all had a single type of cementless THA implanted at a single institution. Healthy subjects without THA (n=150) served as a genetic background. Severity of OL was determined according to the Saleh’s classification. We used the candidate gene approach and overall, 22 cytokine/cytokine receptor SNPs were genotyped by polymerase chain reaction with sequence specific primers (PCR-SSP). Results: The results showed an association of the TNF-238*A allele with severe OL (odds ratio, OR=6.59, p=0.005, population attributable risk percentage, PAR% = 5.2), higher risk of revision (OR=infinity, p=0.017) and poorer survival of THA (p=0.022). In addition, carriers of the IL-6-174*G allele were more frequent among the patients with severe OL (OR=2.51, p=0.007, PAR%=31.5). Finally, the genetic variant IL-2-330*G was associated with lower risk for THA revision (OR=0.44, p=0.02), protection from severe OL (OR=0.55, p=0.043) and longer survival of THA (p=0.018). Conclusions: At least in a Czech population, genetic variants of the pro-inflammatory cytokines TNF-alpha and IL-6 confer susceptibility to severe OL and risk of premature THA failure. Conversely, SNP in the IL-2 gene may protect from development of severe OL and risk of early revision due to OL

Mechanical loading of bone is anabolic, while aseptic loosening of implants is catabolic. In a rat model of mechanically induced aseptic loosening, osteoclast differentiation is increased dramatically but the underlying mechanism is unknown. The objective was to profile molecular pathways in peri-implant bone resorption. Microarrays on cortical bone samples exposed to pressurized fluid flow were performed 3, 6, 12, 24 and 36 hrs, using time 0 as controls. Of a total of 4093 genes that underwent a 1.25-fold change (p<0.05) due to fluid flow only 21 were common for all time points. Signals linked to inflammation and apoptosis were regulated in a biphasic manner at 3 and 12 and/or 24 hrs. The acute response at 3 hrs was associated with increases in the cytokines IL-6, IL-11, LIF and STAT3. Levels of the pro-apoptotic factor TWEAK were higher while those of BOK, a second pro-survival molecule, were lower. There is an early and late rise in RIPK3, which stimulates a form of programmed necrosis. Osteoblast-related genes were clearly suppressed (osteocalcin, Col1a, PTHr1), while those regulating macrophage and osteoclast differentiation (CSF-1, Bach1, HO-1, RANKL, RANK, OPG) were enhanced. These data suggest that mechanical loading of cortical bone stimulates time-dependent expression of genes regulating the survival, necrosis and differentiation of both the myeloid and mesenchymal cell lineages, resulting in an integrated response leading to a rapid increase in osteoclast numbers

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. Methods. In vitro, interleukin-1 beta (IL-1β) was used to establish the mice OA chondrocytes. Cell counting kit-8 evaluated chondrocyte viability. Western blotting analyzed the expression levels of collagen II, aggrecan, SOX9, inducible nitric oxide synthase (iNOS), IL-6, matrix metalloproteinases (MMPs: MMP1, MMP3, and MMP13), and signalling molecules associated with nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) pathways. Immunofluorescence analysis assessed the expression of aggrecan, collagen II, MMP13, and p-P65. In vivo, a destabilized medial meniscus (DMM) surgery was used to induce mice OA knee joints. After injection of DHCA or a vehicle into the injured joints, histological staining gauged the severity of cartilage damage. Results. DHCA prevented iNOS and IL-6 from being upregulated by IL-1β. Moreover, the IL-1β-induced upregulation of MMPs could be inhibited by DHCA. Additionally, the administration of DHCA counteracted IL-1β-induced downregulation of aggrecan, collagen II, and SOX9. DHCA protected articular cartilage by blocking the NF-κB and MAPK pathways. Furthermore, DHCA mitigated the destruction of articular cartilage in vivo. Conclusion. We present evidence that DHCA alleviates inflammation and cartilage degradation in OA chondrocytes via suppressing the NF-κB and MAPK pathways, indicating that DHCA may be a potential agent for OA treatment. Cite this article: Bone Joint Res 2023;12(4):259–273

Anterior cruciate ligament (ACL) injuries have been increasing, especially amongst adolescents. These injuries can increase the risk for early-onset knee osteoarthritis (OA). The consequences of late-stage knee OA include structural joint change, functional limitations and persistent pain. Interleukin-6 (IL-6) is a pro-inflammatory biomarker reflecting knee joint healing, and increasing evidence suggests that IL-6 may play a critical role in the development of pathological pain. The purpose of this study was to determine the relationship between subjective knee joint pain and function, and synovial fluid concentrations of the pro-inflammatory cytokine IL-6, in adolescents undergoing anterior cruciate ligament reconstruction surgery. Seven youth (12-17 yrs.) undergoing anterior cruciate ligament (ACL) reconstruction surgery participated in this study. They completed the Pedi International Knee Documentation Committee (Pedi-IKDC) questionnaire on knee joint pain and function. At the time of their ACL reconstruction surgery, synovial fluid samples were collected through aspiration to dryness with a syringe without saline flushing. IL-6 levels in synovial fluid (sf) were measured using enzyme linked immunosorbent assay. Spearman's rho correlation coefficient was used to determine the correlation between IL-6 levels and scores from the Pedi-IKDC questionnaire. There was a statistically significant correlation between sfIL-6 levels and the Pedi-IKDC Symptoms score (-.929, p=0.003). The correlations between sfIL-6 and Pedi-IKDC activity score (.546, p = .234) and between sfIL-6 and total Pedi-IKDC score (-.536, p = .215) were not statistically significant. This is the first study to evaluate IL-6 as a biomarker of knee joint healing in an adolescent population, reported a very strong correlation (-.929, p=0.003) between IL-6 in knee joint synovial fluid and a subjective questionnaire on knee joint pain. These findings provide preliminary scientific evidence regarding the relationship between knee joint pain, as determined by a validated questionnaire and the inflammatory and healing status of the patient's knee. This study provides a basis and justification for future longitudinal research on biomarkers of knee joint healing in patients throughout their recovery and rehabilitation process. Incorporating physiological and psychosocial variables to current return-to-activity (RTA) criteria has the potential to improve decision making for adolescents following ACL reconstruction to reduce premature RTA thereby reducing the risk of re-injury and risk of early-onset knee OA in adolescents

Aims. Tocilizumab, an interleukin-6 (IL-6) receptor (IL-6R) targeting antibody, enhances the anti-tumour effect of conventional chemotherapy in preclinical models of cancer. We investigated the anti-tumour effect of tocilizumab in osteosarcoma (OS) cell lines. Methods. We used the 143B, HOS, and Saos-2 human OS cell lines. We first analyzed the IL-6 gene expression and IL-6Rα protein expression in OS cells using reverse transcription real time quantitative-polymerase chain reaction (RT-qPCR) analysis and western blotting, respectively. We also assessed the effect of tocilizumab on OS cells using proliferation and invasion assay. Results. The OS cell lines 143B, HOS, and Saos-2 expressed IL-6R. Recombinant human IL-6 treatment increased proliferation of 143B and HOS cells. Tocilizumab treatment decreased proliferation and invasion of 143B, HOS, and Saos-2. Conclusion. In conclusion, we confirmed the production of IL-6 and the expression of IL-6R in OS cells and demonstrated that tocilizumab inhibits proliferation and invasion in OS cells. Cite this article: Bone Joint Res 2020;9(11):821–826

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

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

While cell morphology has been recognized as a fundamental regulator of cell behavior, few studies have measured the complex cell morphological changes of chondrocytes using quantitative cell morphometry descriptors in relation to inflammation and phenotypic outcome. Acute vs. persistent exposure to IL-1β and how IL-1β modulated dynamic changes in cell morphology in relation to the phenotype, donor and OA grade in healthy and osteoarthritis (OA) chondrocytes was investigated. A panel of quantitative cell morphometry descriptors was measured using an automated high-throughput method. Absolute quantification of gene expression was measured by ddPCR followed by correlation analyses. In OA chondrocytes, chronic IL-1β significantly decreased COL2A1, SOX9, and ACAN, increased IL-6 and IL-8 levels and caused chondrocytes to become less wide, smaller, longer, slimmer, less round and more circular, consistent with a de-differentiated phenotype. In healthy chondrocytes, 3 days after acute (72 h) IL-1β exposure, COL1A2 and IL-6 significantly increased but had minor effects on cell morphology. However, in healthy chondrocytes, persistent IL-1β led to more profound effects in all cell morphology descriptors and chondrocytes expressed significantly less COL2A1 and more IL-6 and IL-8 vs. controls and acutely-stimulated chondrocytes. In both OA and healthy chronically-stimulated chondrocytes, area, width and circularity were sensitive to the persistent presence of the IL-1β cytokine. Moreover, there were many significant and strong correlations among the measured parameters, with several indications of an IL-1β-mediated mechanism. Cell morphology combined with gene expression analysis could guide researchers interested in understanding inflammatory effects in the complex domain of cartilage/chondrocyte biology. Use of quantitative cell morphometry could complement classical approaches by providing numerical data on a large number of cells, thereby providing a biological fingerprint for describing chondrocyte phenotype, which could help to understand how changes in cell morphology lead to disease progression

Aims. Rheumatoid arthritis (RA), which mainly results from fibroblast-like synoviocyte (FLS) dysfunction, is related to oxidative stress. Advanced oxidation protein products (AOPPs), which are proinflammatory mediators and a novel biomarker of oxidative stress, have been observed to accumulate significantly in the serum of RA patients. Here, we present the first investigation of the effects of AOPPs on RA-FLSs and the signalling pathway involved in AOPP-induced inflammatory responses and invasive behaviour. Methods. We used different concentrations of AOPPs (50 to 200 µg/ml) to treat RA-FLSs. Cell migration and invasion and the expression levels of tumour necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), matrix metalloproteinase-3 (MMP-3), and MMP-13 were investigated. Western blot and immunofluorescence were used to analyze nuclear factor-κB (NF-κB) activation. Results. AOPPs promoted RA-FLS migration and invasion in vitro and significantly induced the messenger RNA (mRNA) and protein expression of TNF-α, IL-6, MMP-3, and MMP-13 in dose- and time-dependent manners. Moreover, AOPPs markedly activated the phosphorylation of nuclear factor-κB (NF-κB) p65 protein, which triggered inhibitory kappa B-alpha (IκBα) degradation, NF-κB p65 protein phosphorylation, and NF-κB p65 translocation into the nucleus. Furthermore, treatment with a neutralizing antibody specific to receptor for advanced glycation end products (RAGE) significantly suppressed aggressive behaviour and inflammation, decreased TNF-α, IL-6, MMP-3, and MMP-13 expression, and blocked AOPP-induced NF-κB pathway activation. Conclusion. The results indicate that AOPPs can enhance aggressive behaviour and the inflammatory response in RA-FLSs via the RAGE–NF-κB pathway. These results present AOPPs as a new class of potentially important mediators of progressive disease in RA patients. Cite this article: Bone Joint Res 2021;10(4):259–268