Abnormal wear of cobalt-containing metal-on-metal joints is associated with inflammatory pseudotumours. Cobalt ions activate human toll-like receptor 4 (TLR4), which normally responds to bacterial lipopolysaccharide (LPS) in sepsis. Activation of TLR4 by LPS increases the expression of chemokines IL-8 and CXCL10, which recruit leukocytes and activated T-cells, respectively. This study was designed to determine whether cobalt induces a similar inflammatory response to LPS by promoting the expression of IL-8 and CXCL10. A human monocytic cell line, derived from acute monocytic leukaemia, was treated with cobalt ions and expression of IL-8 and CXCL10 measured at mRNA and protein levels. Cobalt-treated macrophages showed a 60-fold increase in IL-8 mRNA, and an eightfold increase in production of the mature chemokine (both p < 0.001); expression of the CXCL10 gene and protein was also significantly increased by cobalt (both p < 0.001). Experiments were also performed in the presence of CLI-095, a TLR4-specific antagonist which abrogated the cobalt-mediated increase in IL-8 and CXCL10 expression. . These findings suggest that cobalt ions induce inflammation similar to that observed during sepsis by the simultaneous activation of two TLR4-mediated signalling pathways. These pathways result in increased production of IL-8 and CXCL10, and may be implicated in pseudotumour formation following metal-on-metal replacement. Cite this article: Bone Joint J 2014; 96-B:1172–7

Complement C5a receptor 1 (C5aR1) has crucial functions in host defense against danger molecules, as does toll-like receptor 2 (TLR2). Both innate immunity receptors interact in immune cells in the context of infectious inflammatory diseases often associated with bone loss, such as periodontitis. C5aR1 plays an important role in bone, as it is expressed on bone cells and strongly upregulated due to bone injury. Importantly, C5aR1-ko mice are protected against arthritis and C5aR1 contributes to bone loss in periodontitis. In contrast, less data exist on the role of TLR2 on osteoblasts, however, it is known that TLR2 is expressed on osteoblasts and contributes to bacterial-induced bone resorption. The aim of this study was to evaluate the interaction of C5aR1 and TLR2 in osteoblasts, including intracellular signaling pathways and gene expression patterns. Primary osteoblasts were isolated from 8–12 week-old WT mice and differentiated for 14 days. Osteoblasts were assessed for expression of C5aR1 and TLR2. Phosphorylation of mitogen-activated protein kinases (MAPK) in response to C5a and Pam3CSK4 (TLR2 agonist) was analyzed by immunoblotting. Gene expression profiling after 30 min and 4 h stimulation of C5a was performed by microarray and candidate genes were validated by quantitative Real-Time PCR (qRT-PCR). Immunoprecipitation was performed using a C5aR1-antibody and C5aR1 and TLR2 were subsequently detected by immunoblotting. Statistics: One way ANOVA p<0.05, n=4–6. We showed that C5aR1 and TLR2 are expressed on osteoblasts and strongly upregulated during differentiation. Via immunoprecipitation, we could show that C5aR1 and TLR2 do physically interact in osteoblasts. We then examined if C5aR1 and TLR2, besides their physical interaction, also act via the same intracellular signaling pathways. Gene expression profiling upon C5a stimulation revealed that the top regulated pathways are related to MAPK and transforming growth factor beta (TGF-β). Respective genes, such as TGF-β (Tgfb1) and its receptor (Tgfbr) were found to be upregulated, and negative MAPK regulators were found to be downregulated, both by microarray analysis and qRT-PCR. Accordingly, we saw a C5aR1- and TLR2-dependent phosphorylation of p38 MAPK. Interestingly, this effect was enhanced and prolonged by costimulation of both receptors. An additive effect of C5aR1 and TLR2 was also seen regarding Cxcl10 levels, which were enhanced compared to C5aR1 or TLR2 stimulation alone. This study shows that C5aR1 and TLR2 interact in osteoblasts, not only physically but also functionally, regarding downstream signaling and target genes. Those data strongly imply a synergistic interplay between the receptors, through which osteoblasts possibly contribute to inflammatory reactions affecting bone

Total hip replacement (THR) is indicated for patients with osteoarthritis where conservative treatment has failed. Metal alloys used in THR implants such as cobalt-chromium (CoCr) have been known to cause pro-inflammatory reactions in patients, therefore leading to the need for costly revision surgery. This study therefore aimed to investigate the role of TLR4 in the activation of a human osteoblast model in response to CoCr particles in vitro. Human osteoblasts (MG-63 cell line) were seeded at a density of 100,000 cells and treated with 0.5, 5, 50mm3 CoCr particles per cell for 24-hours. Trypan blue and the XTT Cell Proliferation Kit II were then used in conjunction with the cells to assess CoCr-induced cytotoxicity. Cells were pre-treated with a commercially available TLR4-specific small molecule inhibitor (CLI-095) for 6 hours. Untreated cells were used as a negative control and lipopolysaccharide (LPS) was used as a positive control. Following treatment the cell supernatant was collected and used for enzyme-linked immunosorbant assay (ELISA) to measure the secretion of interleukin-8 (IL-8), CXCL10, and interleukin-6 (IL-6). Trypan blue and XTT analysis showed that there was no significant changes to cell viability or proliferation at any dose used of CoCr after 24 hours. There was a significant increase in protein secretion of IL-8 (p<0.001), CXCL10 (p<0.001), and IL-6 (p<0.001) in the cells which received the highest dosage of CoCr. This pro-inflammatory secretory response was ameliorated by TLR4 blockade (p<0.001). CoCr particles are not cytotoxic to osteoblasts but they do induce pro-inflammatory changes as characterised by increased secretion of chemokines IL-8, CXCL10, and IL-6. These responses occur via a TLR4-mediated pathway and upon inhibition they can be effectively ameliorated. This is particularly important as TLR4 could be a potential target for pharmacological intervention used in patients experiencing immunological reactions to metal implant debris

Introduction and Objective. Total joint replacement (TJR) is indicated for patients with end-stage osteoarthritis (OA) where conservative treatment has failed. Approximately 1.3 million primary hip replacement surgeries have been recorded in the United Kingdom since 2003 and this number is set to rise due to an increase in obesity as well as an ageing population. Total hip replacement (THR) has a survival rate of 85% at 20 years; the most common reason for failure is aseptic loosening which often occurs secondary to osteolysis caused by immune-mediated inflammation responses to wear debris generated from the materials used in the THR implant. Therefore, by understanding the biological steps by which biomaterials cause immune-mediated reactions it should be possible to prevent them in the future thereby reducing the number of costly revision surgeries required. Materials and Methods. The human osteoblast-like cell line (MG-63) was seeded at a density of 100,000 cell per well of a 6-well plate and treated with and increasing doses (0.5, 5, and 50mm. 3. per cell) of cobalt-chromium (CoCr) particles generated on a six-station pin-on-plate wear generator or commercially available ceramic oxide nanopowders (Al. 2. O. 3. and ZrO. 2. ) for 24 hours. TNF-alpha was used as a positive control and untreated cells as a negative control. Cells were then analysed by transmission electron microscopy (TEM) to determine whether the osteoblasts were capable of phagocytosing these biomaterials. MG-63 cells were used in conjunction with trypan blue and the XTT Cell Proliferation II Kit to assess cytotoxicity of the biomaterials investigated. Cells supernatants were also collected and analysed by enzyme-linked immunosorbant assay (ELISA) to investigate changes in pro-inflammatory protein secretion. Protein extracted from lysed cells was used for western blotting analysis to investigate RANKL protein expression to determine changes to osteolytic activation. Lysed cells were also used for RNA extraction and subsequent cDNA synthesis for real-time quantitative polymerase chain reaction (RT-qPCR) in order to assess changes to pro-inflammatory gene expression. Results. There was no significant change to cellular viability or proliferation in the osteoblasts treated with CoCr, Al. 2. O. 3. or ZrO. 2. when compared to the untreated negative control. TEM images showed clear and distinct intracellular vesicles within the cell cytoplasm which contained CoCr, Al. 2. O. 3. and ZrO. 2. RANKL expression increased at 5 and 50mm. 3. per cell CoCr and 50mm. 3. per cell Al. 2. O. 3. and ZrO. 2. Pro-inflammatory protein secretion of CXCL10, IL-8, and IL-6 all significantly increased at 50mm. 3. per cell CoCr, Al. 2. O. 3. , and ZrO. 2. Similarly to the protein secretion, CXCL10, IL-8, and IL-6 gene expression was significantly upregulated at 50mm. 3. per cell CoCr, Al. 2. O. 3. , and ZrO. 2. Conclusions. Increased in vitro RANKL expression in response to CoCr, Al. 2. O. 3. , and ZrO. 2. may result in disruption of bone metabolism and lead to osteolysis which can contribute to aseptic loosening in vivo. Significant increases in IL-6 are particularly important because as well as being a pro-inflammatory cytokine, IL-6 is also secreted by osteoblasts in order to stimulate mature osteoclast formation to mediate bone breakdown. CXCL10 and IL-8 are chemotactic cytokines and increased secretion in response to implant biomaterials can contribute to ongoing pro-inflammatory responses through the recruitment of monocytes and neutrophils respectively. This is interesting as in vivo data demonstrates increased cellular infiltrate in patients experiencing responses to implant materials. Overall, these findings show clear immune activation as well as altered metabolism of MG-63 osteoblast cells in response to implant wear debris which is in agreement with in vivo clinical reports

Abstract. Objectives. Mesenchymal stromal/stem cells (MSCs) are increasingly recognized as regulators of immune cells during disease or tissue repair. During these situations, the extracellular matrix (ECM) is very dynamic and therefore, our studies aim to understand how ECM influences the activity of MSCs. Methods. Human MSCs cultured on tissue culture plastic (TCP) and encapsulated within collagen type I, fibrin, or mixed Collagen-Fibrin were exposed to low dose TNFα and IFNɣ. Transcription profiles were examined using bulk RNA sequencing (RNAseq) after 24h of treatment. ELISA, Western blot, qPCR and immunofluorescence were employed to validate RNAseq results and to investigate the significance of transcriptional changes. Flow cytometry evaluated monocyte/macrophage phenotype. Results. Previously, we showed that human MSC expression of TNFAIP6 and CXCL10 in 3D environments is significantly upregulated in response to pro-inflammatory stimuli. Here, RNAseq revealed that there were 2,085 highly significant upregulated genes in 3D matrices compared to TCP. Notably, >90% of highly expressed genes (including FOSB, FOS and TNFAIP6) were shared in all hydrogels. Gene ontology confirmed the TNF signalling pathway among the most significantly represented. Protein-protein interaction predictions identified TNF-alpha/NF-kappa B and AP1 pathways as differentially influenced by the hydrogel environment. Using inhibitors to these pathways, NFkB, but not AP1, impacted on the upregulation of TNFAIP6 and CXCL10 in 3D culture. Conditioned media from these studies was added to cultures of human monocytes with distinct changes in the resulting macrophage phenotype. MSCs in a 3D environment promoted a greater acquisition of the M2 repair macrophage phenotype and impacted on the numbers of pro-inflammatory M1 macrophages. Conclusion. These data provide further evidence that the immunomodulatory action of human MSCs can be influenced by the surrounding structural environment. These observations have significance for understanding the events that following skeletal injury and the potential to be exploited in preconditioning MSCs for cell therapy

Mesenchymal stromal cells (MSCs) are promising candidate for cell therapy in osteoarthritis (OA) patients since that they exert anti-inflammatory, immunomodulatory, anti-fibrotic and anti-hypertrophic effects in the joint tissues. However, little is known about the OA milieu factors that could enhance the migration and tissue specific engraftment of exogenously injected MSC for successful regenerative cell therapy. GMP-clinical grade adipose stromal cells (ASC) were evaluated both in normoxic and hypoxic (2%O. 2. ) conditions, with or without OA synovium milieu. We found that both OA synovial fluids and OA synoviocytes derived conditioned medium (CM) contain approximately the same amounts of different cytokines/chemokines (i.e. IL6, CXCL8, CXCL10, CXCL12, CCL2, CCL3, CCL4, CCL5, CCL11). ASC migration was significantly increased by both OA synovium milieu and not affected by normoxic or hypoxic condition. We identify that ASC migration was mainly influenced by different macrophage chemokines (i.e. CCL2, CCL3, CCL4). In hypoxic condition basal GMP-ASC showed an increase of CXCR3 and CCR3, a decrease of CCR1 and CCR5 receptors, while CXCR1, CXCR4, CXCR7, CCR2 and IL6R were not modulated. The addition of OA synovium milieu induced CCR3, CXCR3 and IL6R and decreased CCR1 and not affected CCR2, CCR5, CXCR1, CXCR4, CXCR7 in hypoxic condition. Our data demonstrated that GMP-ASC chemotaxis was mainly induced by macrophage chemokines. Moreover, we evidenced that hypoxia, as better condition to mimic the OA milieu, affected some GMP-ASC cytokine/chemokine receptors, suggesting the involvement of specific chemokine-receptor axis

Aims

This study examined the relationship between obesity (OB) and osteoporosis (OP), aiming to identify shared genetic markers and molecular mechanisms to facilitate the development of therapies that target both conditions simultaneously.

Rheumatoid arthritis (RA) is an autoimmune disease characterized by symmetrical and chronic polyarthritis. Fibroblast-like synoviocytes are mainly involved in joint inflammation and cartilage and bone destruction by inflammatory cytokines and matrix-degrading enzymes in RA. Approaches that induce various cellular growth alterations of synoviocytes are considered as potential strategies for treating RA. However, since synoviocytes play a critical role in RA, the mechanism and hyperplastic modulation of synoviocytes and their motility need to be addressed. In this review, we focus on the alteration of synoviocyte signalling and cell fate provided by signalling proteins, various antioxidant molecules, enzymes, compounds, clinical candidates, to understand the pathology of the synoviocytes, and finally to achieve developed therapeutic strategies of RA.

Cite this article: Bone Joint Res 2021;10(4):285–297.

Aims

The aim of this study was to systematically review the literature for evidence of the effect of a high-fat diet (HFD) on the onset or progression of osteoarthritis (OA) in mice.