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Orthopaedic Proceedings
Vol. 99-B, Issue SUPP_8 | Pages 23 - 23
1 Apr 2017
Lawrence H Deehan D Holland J Kirby J Tyson-Capper A
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Background. Metal-on-metal (MoM) hip arthroplasty has been associated with adverse reactions including pseudotumours, and osteolysis. Tissues surrounding failed MoM hip implants are often infiltrated by inflammatory cells such as monocytes and neutrophils. The mechanisms by which these cells are recruited to the tissues remain unclear. Cobalt from MoM implants activates Toll-like receptor 4 (TLR4), an immune cell surface receptor usually responsible for recognition of bacteria and prevention of sepsis. Activation by bacteria leads to secretion of pro-inflammatory cytokines which guide other immune cells to the site of inflammation. The effect of cobalt on this response is unknown and therefore this study aims to determine the effect of cobalt-mediated TLR4 activation on the migration of inflammatory cells. Methods. A human macrophage cell line (MonoMac 6) was stimulated with a physiologically-relevant range of cobalt ions for 24h with or without pre-treatment with a TLR4 antagonist. Conditioned media was collected and used in a trans-well migration assay to determine its effect on migration of primary monocytes and neutrophils isolated from whole human blood. Migrated cells were stained with haematoxylin and counted at ×40 magnification. Results. Conditioned media from cobalt-treated macrophages caused elevated monocyte and neutrophil migration across all concentrations. Pre-treatment of MonoMac 6 cells with a TLR4 antagonist significantly decreased the response. This suggests that the cytokine profile produced in response to cobalt-mediated TLR4 activation is pro-migratory for immune cells. Conclusions. Cobalt activation of TLR4 leads to secretion of inflammatory cytokines that attract monocytes and neutrophils. This work highlights a potential mechanism by which cobalt ions from failed MoM joints could be involved in inflammatory cell recruitment to the surrounding tissues. The TLR4 signalling pathway represents an exciting area for further investigation as a therapeutic target in the prevention of adverse reactions to cobalt ions. Disclosure. This work is funded by DePuy Synthes Ltd and the Newcastle NIHR Biomedical Research Centre


Orthopaedic Proceedings
Vol. 105-B, Issue SUPP_7 | Pages 88 - 88
4 Apr 2023
Anjum S Kirby J Deehan D Tyson-Capper A
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The most common reason for revision surgery of total hip replacements is aseptic loosening of implants secondary to osteolysis, which is caused by immune-mediated reactions to implant debris. These debris can cause pseudotumour formation. As revision surgery is associated with higher mortality and infection, it is important to understand the pro-inflammatory process to improve implant survival. Toll-like receptor 4 (TLR4) has been shown to mediate immune responses to cobalt ions. Statin use in epidemiological studies has been associated with reduced risk of revision surgery. In-vitro studies have demonstrated the potential for statins to reduce orthopaedic debris-induced immune responses and there is evidence that statins can modulate TLR4 activity. This study investigates simvastatin's effect on orthopaedic biomaterial-mediated changes in protein expression of key inflammatory markers and soluble-ICAM-1 (sICAM-1), an angiogenic factor implicated in pseudotumour formation. Human macrophage THP-1 cells were pre-incubated with 50µM simvastatin for 2-hours or a vehicle control (VC), before being exposed to 0.75mM cobalt chloride, 50μm3 per cell zirconium oxide or LPS as a positive control, in addition to a further 24-hour co-incubation with 50µM simvastatin or VC. Interleukin −8 (IL-8), sICAM-1, chemokine ligand 2 (CCL2), CCL3 and CCL4 protein secretion was measured by enzyme-linked immunosorbent assay (ELISA). GraphPad Prism 10 was used for statistical analysis including a one-way ANOVA. Pre-treatment with simvastatin significantly reduced LPS and cobalt-mediated IL-8 secretion (n=3) and sICAM-1 protein secretion (n=2) in THP-1 cells. Pre-treatment with simvastatin significantly reduced LPS-mediated but not cobalt ion-mediated CCL2 (n=3) and CCL3 protein (n=3) secretion in THP-1 cells. Simvastatin significantly reduced zirconium oxide-mediated CCL4 secretion (n=3). Simvastatin significantly reduced cobalt-ion mediated IL-8 and sICAM-1 protein secretion in THP-1 cells. This in-vitro finding demonstrates the potential for simvastatin to reduce recruitment of leukocytes which mediate the deleterious inflammatory processes driving implant failure


Orthopaedic Proceedings
Vol. 96-B, Issue SUPP_11 | Pages 292 - 292
1 Jul 2014
Lawrence H Deehan D Holland J Kirby J Tyson-Capper A
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Summary. Metal-on-metal hip replacements have been associated with adverse reactions including inflammatory pseudotumours and soft tissue necrosis. We have shown that cobalt can directly activate toll-like receptor 4, an immune receptor causing pro-inflammatory interleukin-8 secretion. This may contribute to adverse reaction development. Introduction. Metal-on-metal hips have the highest failure rate of any joint arthroplasty material. Reasons for failure include the development of pseudotumours, soft tissue necrosis and pain around the affected joint. The adverse reactions appear to be inflammatory as failing joints are often infiltrated by immune cells such as lymphocytes. However the exact cellular and biological mechanisms underlying this inflammation are unknown. Toll-like receptor 4 (TLR4) is found on the surface of immune cells including macrophages and dendritic cells. It is activated by lipopolysaccharide (LPS) from Gram negative bacteria, inducing an immune response against the pathogen through increased secretion of pro-inflammatory cytokines. It has recently been shown that nickel can activate TLR4, causing inflammation. Cobalt, a component of many metal-on-metal joints, is adjacent to nickel in the periodic table and shares a number of nickel's properties. Consequently we hypothesised that cobalt ions from metal-on-metal joints can activate TLR4. Methods. An in vitro cell culture model was developed using human and murine TLR4 reporter cell lines to investigate the effects of metal ions, including cobalt, on TLR4. Real-time PCR was used to examine the effect of cobalt on inflammatory gene expression, including IL-8, CCL-2 and IRAK-2, while an ELISA assay was conducted to investigate IL-8 protein expression in a human macrophage cell line (MonoMac 6). The TLR4 agonist LPS was included as a positive control and as a negative control TLR4 activation was blocked using the chemical agonist CLI-095 (Invivogen, UK). Results. Using human TLR4 reporter cells we show that cobalt at clinically-relevant concentrations can activate human TLR4. This effect appears unique to humans as murine TLR4 is unresponsive to cobalt but still responds to LPS. We also demonstrate that in human macrophages physiologically-relevant concentrations of cobalt cause increased pro-inflammatory IL-8 secretion (p<0.001). IL-8 is involved in perpetuating the immune response by recruiting more inflammatory cells to the site of inflammation. Cobalt-induced IL-8 secretion can be blocked using a TLR4 antagonist (p<0.001) showing that the effect is due to cobalt activation. Cobalt ions also alter gene expression in human macrophages. Cobalt upregulates expression of IL-8 and IRAK2 genes; IRAK2 is a key component of the TLR4 signalling pathway. Interestingly, cobalt causes downregulation of the CCL2 gene whereas it is upregulated in response to LPS. Discussion. In this study we have demonstrated that cobalt ions can activate human TLR4 signalling and in human macrophages this can increase expression of pro-inflammatory IL-8. We have also developed a robust series of assays for determining the effects of metal ions and other orthopaedic materials on the TLR4 signalling pathway. These methods will be used to investigate the immunological effects of additional orthopaedic metals (e.g. chromium, titanium and molybdenum). This work has identified a key pathway involved in the immune response to metal ions which can now be investigated for genetic variability and as a potential therapeutic target


Orthopaedic Proceedings
Vol. 103-B, Issue SUPP_13 | Pages 12 - 12
1 Nov 2021
Anjum S Jamieson S Deehan D Kirby J Tyson-Capper A
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Introduction and Objective. Total joint replacement is indicated for osteoarthritis where conservative treatment has failed, and in the UK the number of patients requiring hip and knee replacements is set to increase with an ageing population. Survival of total hip replacements is around 85% at 20 years with the most common reason for revision being aseptic loosening of the implant secondary to osteolysis, which is caused by immune-mediated reactions to implant debris. These debris can also cause pseudotumour formation. As revision surgery is associated with higher morbidity, mortality, infection rates, venous thromboembolism, resource demand and poorer subsequent function it is important to understand the mechanisms underlying the pro-inflammatory process to improve implant survival. Toll-like receptor 4 (TLR4), an innate immune receptor, has been demonstrated to mediate deleterious immune responses by the Tyson-Capper research group, including inflammatory cytokine interleukin-8 (IL-8) secretion. Statin use in epidemiological studies has been associated with reduced overall risk of revision surgery after hip replacement. In-vitro studies have demonstrated the potential for statins to reduce orthopaedic debris-induced immune responses which can lead to osteolysis and pseudotumour formation. As literature from cardiological investigations demonstrate that statins can reduce the expression and responsiveness of TLR4, this could be an exciting mechanism to exploit to reduce the host immune response to orthopaedic wear debris, thereby improving implant survival by reducing immune mediated osteolysis. This ongoing study investigates simvastatin's effect on cobalt ion-mediated changes in gene and protein expression of interleukin-8 and soluble-ICAM-1 (sICAM-1) which is an angiogenic factor implicated in pseudotumour formation. Materials and Methods. TLR4-expressing human monocyte/macrophage THP-1 cells were pre-incubated with 50μM simvastatin for 2-hours or a vehicle control, before being exposed to exposed to 0.75mM cobalt chloride, in addition to a further 24-hour co-incubation with 50μM simvastatin or vehicle control. IL-8 protein and sICAM-1 secretion was measured by enzyme-linked immunosorbent assay (ELISA). Gene expression changes were quantified by TaqMan-based real time polymerase chain reaction. Results. Pre-treatment with simvastatin significantly reduced cobalt-mediated IL-8 protein secretion (n=3) and sICAM-1 protein secretion (n=2) in THP-1 cells (p-value<0.0001). Work will be undertaken to determine changes in gene expression, the role of TLR4 in these responses and the effect of simvastatin on additional inflammatory markers. Conclusions. Simvastatin significantly reduces cobalt-ion mediated IL-8 and sICAM-1 protein secretion in THP-1 cells. This in-vitro finding demonstrates the potential for simvastatin to reduce recruitment of leukocytes which mediate the deleterious inflammatory processes driving aseptic loosening and pseudotumour formation


Orthopaedic Proceedings
Vol. 103-B, Issue SUPP_13 | Pages 60 - 60
1 Nov 2021
Cazzanelli P Hausmann ON Wuertz-Kozak K
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Introduction and Objective. Intervertebral disc (IVD) degeneration is one of the major contributors to low back pain, the leading cause of disability worldwide. This multifactorial pathological process involves the degradation of the extracellular matrix, inflammation, and cell loss due to apoptosis and senescence. While the deterioration of the extracellular matrix and cell loss lead to structural collapse of the IVD, increased levels of inflammation result in innervation and the development of pain. Amongst the known regulators of inflammation, toll-like receptors (TLRs) and more specifically TLR-2 have been shown to be specifically relevant in IVD degeneration. As strong post-transcriptional regulators, microRNAs (miRNAs) and their dysregulation has been connected to multiple pathologies, including degenerative diseases such as osteoarthritis and IVD degeneration. However, the role of miRNAs in TLR signalling in the IVD is still poorly understood and was hence investigated in this study. Materials and Methods. Human Nucleus pulposus (hNP) and Annulus fibrosus (hAF) cells (n=5) were treated with the TLR-2/6 specific agonist PAM2CSK4 (100 ng/mL for 6 hours) in order to activate the TLR2 signalling pathway. After the activation both miRNA and mRNA were isolated, followed by next-generation sequencing and qPCR analysis of proinflammatory cytokines respectively. Furthermore, cell supernatants were used to analyze the secretion of proinflammatory cytokines with enzyme-linked immunosorbent assay. TLR-2 knockdown (siRNA) cells were used as a control. Statistical analysis was conducted by performing Kolmogorov-Smirnov test and a two-tailed Student's t-test using GraphPad Prism version 9.0.2 for Windows (GraphPad Software, La Jolla California USA). Results. TLR-2 activation resulted in the induction of an inflammatory cell response, with a significant increase in gene expression of interleukin (IL)-6 (525 ± 180 fold change, p < 0.05) and IL-8 (7513 ± 1907 fold change, p < 0.05) and protein secretion of IL-6 (30.5 ± 8.1 pg/mL) and IL-8 (28.9 ± 5.4 pg/mL). TLR-2 activation was furthermore associated with changes in the miRNA profile of hNP and hAF cells. Specifically, we identified 10 differentially expressed miRNAs in response to TLR-2 activation, amongst which were miR-335–3p (1.45 log2 FC, p < 0.05), miR-125b-1–3p (0.55 log2 FC, p < 0.05), and miR-181a-3p (−1.05 log2 FC, p < 0.05). Conclusions. The identified miRNAs are known to be associated with osteoarthritis (miR-335-3p), inflammation and IVD degeneration (mir-125-1-3p and miR-181a-3p), but the link to TLR signalling has not been previously reported. Experiments to validate the identified miRNAs and elucidate their functional role are undergoing. The identification of these miRNAs provides an opportunity to further investigate miRNAs in the context of TLR activation and inflammation and to enhance our understanding of underlying molecular mechanisms behind disc degeneration, inflammation, and TLR dysregulation


Orthopaedic Proceedings
Vol. 100-B, Issue SUPP_14 | Pages 113 - 113
1 Nov 2018
Wang C
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All types of regenerative materials, including metal implants, porous scaffolds and cell-laden hydrogels, interact with the living tissue and cells. Such interaction is key to the settlement and regenerative outcomes of the biomaterials. Notably, the immune reactions from the host body crucially mediate the tissue-biomaterials interactions. Macrophages (as well as monocytes and neutrophils), traditionally best known as defenders, accumulate at the tissue-biomaterials interface and secrete abundant cytokines to create a microenvironment that benefits or inhibits regeneration. Because the phenotype of these cells is highly plastic in response to varying stimuli, it may be feasible to manipulate their activity at the interface and harness their power to mediate bone regeneration. Towards this goal, our team have been working on macrophage-driven bone regeneration in two aspects. First, targeting the abundant, glucan/mannan-recognising receptors on macrophages, we have devised a series of glucomannan polymers that can stimulate macrophages to secrete pro-osteogenic cytokines, and applied them as coating polymer of mesenchymal stem cells-laden hydrogels. Second, targeting the toll-like receptors (TLRs) on macrophages, we have screened TLR-activating polysaccharides and picked up zymosan (beta-glucan) to be modified onto titanium and glass implants. We evaluated both the efficacy of integration and safety of immune stimulation in both in vitro and in vivo models. Our future exploration lies in further elaborating the different roles and mechanisms of macrophages of various types and origins in the regenerative process


Orthopaedic Proceedings
Vol. 101-B, Issue SUPP_2 | Pages 28 - 28
1 Jan 2019
Mawdesley A Tyson-Capper A Kirby J Tipper JL
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Increased revision rates and early failure of Metal-on-Metal (MoM) hip replacements are often due to adverse reaction to metal debris (ARMD). Cobalt is a major component of MoM joints and can initiate an immune response via activation of the innate immune receptor Toll-like receptor 4 (TLR4). This leads to increased secretion of inflammatory cytokines/chemokines e.g. CCL3 and CCL4. The aim of this study was to evaluate whether TLR4-specific neutralising antibodies can prevent cobalt-mediated activation of TLR4. MonoMac 6 (MM6) cells, a human macrophage cell line, were treated with two different TLR4-specific monoclonal antibodies followed by 0.75mM of cobalt chloride (CoCl2). Lipopolysaccharide (LPS), a known TLR4 agonist was used as a positive control. Enzyme-linked immunosorbent assay (ELISA) was used to assess CCL3/CCL4 protein secretion and real time- polymerase chain reaction (RT-PCR) allowed quantification of CCL3/CCL4 gene expression. MM6 cells treated with cobalt and LPS up-regulate CCL3 and CCL4 gene expression and protein secretion. MM6 cells pre-treated with both monoclonal antibodies prior to stimulation with 0.75mM CoCl2 for 16 hours demonstrated significant inhibition of both CCL3 and CCL4 secretion as well as gene expression (both p=<0.0001). One of the antibodies failed to inhibit chemokine expression and secretion in LPS treated cells. This study identifies for the first time the use of TLR4-specific monoclonal antibodies to prevent cobalt activation of TLR4 and subsequent inflammatory response. This finding demonstrates the potential to exploit TLR4 inhibition in the context of MoM joint replacements by contributing to the development of novel therapeutics designed to reduce the incidence of ARMD


Orthopaedic Proceedings
Vol. 99-B, Issue SUPP_9 | Pages 10 - 10
1 May 2017
Mawdesley A Anjum S Lawrence H Deehan D Kirby J Tyson-Capper A
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Background. Increased revision rates and early failure of Metal-on-Metal (MoM) hip replacements are often due to adverse reaction to metal debris (ARMD). ARMD describes numerous symptoms in patients such as pain, osteolysis and soft tissue damage. Cobalt is a major component of MoM joints and can initiate an immune response via activation of the innate immune receptor Toll-like receptor 4 (TLR4). This leads to increased secretion of inflammatory cytokines e.g. interleukin-8 (IL-8). This study investigates whether TLR4-specific antagonists inhibit the inflammatory response to cobalt using IL-8 gene expression and protein secretion as a marker of TLR4 activation. Methods. MonoMac 6 (MM6) cells, a human macrophage cell line, were treated with TLR4-specific antagonists followed by 0.75mM of cobalt chloride. Lipopolysaccharide (LPS), a known TLR4 agonist was used as a positive control. Enzyme-linked immunosorbent assay (ELISA) was used to assess IL-8 protein secretion and real time- polymerase chain reaction (RT-PCR) allowed quantification of IL-8 gene expression. Results. MM6 cells treated with cobalt and LPS up-regulate IL-8 gene expression and protein secretion (n=3). The addition of TLR4-specific antagonists significantly inhibits this up-regulation suggesting the observed effects are TLR4-mediated. MM6 cells stimulated with cobalt (0.75mM) for 16 hours demonstrated a 27-fold increase in IL-8 gene expression (p-value = < 0.0001). When pre-treated with 10μg/ml of a TLR4-specific antagonist fold increase decreased to 6-fold (p-value = < 0.0001). IL-8 secretion decreased from 5000pg/ml to 3000pg/ml (p-value = < 0.0001). Conclusion. TLR4-specific antagonists inhibit cobalt-mediated IL-8 gene expression and protein secretion in MM6 cells. This finding demonstrates the potential to exploit this inhibition in the context of MoM joint replacements by contributing to the development of novel therapeutics designed to improve MoM implant longevity, reduce the incidence of ARMD and prevent subsequent revision surgery


Orthopaedic Proceedings
Vol. 100-B, Issue SUPP_3 | Pages 35 - 35
1 Apr 2018
Hägele Y Rapp A Ignatius A
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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


Orthopaedic Proceedings
Vol. 98-B, Issue SUPP_16 | Pages 16 - 16
1 Oct 2016
Crowe L Akbar M Kitson S Reilly J Kerr S Murrell G McInnes I Gilchrist D Millar N
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Alarmins- also referred to as damage associated molecular patterns (DAMPS)- are endogenous molecules mobilized in response to tissue damage known to activate the innate immune system and regulate tissue repair and remodelling. The molecular mechanisms that regulate inflammatory and remodelling pathways in tendinopathy are largely unknown therefore identifying early immune effectors is essential to understanding the pathology. S100A8 and S100A9 are low molecular weight calcium binding proteins primarily released by activated phagocytes in an inflammatory setting and also secreted as a heterodimeric complex that exhibits cytokine like functions. Based on our previous investigations we sought evidence of S100A8/A9 expression in human tendinopathy and thereafter, to explore mechanisms whereby S100 proteins may regulate inflammatory mediators and matrix regulation in human tenocytes. Torn supraspinatus tendon (established pathology) and matched intact subscapularis tendon (representing ‘early pathology’) biopsies were collected from patients undergoing arthroscopic shoulder surgery. Control samples of subscapularis tendon were collected from patients undergoing arthroscopic stabilisation surgery. S100A8/A9 expression was analysed at transcript and protein level using quantitative RT-PCR and immunohistochemistry, respectively. Primary human tenocytes were cultured from hamstring tendon tissue obtained during hamstring tendon ACL reconstruction. The in vitro effect of recombinant human S100 A8/A9 on primary human tenocytes was measured using quantitative RT-PCR and ELISA. Immunohistochemistry of tendinopathic tissues demonstrated the presence of S100 A8/A9 in diseased tissues compared to control tissue. In addition, early pathological diseased tissue indicated greater S100A9 expression compared with established diseased pathology. These findings were reflected by data obtained at transcript level from diseased tissues. Recombinant human S100A8, A9 and A8/A9 complex led to significant increase in expression of inflammatory mediators, including IL-6 in vitro. Further analysis via quantitative RT-PCR demonstrated recombinant S100A8, A9 and A8/A9 complex treatment on tenocytes, in vitro, had no direct effect on the expression of genes involved in matrix remodelling. The presence of S100A8 and S100A9 in early tendinopathic lesions suggests expression is upregulated in response to cellular damage. S100A8 and S100A9 are endogenous ligands of Toll-like receptors (TLRs) and receptor for advanced glycation end products (RAGE). These receptors have known regulatory effects on immune mediated cytokine production. We propose S100A8 and S100A9 as active alarmins in the early stages of tendinopathy and thus targeting of its downstream signalling may offer novel therapeutic approaches in the management of human tendon disorders


Orthopaedic Proceedings
Vol. 94-B, Issue SUPP_VIII | Pages 57 - 57
1 Mar 2012
Okazaki S Matsumoto H Nagoya S Kaya M Sasaki M Tateda K Kosukegawa I Yamashita T
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Introduction. Although osteonecrosis of the femoral head has been observed in young adult patients with autoimmune diseases such as SLE and MCTD that are treated by corticosteroids, the pathogenesis of the osteonecrosis remains unclear. We established a rat model with osteonecrosis of the femoral head by injecting lipopolysaccharide (LPS) and corticosteroid, and assessed consequences of the histopathological alteration of the femoral head, the systemic immune response, and the lipid synthesis. Methods. Male Wistar rats were given 2 mg/kg LPS intravenously on days 0 and 1 and intramuscularly 20 mg/kg methylprednisolone on days 2, 3, and 4. The animals were sacrificed 1, 2, 3, or 4 weeks after the last injection of the methylprednisolone. Histopathological and biochemical analyses were performed every week. The bone samples were then processed for routine hematoxylin and eosin staining to assess the general architecture and injury of the tissue. The triglyceride and the total cholesterol concentrations in the PRP were measured. The levels of various cytokines (IL-1α, IL-1β, IL-2, IL-4, IL-6, IL-10, GM-CSF, IFN-γ, TNF-α) in blood samples were measured. Results. The body weight of the rats over time decreased for 2 weeks but had recovered by week 4. The plasma triglyceride concentrations had decreased significantly by weeks 2 and 3. The total plasma cholesterol concentrations had increased significantly by week 1 but then decreased significantly by week 4. The plasma concentrations of IL-1?α, IL-2, IL-4, IL-6, IL-10, GM-CSF, IFN-γ and TNF-α had increased significantly by week 1. These cytokines can all be induced by toll-like receptor 4 (TLR4) signaling. We defined osteonecrosis as the diffuse presence of empty lacunae or pyknotic nuclei of osteocytes in the bone trabeculae, accompanied by surrounding bone marrow cell necrosis. Osteonecrosis of the femoral head was observed only in the epiphysis of the femoral head in sacrificed specimen every week. Histological analysis revealed osteocytic death surrounded by necrotic bone marrow with or without repaired tissue. Conclusion. We established a new rat model of corticosteroid-induced femoral head osteonecrosis. The necrosis that is generated in this model is similar to that seen in patients treated with corticosteroid. In particular, the necrotic lesion was exclusively observed in the proximal epiphysis. LPS is known to activate the immune system via the TLR4 signaling pathway. It has been recognized that the unique immunogenic effects of LPS promote autoimmune disease . LPS and methylprednisolone induced osteonecrosis of the femoral head in rats and this was associated with a disruption of the innate immune system and lipid synthesis. These findings suggest that the TLR4 signaling pathway plays an important role in the pathogenesis for osteonecrosis of the femoral head


Bone & Joint Research
Vol. 5, Issue 12 | Pages 594 - 601
1 Dec 2016
Li JJ Wang BQ Fei Q Yang Y Li D

Objectives

In order to screen the altered gene expression profile in peripheral blood mononuclear cells of patients with osteoporosis, we performed an integrated analysis of the online microarray studies of osteoporosis.

Methods

We searched the Gene Expression Omnibus (GEO) database for microarray studies of peripheral blood mononuclear cells in patients with osteoporosis. Subsequently, we integrated gene expression data sets from multiple microarray studies to obtain differentially expressed genes (DEGs) between patients with osteoporosis and normal controls. Gene function analysis was performed to uncover the functions of identified DEGs.


Bone & Joint 360
Vol. 3, Issue 4 | Pages 35 - 38
1 Aug 2014
Hammerberg EM