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.

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.

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.

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.

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.

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.

Background

Adverse reactions to metal debris are implicated in the failure of metal-on-metal hip arthroplasty. The peri-implant tissues are often infiltrated by leukocytes which may cause observed immunological effects, including soft tissue necrosis and osteolysis. Cobalt ions from orthopaedic implants aberrantly activate the innate immune receptor human toll-like receptor-4 (TLR4), leading to inflammatory cytokine release including interleukin-8 (IL-8). IL-8 has been shown to increase expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1). These factors are essential for leukocyte adhesion to endothelium, which is required for leukocyte migration into tissues. This study investigates cobalt's effect on gene and protein changes in IL-8, ICAM-1 and VCAM-1 to determine their potential role in immune cell infiltration of peri-implant tissues.

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.

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.

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.