Introduction

Osteoporosis is a common skeletal disorder characterised by a reduced bone mass and a progressive micro-architectural deterioration in bone tissue leading to bone fragility and susceptibility to fracture. With a progressively aging population, osteoporosis is becoming an increasingly important public health issue. The Wnt/β-catenin pathway is a major signalling cascade in bone biology, playing a key role in regulating bone development and remodelling, with aberrations in signalling resulting in disturbances in bone mass.

Introduction

Improvements in material properties of total joint prostheses and methods of fixation mean that arthroplasty is the most effective means of restoring mobility in osteoarthritic patients. Aseptic loosening is the major cause of long-term failure of prostheses. Cobalt particles may act directly on osteoblasts, decreasing bone formation and potentially playing a role in osteolysis and aseptic loosening.

Introduction: Despite a resurgence in cobalt-chromium metal-on-metal arthroplasty and hip resurfacing, the potential toxicity of cobalt ions in the periprosthetic area remains a cause for concern. Cytotoxic effects have been demonstrated in macrophages with cobalt ions inducing apoptosis and TNF-α secretion. A similar cytotoxic effect has been demonstrated in osteoblast-like cells. However, these studies assessed the acute cellular response to cobalt ions over 48 hours. To date, the effect on osteoblasts of chronic exposure to cobalt ions is unknown.

Aim: In this study we investigated the effect on osteoblasts of chronic exposure to cobalt ions. Specifically we investigated the chemokine response and effect on osteoblast function. We also investigated for a change in osteoblast phenotype to a less differentiated mesenchymal cell type.

Methods. Primary human osteoblasts were cultured and treated with cobalt (10ppm) over 21 days. Secreted chemokines (IL-8, MCP-1, TNF-α) were assayed using enzyme-linked immunosorbent assays (ELISA). Osteoblast function was assessed via alkaline phosphatase activity and calcium deposition. For a change in osteoblast phenotype, osteoblast gene expression was assessed using real time PCR. Immunoflourescent cell staining of actin filaments was used to examine for a change in osteoblast morphology.

Results: Chemokine (IL-8) secretion by osteoblasts was significantly increased after 7 days of stimulation with cobalt ions. In parallel with this, osteoblast function was also significantly inhibited as demonstrated by reduced alkaline phosphatase activity and calcium deposition. Regarding osteoblast phenotype, FSP-1, CTGF and TGF-β gene expression were upregulated after 7 days exposure indicating a transition in osteoblast phenotype to a less differentiated mesenchymal cell type. Immunoflourescent staining of actin filaments also showed a change in osteoblast morphology. Taken together, these data demonstrate cobalt ions induce a change in the osteoblast phenotype to that of a mesenchymal cell type. This is the first study to investigate osteoblast plasticity in the context of periprosthetic osteolysis.

Conclusion: After prolonged exposure to cobalt ions, IL-8 chemokine secretion is increased which attracts neutrophils to the periprosthetic area. Furthermore, osteoblasts no longer function as osteogenic cells as demonstrated by a decrease in osteoblast alkaline phosphatase activity and calcium deposition. Instead, they undergo transition to a mesenchymal cell type as demonstrated by an increase in the expression of genes associated with a mesenchymal cell lineage. Instead of secreting osteoid matrix the new cell type secretes unmineralized collagen. Cobalt ions are not benign and may play an important role in periprosthetic osteolysis by inducing osteoblasts to undergo transition to a less differentiated mesenchymal cell type.

Introduction: Ischaemic preconditioning (IPC) is a well recognised and powerful phenomenon where a tissue becomes more tolerant to prolonged ischaemia when it is first subjected to short bursts of ischaemia/reperfusion. IPC has been most comprehensively studied in cardiothoracic surgery, to date there has been little use of this powerful phenomenon in orthopaedic surgery. In this study, we report on the first clinical trial of IPC on human skeletal muscle, and show the potential of IPC in orthopaedics using global gene expression analysis.

Methods: After local ethics committee approval and informed consent, patients undergoing primary knee arthroplasty were randomly assigned into an IPC group and a control group. Diabetic patients or patients with an ankle/brachial index of less than 1 were excluded.

The IPC consisted of three five-minute periods of tourniquet insufflation on the operative limb, interrupted by five minute periods of reperfusion. The tourniquet was again insufflated and the operation started. The control group simply had tourniquet insufflation as normal prior to the start of surgery.

Muscle samples were taken from the operative knee of all patients at the immediate onset of surgery (t=0), and again, at one hour into the surgery (t=1). Total RNA was extracted from the muscle samples, and the gene expression profiles were determined using microarray technology.

Results: Comparison of IPC and control samples identified 702 transcripts with differences of ≥1.5-fold in their expression. Of these, 137 were altered at t=0 while 565 were altered at t=1. Amongst these changes was an up-regulation in the expression of a number of heat shock proteins (HSPs) in the IPC group as compared to the control group. Notably, there was up-regulation of the well known cytoprotective/anti-apoptotic gene, HSP72, at one hour post IPC (1.5-fold, p=0.039). There was also up-regulation of important oxidative stress defense genes, such as glutathione-S-transferase (1.6-fold, p = 0.021) and superoxide dismutase 2 (3.6-fold, p= 0.048). Microarray analysis also revealed a down-regulation in the expression of genes involved in metabolism, down-regulation of pro-apoptotic genes and up-regulation of genes necessary for transformation to a hypoxia-tolerant state.

Discussion: We present convincing evidence that IPC is beneficial to human skeletal muscle and for the first time show that IPC of human skeletal muscle works in the clinical setting. In this study, the protective effect of IPC involved a down-regulation in the expression of genes associated with metabolism, and an up-regulation in the expression of genes that provide protection from cell stress, oxidative stress and apoptosis. HSPs, and especially HSP72, have well documented roles in cell stress protection. Their presence has been cited by other studies as an indicator of cell adaptation to stress.

Osteoporosis is a common skeletal disorder characterised by a reduced bone mass and a progressive microarchitectural deterioration in bone tissue leading to bone fragility and susceptibility to fracture. The Wnt/β-catenin pathway is a major signaling cascade in bone biology, playing a key role in regulating bone development and remodeling, with aberrations in signalling resulting in disturbances in bone mass.

Our objectives were to assess the gene expression profile of primary human osteoblasts (HOBs) exposed to dexamethasone with a view to identifying key genes driving bone mass regulation and to assess the effects of the Wnt antagonist Dickkopf-1 (Dkk1) on the bone profile of primary human osteoblasts exposed in vitro to dexamethasone.

HOBs were cultured in vitro and exposed to 10–8M dexamethasone over a time course of 4hr, 12hr and 24hr. RNA isolation, cDNA synthesis, in vitro transcription and microarray analysis were performed. Microarray data was validated by quantitative real time RT-PCR. Dkk1 expression was silenced using small interfering RNA (siRNA). Quantitative RT-PCR was performed to confirm gene knockdown. Control and Dex-treated HOBs were compared with respect to bone turnover. Markers of bone turnover analyzed included alkaline phosphatase activity, calcium deposition, osteocalcin expression, along with cell proliferation and cellular apoptosis.

Global changes in HOB gene expression were elicited by dexamethasone.

Development associated gene pathways were co-ordinately dysregulated with the expression profile of key genes of the Wnt Pathway significantly altered. Dkk1 expression in HOBs was increased in response to dexamethasone exposure with an associated reduction in alkaline phosphatase activity, calcium deposition and osteocalcin expression. Silencing of Dkk1 expression, as confirmed by quantitative RT-PCR, was associated with an increase in alkaline phosphatase activity and calcium deposition, along with increased cell proliferation and reduced cellular apoptosis.

Dkk1 is an antagonist of Wnt/β-catenin signalling and plays a key role in regulating bone development and remodeling. Silencing the expression of Dkk1 in primary human osteoblasts has been shown to rescue the effects of dexamethasone-induced bone loss in vitro. The pharmacological targeting of the Wnt/β-catenin signaling pathway offers an exciting opportunity for the development of novel anabolic bone agents to treat osteoporosis and disorders of bone mass.

Introduction: Despite a resurgence in cobalt-chromium metal-on-metal arthroplasty, the potential toxicity of metal ions in the periprosthetic area remains a cause for concern. Studies to date have assessed the acute effect of cobalt ions on osteoblasts over 48 hours. The aim of our study was to determine the response of osteoblasts to cobalt ions over a prolonged period of exposure.

Methods. Primary human osteoblasts were cultured and treated with cobalt (10ppm) over 21 days. Osteoblast function was assessed via alkaline phosphatase activity and calcium deposition. ELISA were used to assess chemokine (IL-8, MCP-1 and TNF-α) secretion. Osteoblast gene expression was assessed using microarray analysis and real time PCR. Immunoflourescent cell staining of actin filaments was used to examine osteoblast morphology.

Results: Chemokine (IL-8) secretion by osteoblasts was significantly increased after 10 days of stimulation with cobalt ions. In parallel with this, osteoblast function was also significantly inhibited as demonstrated by reduced alkaline phosphatase activity and calcium deposition. Regarding osteoblast phenotype, FSP-1, CTGF and TGF-β gene expression were upregulated indicating a transition in osteoblast phenotype. Immunoflourescent staining of actin filaments also showed a change in osteoblast morphology. Taken together, these data show cobalt ions induce a change in the osteoblast phenotype to that of a mesenchymal cell type.

Conclusion: After 10 days of treatment with cobalt ions, osteoblasts no longer function as osteogenic cells. they undergo transition to a mesenchymal cell type. Furthermore, IL-8 secretion is increased which attracts neutrophils to the periprosthetic area thereby contributing to the inflammatory response that characterises osteolysis.

Wear debris is a key factor in the pathophysiology of aseptic loosening of orthopaedic endoprostheses. Cobalt-chromium-molybdenum (Co-CrMo) alloys are used for metal-metal hip implants due to their enhanced wear resistance profiles. Whilst these alloys have widespread clinical application, little is known about their direct effect on osteoblast biology. To address this issue, in this study we have investigated particle-mediated inflammation, as a putative mechanism of aseptic loosening. The effects of Co2+ ions on the bone cellular milieu were assessed in vitro by profiling of classical inflammatory mediators. The inflammatory driver PGE2 was quantified and found to be increased, following osteoblast stimulation with metal ions, suggesting the initiation of a local inflammatory response to metal particle exposure. To determine the biological import of this molecular event, the role of metal ions in recruiting inflammatory cells by chemokine production was assessed. These data demonstrated significant induction of the chemokines, IL-8 and MCP-1 following both 12 and 24 hour exposure to 10ppm of Co2+. In this study, we demonstrate that Co2+ particles can rapidly induce chemotactic cytokines, IL-8 and MCP-1 early stress-responsive chemokines that function in activation and chemotaxis of monocytes, and PGE2, which stimulates bone resorption. We have shown that this induction occurs at a transcriptional level with significantly increased mRNA levels. These data lend further weight to the hypothesis that wear mediated osteolysis, is due, at least in part, to underlying chronic inflammation.