Background: Aseptic loosening remains the most common cause of failure of total hip arthroplasty. Its pathogenesis is based upon the generation of wear debris particles which trigger synovial macrophage activation.

Statins, inhibitors of 3-hydroxy-3 methylglutaryl coenzyme A (HMG-Co-A) reductase, have revolutionised the treatment of hypercholesterolaemia. More recently statins have been shown to have potent anti inflammatory effects. We investigated the effects of cerivastatin in attenuating the activation of human macrophages by polymethylmethacrylate (PMMA) particles.

Methods: Polymethylmethacrylate-particle-stimulated human macrophages were cultured in vitro with cerivastatin at 75 and 150... mols/litre. TNF-α (tumour necrosis factor alpha) and MCP-1 (monocyte chemotactic protein) expression were determined using ELISA. An ERK1/2 inhibitor, UO126 was utilised to identify the mitogen activated protein kinase (MAP-Kinase) pathway involved and western blotting was used to demonstrate the effect of Cerivastatin on this pathway.

Results PMMA-stimulated TNF-α and MCP-1 expression was consistently attenuated by cerivastatin therapy.

PMMA activation was attenuated by the ERK1/2 inhibitor, UO126.

Western blotting confirmed ERK downregulation by cerivastatin, establishing a mechanism for its anti-inflammatory effects.

Conclusion: We have demonstrated the beneficial effects of statins in suppressing particle mediated activation of macrophages and the potential to prevent or treat periprosthetic osteolysis.

The mechanism by which cells die is important in an immune response and its resolution. The role of apoptosis in sepsis and trauma, and its regulation by cytokines is unclear. During the systemic inflammatory response, rates of human neutrophil apoptosis are decreased. Peritoneal macrophage apoptosis has been induced by nitric oxide and Lipopolysaccharide (LPS) in vitro but this has not as yet been demonstrated in vivo.

We examined the induction and effects of macrophage apoptosis in a model of trauma and sepsis.

One hundred female CD-I mice were randomised into four groups: Control, Septic model, challenged with intraperitoneal LPS (1.Img/200ul/mouse), Traumatic model, received hind limb amputation (HLA) and a Combined trauma/septic model. After 24 hrs mice were sacrificed and peritoneal macrophages were assessed for apoptosis by morphology and DNA fragmentation by flow cytometry and DNA gel electrophoresis

Peritoneal lavage from septic models had a decreased percentage of macrophages in comparison to control and trauma groups. The septic model also had a significantly increased incidence of apoptosis in comparison to control and trauma levels. There was no significant difference between control and traumatic groups.

These findings demonstrate that in a murine model of sepsis, lipopolysaccharide induces macrophages apoptosis. Modulation of this immune response may have important roles in the management of trauma patients.

Background: Periprosthetic osteolysis precipitates aseptic component loosening, increases periprosthetic fracture risk and through massive bone loss, complicates revision surgery.

Its pathogenesis is based upon the generation of wear debris particles which trigger synovial macrophage activation. Statins, inhibitors of 3-hydroxy-3 methylglutaryl coenzyme A (HMG-Co-A) reductase, have revolutionised the treatment of hypercholesterolaemia and cardiovascular disease. The antiinflammatory properties of HMG-CoA reductase inhihitors or the statin family are well recognised. We investigated the effects of ceriv-astatin in attenuating the activation of human macrophages by polymethylmethacrylate (PMMA) particles.

Methods: Polymethylmethacrylate-particle-stimulated human macrophages were cultured in vitro with cerivastatin at 75 and 150micromols/litre. TNF- alpha (tumour necrosis factor alpha) and MCP-1 (monocyte chemotactic protein) expression were determined using ELISA. UO126, a Raf/MEK/ERK intracellular transduction pathway inhibitor, was utilised to identify the mitogen activated protein kinase (MAP- Kinase) pathway involved and western blotting was used to demonstrate the effect of cerivastatin on this pathway.

Results Human monocyte/macrophage cultures were activated by PMMA particles evidenced by TNF- alpha and MCP-1 expression(p< 0.05). This activation was consistently attenuated by cerivastatin therapy. Similarily, PMMA activation was attenuated by the Raf/MEK/ERK inhibitor, UO126.

Western blotting confirmed Raf/MEK/ERK down-regulation by cerivastatin, establishing a mechanism for its anti-inflammatory effects.

Conclusion We have demonstrated in vitro, that statins can abrogate particle induced inflammatory responses in a dose dependent manner and this is mediated intra-cellularily through its effect on the Raf/MEK/ERK transduction pathway. We propose that by attenuating this inflammatory response, the associated subsequent osteoclast activation and osteolysis is attenuated. Statins therefore may have role in promoting implant longevity

Background Apoptosis of osteoblasts and osteoclasts regulates bone homeostasis. Skeletal injury in humans results in angiogenic responses primarily mediated by vascular endothelial growth factor(VEGF), a protein essential for bone repair in animal models. Osteoblasts release VEGF in response to a number of stimuli and express receptors for VEGF in a differentiation dependent manner. This study investigates the putative role of VEGF in regulating the lifespan of primary human osteoblasts(PHOB) in vitro.

Methods PHOB were examined for VEGF receptors. Cultures were supplemented with VEGF(0–50ng/mL), a neutralising antibody to VEGF, mAB VEGF(0.3ug/mL) and Placental Growth Factor (PlGF), an Flt-1 receptor-specific VEGF ligand(0–100 ng/mL) to examine their effects on mineralised nodule assay, alkaline phosphatase assay and apoptosis.. The role of the VEGF specific antiapoptotic gene target BCl2 in apoptosis was determined.

Results PHOB expressed functional VEGF receptors. VEGF 10 and 25 ng/mL increased nodule formation 2.3- and 3.16-fold and alkaline phosphatase release 2.6 and 4.1-fold respectively while 0.3ug/mL of mAB VEGF resulted in approx 40% reductions in both. PlGF 50ng/mL had greater effects on alkaline phosphatase release (103% increase) than on nodule formation (57% increase). 10ng/mL of VEGF inhibited spontaneous and pathological apoptosis by 83.6% and 71% respectively, while PlGF had no significant effect. Pretreatment with mAB VEGF, in the absence of exogenous VEGF resulted in a significant increase in apoptosis (14 vs 3%). BCl2 transfection gave a 0.9% apoptotic rate. VEGF 10 ng/mL increased BCl2 expression 4 fold while mAB VEGF decreased it by over 50%.

Conclusions VEGF is a potent regulator of osteoblast lifespan in vitro. This autocrine feedback regulates survival of these cells, mediated via the KDR receptor and expression of BCl2 antiapoptotic gene.

Aims: Pharmacological modulation of skeletal muscle reperfusion injury after trauma associated ischaemia may improve limb salvage rates and prevent the associated systemic sequelae. Resuscitation with hypertonic saline restores the circulating volume and has favourable effects on tissue perfusion and blood pressure. The purpose of our study was to evaluate the effects of hypertonic saline on skeletal muscle ischaemia reperfusion (I/R) injury and the associated endorgan injury.

Methods: Adult male Sprague Dawley rats (n=24) were randomised into three groups: control group, I/R group treated with normal saline and I/R group treated with hypertonic saline. Bilateral hind-limb ischaemia was induced by rubber band application proximal to the level of the greater trochanters for 2.5 hours. Treatment groups received either normal saline or hypertonic saline prior to tourniquet release. Following twelve hours reperfusion, the tibialis anterior muscle was dissected and muscle function assessed electrophysiologically by electrical field stimulation. The animals were then sacrificed and skeletal muscle harvested for evaluation. Lung tissue was also harvested for measurement of wet-to-dry ratio, myeloperoxidase content and histological analysis.

Results: Hypertonic saline significantly attenuated skeletal muscle reperfusion injury as shown by reduced twitch and tetanic contractions of the skeletal muscle (Table). There was also a significant reduction in lung injury as demonstrated by differences in wet-to-dry ratio, myeloperoxidase content and histological analysis.

Conclusion: Resuscitation with hypertonic saline may have a protective role in attenuating skeletal muscle ischaemia reperfusion injury and its associated systemic sequelae.

Aims: Circulating endothelial precursor cells (CEPs) are thought to play a role in angiogenesis. We investigated the angiogenic stress of musculoskeletal trauma on CEP kinetics in trauma patients and their bone marrow progenitor populations in a murine model. Methods: Peripheral blood mononuclear cells (PB-MNCs) were isolated from patients (n=12) on consecutive days following closed lower-limb diaphyseal fractures. CEP levels, deþned by the surface expression patterns of VEGFR2, CD34 and AC133 were determined and cytokine analysis of collected serum was performed. Bonemarrow precursors deþned byLy-6A/E and c-Kit expression were harvested following the traumatic insult from the murine model and quantiþed on ßow cytometry. Human and murine progenitor populations were cultured on þbronectin and examined for markers of endothelial cell lineage (Ulexeuropaeus- agglutinin-1 binding and acetylated-LDL uptake) and cell morphology. Statistical analysis was performed using variance analysis. Results: A consistent increase in human CEPs levels was noted within 72 hours of the initial insult, the percentage increase over day 1 reaching 300% (p=0.008) and returning to normal levels by day 10. Murine bone marrow precursors were mobilisd within 24 hrs peaking at 48hrs (900% p=0.035). On culture, morphologically characteristic endotheliallike cells binding UEA-1 and incorporating LDL were identiþed. Serum VEGF levels increased signiþcantly within 24 hrs of the insult, (p=0.018) preceeding the peak in CEP mobilisation. Conclusion: We propose that musculoskeletal trauma through the release of chemokines such as VEGF, promotes rapid mobilisation of CEPs from born marrow, which have the potential to contribute to reparative neovascularisation. Strategies to enhance CEPs kinetics may accelerate this process and offer a therapeutic role in aberrant fracture healing.

Background: Circulating endothelial precursor cells (CEPS) are thought to play a role in postnatal angiogenesis. We investigated the angiogenic stress of musculoskeletal trauma on CEP kinetics in trauma patients and their bone marrow progenitor populations in a murine model.

Methods: Peripheral blood mononuclear cells (PB-MNCs) were isolated from patients (n=12) on consecutive days following closed lower-limb diaphyseal fractures. CEP levels, defined by the surface expression patterns of VEGFR2, CD34 and AC133 were determined and cytokine analysis of collected serum was performed. Bone marrow precursors defined by Ly-6A/E and c-Kit expression were harvested following traumatic insult from the murine model and quantified on flow cytometry. Human and murine progenitor populations were cultured on fibronectin and examined for markers of endothelial cell linage (Ulexeuropaeus- agglutinin- 1 binding and acetylated-LDL uptake) and cell morphology. Statistical analysis was performed using variance analysis.

Results: A consistent increase in human CEPs levels was noted within 72 hours of the initial insult, the percentage increase over day 1 reaching 300%.

Conclusion: We propose that musculoskeletal trauma through the release of chemokines such as VEGF, promotes rapid mobilisation of CEP from born marrow, which have the potential to contribute to reparative neovascularisation. Strategies to enhance CEPs kinetics may accelerate this process and offer a therapeutic role in aberrant fracture healing.

To determine whether systemic nitric oxide production in tourniquet-induced skeletal muscle ischaemia-reper-fusion injury (SMRI) is dependent on release of vascular endothelial growth factor (VEGF), a modulator of nitric oxide cytoprotection in myocardial ischaemia-reperfusion injury.

Mice were randomised (n=10 per group) into: time controls (no tourniquet) and test animals (bilateral hindlimb tourniquet ischaemia). Blood samples were collected in test animals prior to ischaemia and after reper-fusion. In controls, blood samples were collected at the same corresponding time points. Serum VEGF, nitric oxide metabolites (nitrite and nitrate) and the proinflammatory cytokine tumour necrosis factor (TNF)-α (an indicator of systemic inflammation) were determined. At the end of reperfusion, the lungs and muscle (right gastrocnemius) were harvested and tissue injury determined by measuring myeloperoxidase (MPO) activity, a marker of neutrophil infiltration. Data are presented as mean ± SEM and statistical comparison was performed using one-way analysis of variance (ANOVA) with significance attributed to P < 0.05.

In comparison to control animals, muscle (4.9±0.3 versus 4±0.03 units/g of wet tissue; P=0.02) and lung (16.7±1.9 versus 10.4±0.5; P=0.005) MPO activity at the end of repercussion was significantly greater in test animals. The table shows the results with respect to serum cytokine levels and nitricxide metabolites.

These data demonstrate that SMRI results in local and systemic proinflammatory responses. In contrast to myocardial ischaemia-reperfusion injury, nitric oxide production in tourniquet-induced SMRI is VEGF-independent. Alternative mechanisms for nitric oxide production in tourniquet-controlled extremity surgery requires further evaluation.

Introduction: Limb reperfusion in patients following pneumatic tourniquet-controlled surgery is associated with nitric oxide (NO) generation. Meanwhile, NO mediates vascular endothelial growth factor (VEGF)-cytoprotection in myocardial ischaemia-reperfusion injury. In addition, VEGF is contributory in attenuating skeletal muscle ischaemia-reperfusion injury (SMRI). Whether this effect of VEGF is NO-mediated in SMRI is unknown. We investigate whether systemic nitric oxide production in tourniquet-induced SMRI is dependent on VEGF release.

Methods: Anaesthetised male C57BL/6 mice were randomised (n=10 per group) into two groups: time controls (no tourniquet) and test animals with bilateral hindlimb tourniquets (SMRI; 2 hours of ischaemia, 2 hours of reperfusion). Blood samples were collected in test animals prior to ischaemia and after 2 hours of reperfusion. In controls, blood samples were collected at the same corresponding time points. Serum VEGF, nitric oxide metabolites (nitrite and nitrate) and the proinflammatory cytokine tumour necrosis fractor (TNF)-α (an indicator of systemic inflammation) were determined. At the end of reperfusion, the lungs and muscle (right gastrocnemius) were harvested and tissue injury determined by measuring myeloperoxidase (MPO) activity, a marker of neutrophil infiltration. Data are presented as mean ± SEM and statistical comparison was performed using one-way analysis of variance (ANOVA) with significance attributed to P,0.05.

Results: In comparison to control animals, both the muscle (4.9±0.3 versus 4±0.03 units/g of wet tissue; P=0.02) and lung (16.7±1.9 versus 10.4±0.5; P=0.005) MPO activity at the end of reperfusion was significantly greater in test animals.

Conclusions: Our data demonstrates that SMRI results in local and systemic proinflammatory responses. In contrast to myocardial ischaemia-reperfusion injury, nitric oxide production in tourniquet-induced SMRI is VEGF-independent. Alternative mechanisms for nitric oxide production in tourniquet-controlled limb surgery requires further evaluation.

Background: Low molecular weight heparins (LMWH) are of undoubted efficacy as thromboprophylaxis in orthopaedic surgical practice. However, prolonged dosage inhibits bone nodule formation in vitro and we have previously reported that daily dosing significantly delays fracture healing. To further investigate these phenomena we hypothesised that LMWH’s would reduce osteoblast survival and thus bone formation by inducing programmed cell death (apoptosis).

Methods: Primary human osteoblasts were isolated from femoral heads excised during hip arthoplasty and cultured to passage 3–5. These were examined for VEGF receptor expression using a biotinylated binding assay on flow cytometry. Osteoblasts were grown to confluence and then incubated for 24 hours in control medium or medium treated with enoxaparin (200 – 2X10(−4) IU/mL) or combination of enoxaparin (200 – 2X10 (−4) IU/mL) and VEGF (1ng/ml). Apoptosis was determined by measuring cytosolic histone-associated DNA fragmentation using an enzyme linked immunosorbant assay. Results were confirmed by DNA fragmentation analysis on agarose gel electrophoresis. Cell functional viability was measured by a tetrazolium bioreduction colorimetric assay.

Results: Data is expressed as percentage of control apoptosis or viability, illustrates mean ± s.e.m. and n=4 experiments in each case. ANOVA was employed for statistical analysis; *versus control, #versus enoxaparin treated; p< 0.05 was considered significant.

Conclusions: Therapeutic doses of LMWH attenuate osteo-blast survival by inducing significant apoptosis. This effect is partly abrogated by VEGF, which independently enhances osteoblast viability, thus delaying spontaneous and enoxaparin induced apoptosis. These findings may explain the bone resorptive effects of prolonged LMWH therapy and suggest a potential therapeutic role for VEGF in conditions of delayed bone formation.