Epidemiological studies have shown that accumulated mechanical stress is a risk factor for the development of osteoarthritis (OA). This debilitating progressive clinical condition affects a broad spectrum of patients and will ultimately lead to definitive arthroplasty surgery as the endpoint treatment option in many cases. The aim of this study is to establish a graded murine model of OA by medial meniscotibial destabilisation of the knee joint and in phase two, to investigate the migration and engraftment of radioisotope labeled mesenchymal stem cells (MSCs) at varying points of disease progression. The first phase of the study was to establish the murine model, an Irish first. All procedures were performed aseptically under general anaesthesia via a midline medial parapatellar approach on a murine fracture table. Microsurgical dissection was performed through necropsy analysed layers to the joint space and the meniscotibial ligament identified and transected. Validated histopathological analysis was performed at two, four, eight and twelve weeks postoperatively. The results showed a gradation of OA changes from mild unicondylar changes at two weeks, moderate unicompartmental change at four, severe unicompartmental change at eight and severe bicompartmental change at twelve weeks post-operatively. In vivo Bazooka-Single Photon Emission Computed Tomography (SPECT) (Phase 2) imaging studies are currently ongoing following the model establishment.

Introduction

Ischaemia reperfusion injury (IRI) is a very common metabolic insult in orthopaedics. It is often a subtle clinical event such as after brief tourniquet use, however severe injury, even multi-organ failure or death may result from prolonged tourniquet-use, crush injuries, vascular trauma or the release of compartment-syndrome. IRI is mediated by leukocyte infiltration and oxidatively-induced endothelial disruption. Antioxidants clearly attenuate or prevent this effect in animal models.

Background

70% of Breast Cancer patients develop metastatic bone deposits, predominantly spinal metasases. Adult Mesenchymal Stem Cells (MSCs) are multiprogenitor stem cells found within the bone marow which have the ability to self renew and differentiate into multiple cell types. MSCs home specifically to tumour sites, highlighting their potential as delivery vehicles for therapeutic agents. However studies show they may also increase tumour metastatic potential.

Over 80% of patients with advanced breast cancer will develop bone metastases for which there is no cure. Although thought to involve a complex cascade of cell-cell interactions, the factors controlling the development of bone metastases are still poorly understood. Osteoblasts may have an important role in mediating homing and proliferation of breast cancer cells to the bony environment.

This study aimed to examine the potential role osteoblasts have in the migration of circulating tumour cells to bone and the factors involved in this attraction.

Culture of osteoblasts and MDA-MB-231 breast cancer cells was performed. Breast cancer cell migration in response to osteoblasts was measured using Transwell Migration Inserts. Potential mediators of cell migration were detected using ChemiArray & ELISA assays. A luminometer based Vialight assay was used to measure breast cancer cell proliferation in response to factors secreted by osteoblasts.

There was a 3-4 fold increase of MDA-MB-231 migration in response to osteoblasts. ChemiArray analysis of osteoblast-conditioned medium revealed a range of secreted chemokines including IL-6 & 8, TIMP 1 & 2 and MCP-1. Initially, MCP-1 was quantified at 282 pg/ml, but rose to over 9000 pg/ml when osteoprogenitor cells were differentiated into mature osteoblasts. Inclusion of a monoclonal antibody to MCP-1 in osteoblast-conditioned medium resulted in a significant decrease in breast cancer cell migration to osteoblasts. There was no significant change in proliferation of MDA-MB 231 cells when exposed to osteoblast-conditioned medium.

Osteoblasts are capable of inducing breast cancer cell migration mediated at least in part by chemokine secretion. MCP-1 produced by the osteoblasts was shown to play a central role in mediating homing of the breast cancer cells. Increased understanding of the pathways involved in the development of bone metastases may provide new targets for therapeutic intervention.

Introduction: Ischaemia-reperfusion-injury (IRI) is one of the most common metabolic insults in orthopaedic practice. It is often a mild insult after brief tourniquet use with minimal clinical implications; but much more severe insults may result from excessive tourniquet-times, vascular trauma or release of compartment-syndrome. It is mediated largely by oxidatively-induced endothe-lial disruption and leukocyte infiltration. Antioxidants attenuate or prevent this effect in animal models.

Hypothesis: That IRI can be attenuated using established antioxidant medications (ascorbate and n-acetyl-cysteine) in the controlled setting of elective knee arthroscopy.

Methods: A EudraCT registered, prospective, randomized-controlled trial was performed. Patients (n=24) undergoing elective knee arthroscopy were randomized to one of 3 groups (IV NAC/oral ascorbate/placebo). Full blood counts, a broad array of cytokines and adhesion molecules, physiological response, pain scores and analgesia were recorded pre-operatively and at 3 postoperative time-points (10mins, 2hours, 4hours).

Results: Physiological response, analgesia and VAS did not differ. Systemic leukocytes and neutrophils were increased (p=0.001) indicating a measurable reperfusion injury. Ascorbate tended to inhibit ICAM-1 (p=0.10) and IFN-gamma (p=0.080). NAC inhibited VCAM-1 (p=0.003) and tended to inhibit ICAM-1 (p=0.094). Selectins responded in a similar pattern but not significantly. NAC tended to increase circulating leukocytes (0.093), neutrophils (0.12) and monocytes (0.04) and also induced a transient early increase in IFN-gamma (p=0.022).

Conclusions: Elevated circulating leukocytes indicate reduced leukocyte trapping and infiltration due to reduced adhesion molecule expression. NAC attenuates IRI resulting from tourniquet use in knee arthroscopy. The study was underpowered to confirm the efficacy of ascorbate in this setting. Further studies are necessary on the effects of these substances in more extreme ischaemic insults in which they may confer significant local and systemic benefits for the patient. Ascorbate and NAC act at different points in the inflammatory cascade and their potential synergistic effects warrant investigation.

Background: 70% of Breast Cancer patients develop metastatic bone deposits, predominantly spinal metasases. Adult Mesenchymal Stem Cells (MSCs) are multiprogenitor stem cells found within the bone marow which have the ability to self renew and differentiate into multiple cell types. MSCs home specifically to tumour sites, highlighting their potential as delivery vehicles for therapeutic agents. However studies show they may also increase tumour metastatic potential.

Aims: The aim of this study was to investigate interactions between MSCs and breast cancer cells to further elucidate their role in the tumour microenvironment and hence understand factors involved in stimulating the formation of bone metastases.

Methods: MSCs harvested from the iliac crest of healthy volunteers were grown for collection of conditioned medium (CM), containing all factors secreted by the cells. Breast cancer cell lines (T47D, SK-BR3) were then cultured in MSC CM +/− antibodies to TGFβ, VEGF, MCP-1 and CCL5 for 72hrs. Cell proliferation was assessed using an Apoglow^® assay and RNA harvested for analysis of changes in Epithelial Mesenchymal Transition specific gene expression: N-Cadherin, E-Cadherin, Vimentin, Twist, Snail.

Results: A significant down regulation of breast cancer cell proliferation in the presence of MSC secreted factors was observed (p< 0.05). There was a dramatic increase in expression of EMT specific genes in both cell lines following exposure to MSC-secreted factors. Inclusion of antibodies to TGF, VEGF, MCP-1 and CCL5 inhibited the effect seen, suggesting these paracrine factors played a role in the elevated expression levels.

Conclusion: MSCs clearly have a distinct paracrine effect on breast cancer epithelial cells, mediated at least in part through secretion of growth factors and chemokines. These factors play an important role in the metastatic cascade and may represent potential therapeutic targets to inhibit MSC-breast cancer interactions, helping to prevent the formation of bone metastases in cancer.

Objectives: Ischaemia reperfusion injury (IRI) is one of the most common metabolic insults in orthopaedic clinical practice. Oral ascorbate and both oral and intravenous n-acetylcysteine (NAC) have shown definitive beneficial effects in animal skeletal muscle IRI models. The authors hypothesized that a similar protective effect could be demonstrated in a well designed clinical trial.

Materials and Methods: A EudraCT registered, prospective, randomized, controlled, double blind trial was performed to assess the hypothesis. Ethical approval was obtained from the competent authority. Patients (n=18) undergoing elective knee arthroscopy were randomised to one of 3 groups. The NAC group received IV NAC and preoperative oral placebo. The ascorbate group received oral ascorbate and IV placebo. The placebo group received both oral and IV placebo. Anaesthetic protocols were standardized across all groups. Phlebotomy was performed preoperatively and at 3 post-operative time points. IL-1, 2, 6 and 10, ICAM, VCAM, Selectins, TNF-alpha and malondialdehyde (MDA) were measured in systemic and local blood samples. Physiological parameters were recorded in the peri-operative period. Post-operative analgesic requirements and visual analogue scores were recorded. Leg oedema was measured using volumetric analysis and figure-of-eight tape measurement.

Results: There were no differences between the groups pre-operatively. In the post-operative period the analgesic requirements were lower in the NAC group compared to ascorbate and placebo groups. CRP and d-dimers were found to peak in the early post operative period. White cell counts decreased in all groups in the early post-operative period, with a lesser reduction in the NAC and ascorbate groups. Elevation of MDA was noted in all groups but was significantly less in the NAC group. There was a trend towards increasing IL-6 and IL-8. There was a trend towards decreasing TNF-alpha and IL-1.

Conclusions: Ascorbate and NAC appear to attenuate the inflammatory response to IRI in a clinical model. These cheap, readily available medications which are acceptable to patients and doctors alike appear offer a potential benefit to patients. Further studies are required to clarify the extent of the benefit and to examine the role of these medications in trauma and in the setting of more extensive ischaemic insults.