Diatoms are unicellular microalgae whose cell walls are composed of remarkably uniform, hierarchical micro/nanopatterned, amorphous biosilica that cannot be replicated synthetically. Each species hosts its own unique morphology which is identically replicated generation-to-generation. There are currently estimated to be over 200,000 different diatom species, each with their own unique shape and morphology. This offers a huge array of surface topographies, particle sizes and shapes, each with the same silica precursor. Our research to date has shown that diatom-biosilica is non-cyctotoxic to J774.2 macrophages and hBMSC cells and does not invoke an immunological response or organ toxicity (kidney, spleen and liver) when tested in a murine model. Before testing diatom-biosilica
Ischaemic preconditioning protected skeletal myotubes against the effects of ischaemia-reperfusion in vitro. This protection was associated with increased Nrf2 signalling. Ischaemic preconditioning (IPC) is a well recognised and powerful phenomenon where a tissue becomes more tolerant to a period of prolonged ischaemia when it is first subjected to short bursts of ischaemia/reperfusion. While much is known about the ability of ischaemic preconditioning to protect myocardial tissue against ischaemia-reperfusion injury, its potential to confer benefit in an orthopaedic setting by protecting skeletal muscle remains relatively unexplored to date. One mechanism by which ischaemic preconditioning may induce protection is through a reduction in oxidative stress. Reactive oxygen species (ROS) are generated both during prolonged ischaemia and also upon reperfusion by infiltrating neutrophils, thereby leading to an increase in oxidative stress. The transcription factor, NF-E2-related factor 2 (Nrf2), is a key regulator of the cells response to oxidative stress as it regulates the expression of a network of anti-oxidant/detoxifying enzymes. Nrf2 signalling has recently been shown to protect against ischaemia-reperfusion injury in both a kidney cell line and in liver biopsies, indicating that this transcription factor may play a key role in the protection provided by ischaemic preconditioning. To date, the involvement of Nrf2 in the response of skeletal muscle to ischaemia-reperfusion has not been investigated. Thus, the aims of this study were to investigate the ability of ischaemic preconditioning to protect skeletal myotubes against ischaemia-reperfusion and to determine the role of Nrf2 signalling in this protection.Summary Statement
Introduction
Recent guidelines have been published by the Association of Neurophysiological Scientists / British Society for Clinical Neurophysiology (ANS/BSCN) regarding the use of intra-operative neurophysiological monitoring (IOM) during spinal deformity procedures. We present our unit's experience with IOM and the compliance with national guidelines. All patients undergoing intra-operative spinal cord monitoring during adult and paediatric spinal deformity surgery between Jan 2009 and Dec 2012 were prospectively followed. The use of somatosensory-evoked potentials (SSEPs) and motor-evoked potentials (MEPs) was recorded and monitoring outcomes were compared to post-operative clinical neurological outcomes. Compliance with the national ANS/BSCN guidelines was assessed.Aim:
Method:
Ischaemic preconditioning (IPC) is a phenomenon whereby tissues develop an increased tolerance to ischaemia and subsequent reperfusion if first subjected to sublethal periods of ischaemia. Despite extensive investigation of IPC, the molecular mechanism remains largely unknown. Our aim was to show genetic changes that occur in skeletal muscle cells in response to IPC. We established an in-vitro model of IPC using a human skeletal muscle cell line. Gene expression of both control and preconditioned cells at various time points was determined. The genes examined were HIF-1?, EGR1, JUN, FOS, and DUSP1. HIF-1? is a marker of hypoxia. EGR1, JUN, FOS and DUSP1 are early response genes and may play a role in the protective responses induced by IPC. Secondly, the expression of HSP22 was examined in a cohort of preconditioned total knee arthroplasty patients.Objectives
Methods
Matrix metalloproteinases (MMP) play a key role in cartilage degradation in osteoarthritis. Statins are a potential suppressor of MMPs. The aim of this research was to assess the efficacy of Pravastatin in suppressing MMP gene and protein expression in an in vitro model. We stimulated normal human chondrocytes with IL-1b for 6 hours to induce MMP expression and then treated with Pravastatin (1, 5 & 10 mM) for a further 18 hours. Cells stimulated with IL-1b but not treated with Pravastatin served as controls. Real-time PCR was used to assess expression of MMP-3 and MMP-9 mRNA. MMP enzyme activity was assessed using a fluorescent MMP-specific substrate. Staistical analysis was performed using ANOVA.Introduction
Methods
Ischaemic preconditioning (IPC) is a phenomenon whereby tissues develop an increased tolerance to ischaemia and subsequent reperfusion if first subjected to sublethal periods of ischaemia. Despite extensive investigation of IPC, the molecular mechanism remains largely unknown. Our aim was to show genetic changes that occur in skeletal muscle cells in response to IPC. Firstly, we established an in-vitro model of IPC using a human skeletal muscle cell line. Gene expression of both control and preconditioned cells at various time points was determined. The genes examined were HIF-1 alpha, EGR1, JUN, FOS, and DUSP1. HIF-1 alpha is a marker of hypoxia. EGR1, JUN, FOS and DUSP1 are early response genes and may play a role in the protective responses induced by IPC. Secondly, the expression of HSPB8 was examined in a cohort of preconditioned total knee arthroplasty patients.OBJECTIVES
METHODS
Local anaesthetic has been reported to have a detrimental effect on human chondrocytes both Human chondrocytes were grown under standard conditions. Cells were exposed to either lignocaine (0.5, 1, 2%), levobupivacaine (0.125, 0.25, 0.5%), bupivacaine (0.125, −.25, 0.5%) or ropivacaine (0.1875, 0.375, 0.75%) for 15 minutes. Cells were also exposed to a local anesthetic agent with the addition of magnesium (10, 20, or 50%). Cells exposed to media or saline served as controls. The MTS assay was used to assess cell viability 24-hours after exposure.Introduction
Methods
Ischaemic preconditioning (IPC) is a phenomenon whereby a tissue is more tolerant to an insult if it is first subjected to short bursts of sublethal ischaemia and reperfusion. The potential of this powerful mechanism has been realised in many branches of medicine where there is an abundance of ongoing research. However, there has been a notable lack of development of the concept in Orthopaedic surgery. The routine use of tourniquet-controlled limb surgery and traumatic soft tissue damage are just two examples of where IPC could be utilised to beneficial effect in Orthopaedic surgery. We conducted a randomized controlled clinical trial looking at the role of a delayed remote IPC stimulus on a cohort of patients undergoing a total knee arthroplasty (TKA). We measured the effect of IPC by analysing gene expression in skeletal muscle samples from these patients. Specifically we looked at the expression of Heat shock protein-90 (HSP-90), Catalase and Cyclo-oxygenase-2 (COX-2) at the start of surgery and at one hour into surgery. Gene analysis was performed using real time polymerase chain reaction amplification. As a second arm to the project we developed an in-vitro model of IPC using a human skeletal muscle cell line. A model was developed, tested and subsequently used to produce a simulated IPC stimulus prior to a simulated ischaemia-reperfusion (IR) injury. The effect of this on cell viability was investigated using crystal violet staining.Introduction
Methods
Local anaesthetic has been reported to have a potentially detrimental effect on human chondrocytes both in vitro and in vivo. Due to chondroproliferative effects, magnesium may be an alternative intra-articular analgesic agent following arthroscopy. We aimed to examine the dose response effect of commonly used local anaesthetics on chondrocyte viability and also to report on the effect of adding magnesium to the local anesthetic agent. Human chondrocytes were grown under standard culture conditions. Cells were exposed to either lignocaine (0.5, 1, 2%), levobupivacaine (0.125, 0.25, 0.5%), bupivacaine (0.125, 0.25, 0.5%) or ropivacaine (0.1875, 0.375, 0.75%) for 15 minutes. Cells were also exposed to a local anesthetic agent with the addition of magnesium (10, 20, or 50%). Cells exposed to culture media or saline served as controls. The MTS assay was used to assess cell viability 24 hours after exposure. One-way ANOVA were used to test for statistical significance.Introduction
Methods
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.
