Background. Membrane type 1 matrix metalloproteinase (MT1-MMP) plays a role in the progression of several common solid cancers. Given that osteosarcoma features extensive local invasion and haematogenous metastases, we hypothesised that osteosarcoma cells utilise MT1-MMP to drive these processes. Moreover, since hypoxia regulates MT1-MMP expression in breast cancer we investigated the effects of hypoxia on MT1-MMP expression in osteosarcoma cells. Aims. Examination of MT1-MMP expression in osteosarcoma biopsy tissue in relation to clinical outcome. Assessment of MT1-MMP, together with hypoxia inducible factors HIF-1α and HIF-2α expression in a panel of osteosarcoma cell lines under normoxia and hypoxia. Methods. Immunohistochemistry: Formalin-fixed and paraffin embedded osteosarcoma biopsy samples from 71 patients were immunostained for MT1-MMP, HIF-1α and -2α and the data correlated with patient survival. Confocal microscopy: following 24 hours culture in 20% versus 1% oxygen, a panel of osteosarcoma cell lines were analysed for the subcellular distribution of MT1-MMP, HIF-1α and -2α. Subcellular fractionation: following 48 hours culture in 20% versus 1% oxygen, the U2OS cell line was fractionated and the compartmental lysates immunoblotted for MT1-MMP, HIF-1α and -2α. Results. Immunohistochemistry showed MT1-MMP immunopositive cytoplasmic and nuclear staining. Biopsy samples with the highest MT1-MMP and HIF-2α intranuclear staining correlated with reduced patient survival: HR 16.10; (95% CI: 5.1–40.3); p< 0.0001. In vitro studies confirmed the intranuclear MT1-MMP presence with an increased nuclear fraction in hypoxia and evidence of nuclear co-localisation with HIF-2α. Conclusions. MT1-MMP expression in osteosarcoma tissue correlates with patient survival. The functional significance of the increased intranuclear presence in hypoxia warrants further investigation

This is quite an innovative study that should lead to a multicentre validation trial. We have developed an FDG-PET/MRI texture-based model for the prediction of lung metastases (LM) in newly diagnosed patients with soft-tissue sarcomas (STSs) using retrospective analysis. In this work, we assess the model performance using a new prospective STS cohort. We also investigate whether incorporating hypoxia and perfusion biomarkers derived from FMISO-PET and DCE-MRI scans can further enhance the predictive power of the model. A total of 66 patients with histologically confirmed STSs were used in this study and divided into two groups: a retrospective cohort of 51 patients (19 LM) used for training the model, and a prospective cohort of 15 patients (two patients with LM, one patient with bone metastases and suspicious lung nodules) for testing the model. In the training phase, a model of four texture features characterising tumour sub-region size and intensity heterogeneities was developed for LM prediction from pre-treatment FDG-PET and MRI scans (T1-weighted, T2-weighted with fat saturation) of the retrospective cohort, using imbalance-adjusted bootstrap statistical resampling and logistic regression multivariable modeling. In the testing phase, this multivariable model was applied to predict the distant metastasis status of the prospective cohort. The predictive power of the obtained model response was assessed using the area under the receiver-operating characteristic curve (AUC). In the exploratory phase of the study, we extracted two heterogeneity metrics from the prospective cohort: the area under the intensity-volume histogram of pre-treatment DCE-MRI volume transfer constant parametric maps and FMISO-PET hypoxia maps (AU-IVH-Ktrans, AU-IVH-FMISO). The impact of the addition of these two individual metrics to the texture-based model response obtained in the testing phase was first investigated using Spearman's correlation (rs), and lastly using logistic regression and leave-one-out cross-validation (LOO-CV) to account for overfitting bias. First, the texture-based model reached an AUC of 0.94, a sensitivity of 1, a specificity of 0.83 and an accuracy of 0.87 when tested in the prospective cohort. In the exploratory phase, the addition of AU-IVH-FMISO did not improve predictive power, yielding a correlation of rs = −0.42 (p = 0.12) with lung metastases, and a relative change in validation AUC of 0% in comparison with the texture-based model response alone in LOO-CV experiments. In contrast, the addition of AU-IVH-Ktrans improved predictive power, yielding a correlation of rs = −0.54 (p = 0.04) with lung metastases, and a change in validation AUC of +10%. Our results demonstrate that texture-based models extracted from pre-treatment FDG-PET and MRI anatomical scans could be successfully used to predict distant metastases in STS cancer. Our results also suggest that the addition of perfusion heterogeneity metrics may contribute to improving model prediction performance

Purpose. Traumatic articular cartilage (AC) defects are common in young adults and frequently progresses to osteoarthritis. Matrix-Induced Autologous Chondrocyte Implantation (MACI) is a recent advancement in cartilage resurfacing techniques and is a variant of ACI, which is considered by some surgeons to be the gold standard in AC regeneration. MACI involves embedding cultured chondrocytes into a scaffold that is then surgically implanted into an AC defect. Unfortunately, chondrocytes cultured in a normoxic environment (conventional technique) tend to de-differentiate resulting in decreased collagen II and increased collagen I producing in a fibrocartilagous repair tissue that is biomechanically inferior to AC and incapable of withstanding physiologic loads over prolonged periods. The optimum conditions for maintenance of chondrocyte phenotype remain elusive. Normal oxygen tension within AC is <7%. We hypothesized that hypoxic conditions would induce gene expression and matrix production that more closely characterizes normal articular chondrocytes than that achieved under normoxic conditions when chondrocytes are cultured in a collagen scaffold. Method. Chondrocytes were isolated from Outerbridge grade 0 and 1 AC from four patients undergoing total knee arthroplasty and embedded within 216 bovine collagen I scaffolds. Scaffolds were incubated in hypoxic (3% O2) or normoxic (21% O2) conditions for 1hr, 21hr and 14 days. Gene expression was determined using Q-rt-PCR for col I/II/X, COMP, SOX9, aggrecan and B actin. Matrix production was determined using glycosaminoglycan (GAG) content relative to cell count determined by DNA quantification. Cell viability and location within the matrix was determined by Live/Dead assay and confocal microscopy. Statistical analysis was performed using a two-tailed T-test. Results. Chondrocytes cultured under hypoxic conditions showed an upregulation of all matrix related genes compared to normoxic conditions noted most markedly in col II, COMP and SOX9 expression. There were similar numbers of chondrocytes between hypoxic and normoxic groups (P=0.68) but the chondrocytes in the hypoxic group produced more GAG per cell (P= 0.052). Viable cells were seen throughout the matrix in both groups. Conclusion. Important matrix related genes (col II, COMP, SOX9) were most significantly upregulated in hypoxic conditions compared to normoxic conditions. This was supported by an increase in GAG production per cell in hypoxic conditions. The results indicate that hypoxia induces an upregulation in the production of extracellular matrix components typical of AC with only modest increases in col I (possibly related to the col I based scaffold used in this experiment). These results indicate that hypoxic conditions are important for the maintenance of chondrocyte phenotype even when the cells are cultured in a 3D environment. In conclusion, hypoxic culture conditions should be used to help maintain chondrocyte phenotype even when culturing these cells in a 3D scaffold

Despite advances in treating acute spinal cord injury (SCI), measures to mitigate permanent neurological deficits in affected patients are limited. Augmentation of mean arterial blood pressure (MAP) to promote blood flow and oxygen delivery to the injured cord is one of the only currently available treatment options to potentially improve neurological outcomes after acute spinal cord injury (SCI). However, to optimize such hemodynamic management, clinicians require a method to measure and monitor the physiological effects of these MAP alterations within the injured cord in real-time. To address this unmet clinical need, we developed a series of miniaturized optical sensors and a monitoring system based on multi-wavelength near-infrared spectroscopy (MW-NIRS) technique for direct transdural measurement and continuous monitoring of spinal cord hemodynamics and oxygenation in real-time. We conducted a feasibility study in a porcine model of acute SCI. We also completed two separate animal studies to examine the function of the sensor and validity of collected data in an acute experiment and a seven-day post-injury survival experiment. In our first animal experiment, nine Yorkshire pigs underwent a weight-drop T10 vertebral level contusion-compression injury and received episodes of ventilatory hypoxia and alterations in MAP. Spinal cord hemodynamics and oxygenation were monitored throughout by a transdural NIRS sensor prototype, as well as an invasive intraparenchymal (IP) sensor as a comparison. In a second experiment, we studied six Yucatan miniature pigs that underwent a T10 injury. Spinal cord oxygenation and hemodynamics parameters were continuously monitored by an improved NIRS sensor over a long period. Episodes of MAP alteration and hypoxia were performed acutely after injury and at two- and seven-days post-injury to simulate the types of hemodynamic changes patients experience after an acute SCI. All NIRS data were collected in real-time, recorded and analyzed in comparison with IP measures. Noninvasive NIRS parameters of tissue oxygenation were highly correlated with invasive IP measures of tissue oxygenation in both studies. In particular, during periods of hypoxia and MAP alterations, changes of NIRS-derived spinal cord tissue oxygenation percentage were significant and corresponded well with the changes in spinal cord oxygen partial pressures measured by the IP sensors (p < 0.05). Our studies indicate that a novel optical biosensor developed by our team can monitor real-time changes in spinal cord hemodynamics and oxygenation over the first seven days post-injury and can detect local tissue changes that are reflective of systemic hemodynamic changes. Our implantable spinal cord NIRS sensor is intended to help clinicians by providing real-time information about the effects of hemodynamic management on the injured spinal cord. Hence, our novel NIRS system has the near-term potential to impact clinical care and improve neurologic outcomes in acute SCI. To translate our studies from bench to bedside, we have developed an advanced clinical NIRS sensor that is ready to be implanted in the first cohort of acute SCI patients in 2022

Aim. Osteomyelitis is a difficult-to-treat disease with high chronification rates. The surgical amputation of the afflicted limb sometimes remains as the patients’ last resort. Several studies suggest an increase in mitochondrial fission as a possible contributor to the accumulation of intracellular reactive oxygen species and thereby to cell death of infectious bone cells. The aim of this study is to analyze the ultrastructural impact of bacterial infection and its accompanying microenvironmental tissue hypoxia on osteocytic and osteoblastic mitochondria. Method. 19 Human bone tissue samples from patients with osteomyelitis were visualized via light microscopy and transmission electron microscopy. Osteoblasts, osteocytes and their respective mitochondria were histomorphometrically analyzed. The results were compared to the control group of 5 non-infectious human bone tissue samples. Results. The results depicted swollen hydropic mitochondria including depleted cristae and a decrease in matrix density in the infectious samples as a common finding in both cell types. Furthermore, perinuclear clustering of mitochondria could also be observed regularly. Additionally, increases in relative mitochondrial area and number could be found as a sign for increased mitochondrial fission. Conclusions. The results show that mitochondrial morphology is altered during osteomyelitis in a comparable way to mitochondria from hypoxic tissues. This suggests that manipulation of mitochondrial dynamics in a way of inhibiting mitochondrial fission may improve bone cell survival and exploit bone cells regenerative potential to aid in the treatment of osteomyelitis

Cellular therapies play an important role in tendon tissue engineering with tenocytes being described as the most prominent cell population if available in large numbers. However, in vitro expansion of tenocytes in standard culture leads to phenotypic drift and cellular senescence. Recent work suggests that maintenance of tenogenic phenotype in vitro can be achieved by recapitulating different aspects of the native tendon microenvironment. One approach used to modulate the in vitro microenvironment and enhance extracellular matrix (ECM) deposition is macromolecular crowding (MMC). MMC is based on the addition of inert macromolecules to the culture media mimicking the dense extracellular matrix. In addition, as tendon has been described to be a relatively avascular and hypoxic tissue and low oxygen tension can stimulate collagen synthesis and cross-linking, we venture to assess the synergistic effect of MMC and low oxygen tension on human tenocyte phenotype maintenance by enhancing synthesis and deposition of tissue-specific ECM. Human tendons were kindly provided from University Hospital Galway, after obtaining appropriate licenses, ethical approvals and patient consent. Afterwards, tenocytes were extracted using the migration method. Experiments were conducted at passage three. Optimization of MMC conditions was assessed using 50 to 500 μg/ml carrageenan (Sigma Aldrich, UK). For variable oxygen tension cultures, tenocytes were incubated in a Coy Lab (USA) hypoxia chamber. ECM synthesis and deposition were assessed using SDS-PAGE (BioRad, UK) and immunocytochemistry (ABCAM, UK) analysis. Protein analysis for Scleraxis (ABCAM, UK) was performed using western blot. Gene analysis was conducted using a gene array (Roche, Ireland). Cell morphology was assessed using bright-field microscopy. All experiments were performed at least in triplicate. MINITAB (version 16, Minitab, Inc.) was used for statistical analysis. Two-sample t-test for pairwise comparisons and ANOVA for multiple comparisons were conducted. SDS-PAGE and immunocytochemistry analysis demonstrated that human tenocytes treated with the optimal MMC concentration at 2% oxygen tension showed increased synthesis and deposition of collagen type I, the major component of tendon ECM. Moreover, immunocytochemistry for the tendon-specific ECM proteins collagen type III, V, VI and fibronectin illustrated enhanced deposition when cells were treated with MMC at 2% oxygen tension. In addition, protein analysis revealed elevated dexpression of the tendon-specific protein Sclearaxis, while a detailed gene analysis revealed upregulation of tendon-related genes and downregulation of trans-differentiation markers again when cells cultured with MMC at 2% oxygen tension. Finally, low oxygen tension and MMC did not affect the metabolic activity, proliferation and viability of human tenocytes. Collectively, results suggest that the synergistic effect of MMC and low oxygen tension can accelerate the formation of ECM-rich substitutes, which stimulates tenogenic phenotype maintenance. Currently, the addition of substrate aligned topography together with MMC and hypoxia is being investigated in this multifactorial study for the development of an implantable device for tendon regeneration

Purpose. Bone marrow multi-potent stromal cells represent a heterogenous source of cells with great promise in joint cartilage regenerative medicine. However, due to their low numbers upon harvesting, MSCs need to be expanded without compromising their capacity to form chondrocytes (cartilage cells). To date there is no consensus on how to expand MSCs in order to maximize their potential for cartilage repair and nor are there any specific cell signatures of MSCs with chondrogenic propensity. Emerging evidence suggest that marrow stem cells exist in a hypoxic microenvironment. On this basis and in addition to cartilages natural existence in hypoxic environment (1–7% O2), we hypothesized that MSC expansion under hypoxia will result in the enrichment of MSCs with predilection to chondrocytes compared to expansion under the conventional culture conditions of 21% O2. Method. Bone marrow was harvested from the iliac crest of 4 donors (mean age 43.5 years) post informed consent and local ethical approval. Fifteen million mono-nucleated (MNCs) cells were seeded into T150cm2 culture flasks in the presence of alpha MEM plus 10% FBS and 5 ng/ml FGF2. Similarly, 0.25 million MNCs were seeded in 10cm petri dishes for colony forming unit-fibroblastic (CFU-f) assay. The seeded flasks and petri dishes were cultured under normoxia (21% O2) and hypoxia (3% O2). Petri dished cells were cultured for 14 days and those in flasks were cultured until passage 2 (P2). Developed cell colonies per dish were revealed after crystal violet staining. Colony counts and diameters were recorded. P2 cells were treated with a panel of antibodies for cell surface marker analysis by fluorescent activated cell sorting (FACS) flow cytometry. P2 cell pellets were formed and induced towards cartilage in a defined serum free medium containing TGFβ1. Pellets were cultured for 3 weeks under normoxia and were then processed for histological, biochemical and gene expression analyses. Results. The mean number of cell colonies was 1.25-fold higher after hypoxia culture relative to normoxia. There were no differences in colony diameters. A panel of common protein signatures (CD29, CD90, CD105 and CD151) for stem cells declined in expression after expansion in hypoxia. However, other signatures (CD13, CD34 and CD44) expression level increased under hypoxia, whilst CD73 expression was unchanged. Pellets from hypoxia-expanded MSCs showed on average a 1.4-fold increase in chondrogenic capacity as judged by glycosaminoglycan (GAG) matrix per DNA content relative to normoxia pellets. The gene expression of collagen II, SOX9, aggrecan and matrillin-3 increased by 1.2-, 2-, 1.3- and 1.5-fold, respectively, in pellets formed from hypoxia-expanded stem cells relative to their normoxia counterparts. Conclusion. Expansion of stem cells under hypoxia potentiates their capacity to form cartilage with improved cartilage properties. However, there is a need for signatures to identify stem cells with propensity to form cartilage

Objectives. 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. Methods. 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. Results. HIF-1? was upregulated following 1 and 2 hours of simulated ischaemia (p = 0.076 and 0.841 respectively) verifying that hypoxic conditions were met. Expression of EGR1, FOS and DUSP1 were upregulated and peaked after 1 hour of hypoxia (p = 0.001, < 0.00, and 0.038 respectively). cFOS was upregulated at 2 and 3 hours of hypoxia. IPC prior to simulated hypoxia resulted in a greater level of upregulation of EGR1, JUN and FOS genes (p = < 0.00, 0.047, and < 0.00 respectively). HSP22 was not significantly upregulated following IPC using the hypoxic model. It was, however, upregulated on an mRNA level in total knee arthroplasty patients (p = 0.15). Conclusion. This study has validated the use of our hypoxic model for studying IPC in-vitro. IPC results in a greater upregulation of protective genes in skeletal muscle cells exposed to hypoxia than in control cells. We have demonstrated hitherto unknown molecular mechanisms of IPC in cell culture and in patients undergoing TKA

Aim. Determine the incidence of surgical site infections (SSI) after intramedullary nailing (IN) of femoral and tibial diaphyseal fractures and evaluate possible risk factors. Method. Prospective observational cohort study. SSI was defined according to CDC-NHSN criteria and surveillance period for the occurrence of infection was 12 months instead of the 90 days currently recommended. Incidence was calculated as the ratio between the number of patients with SSI and total number of patients. Analysis of potential risk factors included patients-related factors (age, gender, body mass index, active foci of infection, immunosuppressive conditions, ASA score, alcohol or illicit drug abuse, smoking, polytrauma, etiology of fracture, type of fracture if closed or open, classification of fracture according to Müller AO, Tcherne classification for closed fractures, Gustilo-Anderson classification and duration of bone exposure for open fractures, previous stay in other healthcare services, use of external fixator, previous surgical manipulation at same topography of fracture, use of blood products); environmental and surgical-related factors (surgical wound classification, duration of surgery, hair removal, intraoperative contamination, antimicrobial use, presence of drains, hypothermia or hypoxia in the perioperative period, type of IN used, reaming, need for muscle or skin flap repair, use of negative pressure therapy) and microbiota-related factors (presence of preoperative colonization by Staphylococcus aureus or Acinetobacter baumannii). Results. 221 patients were included and completed the 12-month follow-up period. Incidence of SSI was 11.8% after 12-month follow-up, but would be 8.59% if used the 90-day vigilance period currently recommended. In the initial analysis by unadjusted logistic regression, following factors were associated SSI: Müller AO classification of the fracture morphology groups 2 or 3, previous use of external fixator, presence of drains, use of negative pressure therapy and need for muscle or skin flap repair. Preoperative colonization by S. aureus or A. baumannii was not associated with occurrence of infection. In the multiple logistic regression-adjusted analysis, only previous use of external fixator and need for muscle or skin flap repair remained associated with SSI. Conclusions. Incidence of SSI associated with IN for femoral and tibial diaphyseal fractures was 11.8%, but currently recommended vigilance period would be less sensitive for SSI detection after fracture fixation. Previous use of external fixators and need for muscle or skin flap repair were factors associated with occurrence of IN related infection

Tourniquet use in total knee arthroplasty is convenient for the surgeon and provides a bloodless field for expeditious surgery and a dry field for cementation, but can best be described as an orthopaedic tradition. It is logical for complex anatomy of ligament, nerve, and vessel surgery but it may not be necessary for total knee replacement. In one recent randomised trial, the absence of the tourniquet was not found to affect the quality of cement fixation. There are numerous potential downsides to the use of a tourniquet including decrease range of motion, delayed recovery, increased pain, wound complications, micro-emboli, neuropathy, and increased VTE. There are also a number of complications associated with the use of a tourniquet including arterial thrombosis, skin irritation below the tourniquet, post-operative hyperemia, blood loss, less accurate intra-operative assessment, and it complicates intravenous drug administration. Studies of range of motion have shown that when there is a difference noted, the range of motion is consistently better without tourniquet use. When a tourniquet is utilised it has been found to be advantageous to only use of tourniquet for a minimal amount of the case, typically when cementing is performed. Functional strength has also been found to be improved without the use of a tourniquet. This was attributed to muscle damage, tourniquet-induced ischemia, and compressive injury. Increased peri-operative pain has also been reported in randomised trials associated with the use of a tourniquet. Edema, swelling, and limb girth issues have also been noted to be associated with tourniquet use. Exsanguinating a limb will result in swelling approximately 10% of the original volume half due to a return of blood, and half due to reactive hyperemia. Longer tourniquet times are also associated with increased wound drainage and more wound hypoxia. Tourniquet use has also been associated with embolic phenomenon with several times greater risk of large emboli associated with tourniquet use. A number of complications have been associated with tourniquet use including thromboembolic complications. In one study where quantitative MRI was utilised on both thighs after unilateral total knee replacement with and without a tourniquet, the tourniquet group showed more atrophy with a loss of 20% of the volume compared to the normal side in total knees performed with a tourniquet which also performed clinically worse. There is a small but substantial risk of arterial thrombosis particularly in patients that have atherosclerotic plaque. Ironically there is a risk of increased post-operative blood loss due to the post-tourniquet “blush” as the blood pressure and pain increase hours after a surgical procedure is completed. There is also difficulty in identifying and coagulating posterior and lateral geniculate vessels with the components in place. Utilizing a tourniquet also interferes with intra-operative assessment of patella tracking, range of motion, ligament stability, and gap balancing. Randomised clinical trials have concluded that there is less pain and quicker recovery without the use of a tourniquet. There have also been reports of less swelling, increased range of motion, less analgesic use and better clinical outcome when a tourniquet is not utilised. A meta-analysis of systematic reviews favored not utilizing a tourniquet due to the decrease in complication rate and the improvement in clinical results. While it is standard practice in the US to utilise a tourniquet, the strong consensus of the literature on the subject favors either not using a tourniquet or minimizing the use of a tourniquet for the period of time necessary for a very dry field for cement fixation

Tourniquet use in total knee arthroplasty (TKA) is convenient for the surgeon and provides a bloodless field for expeditious surgery and a dry field for cementation, but can best be described as an orthopaedic tradition. It is logical for complex anatomy of ligament, nerve, and vessel surgery but it may not be necessary for TKA. In one recent randomised trial, the absence of the tourniquet was not found to affect the quality of cement fixation. There are numerous potential downsides to the use of a tourniquet including decrease range of motion, delayed recovery, increased pain, wound complications, micro-emboli, neuropathy, and increased VTE. There are also a number of complications associated with the use of a tourniquet including arterial thrombosis, skin irritation below the tourniquet, post-operative hyperemia, blood loss, less accurate intra-operative assessment, and it complicates intravenous drug administration. Studies of range of motion have shown that when there is a difference noted, the range of motion is consistently better without tourniquet use. When a tourniquet is utilised it has been found to be advantageous to only use the tourniquet for a minimal amount of the case, typically when cementing is performed. Functional strength has also been found to be improved without the use of a tourniquet. In a recent randomised trial, tourniquet use was associated with decreased quad strength at 3 weeks that persisted at 3 months. This was attributed to muscle damage, tourniquet-induced ischemia, and compressive injury. Increased peri-operative pain has also been reported in randomised trials associated with the use of a tourniquet. Edema, swelling, and limb girth issues have also been noted to be associated with tourniquet use. Exsanguinating a limb will result in swelling approximately 10% of the original volume half due to a return of blood, and half due to reactive hyperemia. Longer tourniquet times are also associated with increased wound drainage and more wound hypoxia as measured by transcutaneous oxygen levels. Tourniquet use has also been associated with embolic phenomenon with several times greater risk of large emboli. In one study where quantitative MRI was utilised on both thighs after unilateral total knee replacement with and without a tourniquet, the tourniquet group showed more atrophy with a loss of 20% of the volume compared to the normal side in total knees performed with a tourniquet which also performed clinically worse. There is a small but substantial risk of arterial thrombosis particularly in patients that have atherosclerotic plaque. Ironically there is a risk of increased post-operative blood loss due to the post-tourniquet “blush” as the blood pressure and pain increase hours after a surgical procedure is completed. There is also difficulty in identifying and coagulating posterior and lateral geniculate vessels with the components in place. Utilizing a tourniquet also interferes with intra-operative assessment of patella tracking, range of motion, ligament stability, and gap balancing. Randomised clinical trials have concluded that there is less pain and quicker recovery without the use of a tourniquet. There have also been reports of less swelling, increased range of motion, less analgesic use and better clinical outcome when a tourniquet is not utilised. A meta-analysis of systematic reviews favored not utilizing a tourniquet due to the decrease in complication rate and the improvement in clinical results. While it is standard practice in the US to utilise a tourniquet, the strong consensus of the literature on the subject favors either not using a tourniquet or minimizing the use of a tourniquet for the period of time necessary for a very dry field for cement fixation

Osteocytes (OCY) are the end stage differentiation cells of the osteoblast lineage, and are incorporated in the bone matrix during bone formation. In doing so, OCY control the mineralisation of osteoid. OCY form a dense inter-connected network of cell bodies and cell processes throughout the mineralised matrix of bone. OCY viability depends on interstitial fluid flow along the OCY canaliculi, driven by pulsatile blood flow and loading of the skeleton. Maintenance of the density and viability of OCY are essential for bone health because OCY perform many important functions in bone. Firstly, OCY appear to initiate bone repair of bone microdamage. Secondly, OCY are almost certainly the cells, which initiate new bone formation in response to increased loading of bone. Thirdly, OCY are able to regulate the amount of new bone formation in bone remodelling cycles, at least in part by the production of a molecule called sclerostin (SCL). Mutations in the SCL gene, or deletion of the SCL gene in transgenic mice, are associated with particularly dense, fracture resistant bones. This information has led to development of anti-SCL antibodies as a potential anabolic therapy for bones. Bone loss in ovariectomised aged rats was shown recently to be reversed by treatment with neutralising SCL antibodies. There is also some data to suggest that these antibodies may promote fracture healing. Reduced OCY viability and/or density have been reported in association with osteoporotic fracture. OCY viability seems to be dependent on skeletal loading, adequate skeletal blood flow and estrogen in females. OCY viability is adversely affected by hypoxia, unloading of the skeleton and pharmacobiology, such as chronic exposure to glucocorticoids. Both micro and macro-fractures result in disruption of the OCY network, as do procedures such as drilling and cutting of bone. Because of the important roles of OCY in bone, new approaches to bone health may require the identification of agents to protect these cells from harmful influences in disease and ageing

Introduction. Pedicle Subtraction osteotomy (PRO) in correction of severe spinal deformities is well established. Prospective analysis of its efficacy in complex spinal deformities is sparse in literature. Aims and objectives. To assess the role of PRO in correction of uniplanar and multiplanar spinal deformity and to assess the role of revision PRO in failed corrections. Material and methods. 50 patients were operated between 1996-2007 and followed up for 2 years (2-6). 27 had uniplanar kyphosis (60-128 degrees) and kyphoscoliosis was seen in 10. Failed corrections were seen in 11 uniplanar and 2 multiplanar deformities. The average pre-operative kyphosis and sagittal balance was 78.7 degrees and 22 mm (7-30) respectively. Scoliotic deformity ranged from 97-138 (average 108 degrees) and the coronal imbalance from 10-55 (average 24mm). Deformity distribution was upper dorsal 5, mid dorsal 22, dorso-lumbar 18 and lumbar 5. A single posterior approach sufficed in 47 cases while 3 required an anterior approach for reconstruction. 13 patients had pre-operative neurological deficit (bedridden 10, ambulatory 3). The average surgical time required was 300 minutes and blood loss was 800cc. The anterior defect reconstructed averaged 16.5mm (5-28). Results. Pulmonary complications occurred in 8 (21%), (embolism 1, pneumonia 2, hypoxia 5). Wound infection required debridement in 3 (8%). Failed corrections were seen in 10 (3 out of 37 in our series, 8%) due to failure of construct 2, severe disease 2, infection (active 2, quiescent 4). Neurological deterioration occurred in 1(2%), medial pedicle wall perforation. 12 patients regained ambulation (independent 7, support 5). Post-operative kyphosis and sagittal balance was 36.5 (10-108) and 10mm (5-20) respectively. Average correction was: sagittal 46.4%, coronal 37.5% and revisions 58%. The correction of kyphosis and sagittal balance was statistically comparable between primary and revision cases (p >0.05). Conclusions. PRO offers an excellent single stage decompression and controlled correction of kyphosis