A study of cortical bone microdamage and crack morphology utilising confocal microscopy and sequential labelling

The formation and accumulation of microdamage in bone plays an important role in the occurrence of stress and fragility fractures as well as in the initiation of bone remodelling. In this study a novel technique is presented for the investigation of bone microdamage and crack morphology using laser scanning confocal microscopy and sequential labelling with chelating fluorochromes. Compact tension fracture specimens machined from bovine tibial cortical bone, were mechanically tested in a wedge loaded crack-propagating tool. Sequential labelling with xylenol orange and calcein allowed for the crack propagation and microdamage progression to be assessed at each stage using confocal microscopy. Both twodimensional confocal images and three-dimensional z-series reconstructions displayed the formation of a microdamage process zone and wake surrounding the main crack. Further imaging demonstrated the significance of the bone microstructure, such as the vasculature and osteocytes, in the distribution of the microdamage.J. Codrington, J. Kuliwaba, K. Zarrinkalam and N. Fazzalarihttp://www.cp2009.unipr.it/http://www.gruppofrattura.it/index.php?option=com_jombib&task=showbib&id=105

New insights into the propagation of fatigue damage in cortical bone using confocal microscopy and chelating fluorochromes

Fatigue damage in bone occurs in the form of microcracks and plays an important role in the initiation of bone remodelling and in the occurrence of stress and fragility fractures. The process by which fatigue microcracks in bone initiate and grow remains poorly understood. The aim of this study was to investigate the microscopic tissue changes associated with microcracks during crack propagation in cortical bone and the influence of bone microstructure on this process. Cracks were mechanically initiated and extended longitudinally in a two-stage process, in six bovine tibial compact tension specimens. The sequential application of chelating fluorochromes, xylenol orange followed by calcein, allowed the nature of microcrack damage at different stages of propagation to be monitored by laser scanning confocal microscopy. Specimens were imaged at a focal plane 20 microm below the samples' surface, or as a series of z-plane images collected to a maximal depth of 200 microm and 35 microm for x 4 and x 40 objectives, respectively. Z-series image stacks were then reconstructed using Amira 3.0 software. Confocal images showed that xylenol orange localised to the crack surface and did not migrate into the crack's extension following further mechanical propagation. Similarly, calcein stained the extended crack's surface and displayed minimal incorporation within the original crack. High resolution confocal images provided a detailed visual description of the crack's 'process zone', and 'process zone wake'. Additionally, an 'interface region' was revealed, displaying a clear distinction between the end of the first crack and the commencement of its extension. Confocal images of the interface region demonstrated that the extended crack forms a continuum with the pre-existing crack and propagates through the former process zone. Upon viewing the three-dimensional reconstructed images, we found evidence suggesting a submicroscopic tissue involvement in fatigue damage, in addition to the potential influence of vascular canals and osteocyte lacunae on its propagation through the bone matrix. This study has provided new insights into the process of fatigue damage growth in bone and factors influencing its progression through the bone matrix. Confocal microscopy in combination with sequential chelating fluorochrome labelling is a valuable technique for monitoring microcrack growth in bone.K.H. Zarrinkalam; Dr J.S. Kuliwaba; R.B. Martin; M.A.B. Wallwork; N.L. Fazzalar

Reversal of established bone pathology in MPS VII mice following lentiviral-mediated gene therapy

Abstract not availableAinslie L.K. Derrick-Roberts, Kavita Panir, Carmen E. Pyragius, Krystyna H. Zarrinkalam, Gerald J. Atkins, Sharon Byer

Glucosylceramide accumulation is not confined to the lysosome in fibroblasts from patients with Gaucher disease

Gaucher disease (GD) is an inborn error of glycosphingolipid metabolism resulting from a deficiency of the lysosomal enzyme beta-glucosidase leading to the accumulation of glucosylceramide (GC) in lysosomes of affected cells. In order to determine the effect of GC accumulation on intracellular lipid content in fibroblasts from patients with GD, we measured individual species of ceramide, di- and trihexosylceramide, sphingomyelin, phosphatidylcholine, phosphatidylinositol and phosphatidylglycerol using electrospray ionisation-tandem mass spectrometry. The different subspecies of each lipid class correlated with each other and were summed to give total lipid concentrations. In addition to GC, we also noted secondary elevations in other lipids, especially in type 2 GD. Sub-cellular fractionation showed that GC was not confined to the lysosome but increased throughout the cell. The sequelae of extra-lysosomal accumulation may have implications in the pathogenic mechanisms of GD by interaction with biochemical and metabolic pathways located outside the lysosome. The elevation of ceramide in confluent type 2 GD fibroblasts redistributed from its primary site of accumulation in the lysosome to the endosomal region at four-weeks post-confluence. The accumulation of lipids in the endosome and lysosome suggests both impaired trafficking of lipids and reduced capacity of the lysosome to degrade lipids.Maria Fuller, Tina Rozaklis, Melanie Lovejoy, Krystyna Zarrinkalam, John J. Hopwood and Peter J. Meiklehttp://www.elsevier.com/wps/find/journaldescription.cws_home/622920/description#descriptio

Audit of CT referral in patients with suspected aneurysmal subarachnoid haemorrhage in a tertiary hospital

Exhibit abstract E22 – Educational ExhibitR. Mohindra, S. Constantine, K. Tew, M.A.R. Reid, M. Nottage, N. Zarrinkala

Exploring the Potential of User Modeling Based on Mind Maps

Mind maps have not received much attention in the user modeling and recommender system community, although mind maps contain rich information that could be valuable for user-modeling and recommender systems. In this paper, we explored the effectiveness of standard user-modeling approaches applied to mind maps. Additionally, we develop novel user modeling approaches that consider the unique characteristics of mind maps. The approaches are applied and evaluated using our mind mapping and reference-management software Docear. Docear displayed 430,893 research paper recommendations, based on 4,700 user mind maps, from March 2013 to August 2014. The evaluation shows that standard user modeling approaches are reasonably effective when applied to mind maps, with click-through rates (CTR) between 1.16% and 3.92%. However, when adjusting user modeling to the unique characteristics of mind maps, a higher CTR of 7.20% could be achieved. A user study confirmed the high effectiveness of the mind map specific approach with an average rating of 3.23 (out of 5), compared to a rating of 2.53 for the best baseline. Our research shows that mind map-specific user modeling has a high potential, and we hope that our results initiate a discussion that encourages researchers to pursue research in this field and developers to integrate recommender systems into their mind mapping tools

Skeletal response to lentiviral mediated gene therapy in a mouse model of MPS VII

Mucopolysaccharidosis VII (MPS VII) is an autosomal recessive, lysosomal storage disorder caused by β-glucuronidase (GUSB) deficiency, resulting in the accumulation of glycosaminoglycans (GAGs), in a variety of cell types. Severe, progressive skeletal pathology, termed dysostosis multiplex, is a prominent clinical feature of MPS VII. We have evaluated a gene therapy protocol for its efficacy in preventing the development and progression of bone pathology in MPS VII mice treated with a lentiviral vector at birth or at 7 weeks. Two weeks after injections, high levels of vector expression were observed in liver, spleen and bone marrow and to a lesser extent in kidney, lung and heart. Widespread clearance of GAG storage was observed in somatic tissues of both groups and some clearance of neuronal storage was observed in mice treated from birth. Micro-CT analysis demonstrated a significant decrease in vertebral and femoral bone mineral volume, trabecular number, bone surface density and cortical bone thickness in both treatment groups. Lumbar and femoral bone lengths were significantly decreased in untreated MPS VII mice, while growth plate heights were increased and these parameters did not change upon treatment. Small improvements in performance in the open field and rotarod behaviour tests were noted. Overall, systemic lentiviral-mediated gene therapy results in a measurable improvement in parameters of bone mass and architecture as well as biochemical and enzymatic correction. Conversely, growth plate chondrocytes were not responsive to treatment, as evidenced by the lack of improvement in vertebral and femoral bone length and growth plate height.Carmen E. Macsai, Ainslie L.K. Derrick-Roberts, Xiaodan Ding, Krystyna H. Zarrinkalam, Chantelle McIntyre, Paul H. Anderson, Don S. Anson and Sharon Byer