Professor Bruce Milthorpe

Green, D.W., Li, G., Milthorpe, B.K. & Ben-Nissan, B. 2012, 'Adult stem cell coatings for regenerative medicine', Materials Today, vol. 15, no. 1-2, pp. 60-66.
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Stem cells can become potent tools for the treatment of degenerative disorders such as heart failure, eye disease and osteoarthritis. Housing stem cells inside a hydrogel coating, directly deposited around them individually and in groups, may be an important solution to the problem of increasing stem cell viability and protection in cultivation. Such coatings can target regulatory proteins and genes for maintenance, differentiation and development into tissues. Already polymer coatings are being applied directly to protect insulin producing pancreatic islet cells in the hope of treating type I diabetes. Here, we review current emerging developments in adult mesenchymal stem cell nanocoating and microcoating techniques and assess their unique practical engineering, biological and potential clinical advantages.

Jones, A.C., Arns, C.H., Hutmacher, D.W., Milthorpe, B.K., Sheppard, A.P. & Knackstedt, m.A. 2009, 'The correlation of pore morphology, interconnectivity and physical properties of 3D ceramic scaffolds with bone ingrowth', Biomaterials, vol. 30, no. 7, pp. 1440-1451.
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In the design of tissue engineering scaffolds, design parameters including pore size, shape and interconnectivity, mechanical properties and transport properties should be optimized to maximize successful inducement of bone ingrowth. In this paper we describe a 3D micro-CT and pore partitioning study to derive pore scale parameters including pore radius distribution, accessible radius, throat radius, and connectivity over the pore space of the tissue engineered constructs. These pore scale descriptors are correlated to bone ingrowth into the scaffolds. Quantitative and visual comparisons show a strong correlation between the local accessible pore radius and bone ingrowth; for well connected samples a cutoff accessible pore radius of similar to 100 mu M is observed for ingrowth. The elastic properties of different types of scaffolds are simulated and can be described by standard cellular solids theory: (E/E(0))-(rho/rho(s))(n). Hydraulic conductance and diffusive properties are calculated; results are consistent with the concept of a threshold conductance for bone ingrowth. Simple simulations of local flow velocity and local shear stress show no correlation to in vivo bone ingrowth patterns. These results demonstrate a potential for 3D imaging and analysis to define relevant pore scale morphological and physical properties within scaffolds and to provide evidence for correlations between pore scale descriptors, physical properties and bone ingrowth.

Lord, M.S., Pasqui, D., Barbucci, R. & Milthorpe, B.K. 2009, 'Protein adsorption on derivatives of hyaluronic acid and subsequent cellular response', Journal of Biomedical Materials Research Part A, vol. 91A, no. 3, pp. 635-646.
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The modulation of biological interactions with artificial surfaces is a vital aspect of biomaterials research. Serum protein adsorption onto photoreactive hyaluronic acid (Hyal-N3) and its sulfated derivative (HyalS-N3) was analyzed to determine extent of protein interaction and protein conformation as well as subsequent cell adhesion. There were no significant (p < 0.01) differences in the amount of protein adsorbed to the two polymers; however, proteins were found to be more loosely bound on HyalS-N3 compared with Hyal-N3. Fibronectin was adsorbed onto HyalS-N3 in such an orientation as to allow the availability of the cell binding region, while there was more restricted access to this region on fibronectin adsorbed onto Hyal-N3. This was confirmed by reduced cell adhesion on fibronectin precoated Hyal-N3 compared with fibronectin precoated HyalS-N3. Minimal cell adhesion was observed on albumin and serum precoated Hyal-N3. The quartz crystal microbalance confirmed that specific cell-surface interactions were experienced by cells interacting with the fibronectin precoated polymers and serum precoated HyalS-N3

Milthorpe, B.K. 2009, 'In Vitro Adsorption of Tear Proteins to Hydroxyethyl Methacrylate-Based Contact Lens Materials', Eye and Contact Lens: Science and Clinical Practice, vol. 35, no. 6, pp. 320-328.
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Investigations of polymer interactions in single protein solutions is a necessary step in the elucidation of in vivo early binding events during protein deposition on hydroxyethyl methacrylate-based contact lens materials. Quantity and tenacity of binding of significant tear components to groups I and IV contact lenses was assessed. Competitive binding by these components was also examined. METHODS: Adsorption on FDA groups I and IV hydrogel lenses was monitored using I-labeled protein. Lenses were incubated in increasing concentrations of radiolabeled single species proteins in solution. For competition experiments, concentration of each radiolabeled protein was held constant and the adsorption/sorption challenged with increasing concentrations of nonlabeled proteins. Lenses were soaked in phosphate-buffered saline to determine desorption. RESULTS: Group IV lenses bound large amounts of lysozyme, whereas group I lenses bound highest amounts of albumin. Albumin binding to both lens types was relatively strong and could not be competed from binding by other proteins lysozyme, lactoferrin, and mucin. Mucin at high concentrations tended to positively cooperate with the binding of lactoferrin and albumin to all lenses. CONCLUSIONS: Binding of proteins to hydroxyethyl methacrylate-based hydrogel lens surfaces is affected by charge and polymer components, and perhaps manufacturing processes. Albumin binds strongly to lens surfaces, and this may play an adverse role during contact lens wear.

Lord, M.S., Modin, C., Foss, M., Duch, M., Simmons, A., Pedersen, F.S., Besenbacher, F. & Milthorpe, B.K. 2008, 'Extracellular matrix remodelling during cell adhesion as monitored by the quartz crystal microbalance', Biomaterials, vol. 29, no. 17, pp. 2581-2587.
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A cell's ability to remodel adsorbed protein layers on surfaces is influenced by the nature of the protein layer itself. Remodelling is often required to accomplish cellular adhesion and extracellular matrix formation which forms the basis for cell spreading, increased adhesion and expression of different phenotypes. The adhesion of NIH3T3 (EGFP) fibroblasts to serum protein (albumin or fibronectin) precoated tantalum (Ta) and oxidised polystyrene (PSox) surfaces was examined using the quartz crystal microbalance with dissipation (QCM-D) monitoring and fluorescence microscopy. The cells were either untreated or treated with cycloheximide to examine the contribution of endogenous protein production during cell adhesion to the QCM-D response over a period of 2 h. Following adsorption of albumin onto Ta and PSox there was no difference detected between the response to seeding untreated and cycloheximide treated cells. The QCM-D was able to detect differences in the untreated cellular responses to fibronectin versus serum precoated Ta and PSox substrates, while cycloheximide treatment of the cells produced the same QCM-D response for fibronectin and serum precoatings on each of the materials. This confirmed that the process of matrix remodelling by the cells is dependent on the underlying substrate and the preadsorbed proteins and that the QCM-D response is dominated by changes in the underlying protein layer. Changes in dissipation correspond to the development of the actin cytoskeleton as visualised by actin staining.

Lord, M.S., Pasqui, D., Barbucci, R. & Milthorpe, B.K. 2008, 'Protein Adsorption on Derivatives of Hyaluronan', Macromolecular Symposia, vol. 266, no. 1, pp. 17-22.
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Serum protein adsorption and fibroblast cell adhesion on photo reactive hyaluronic acid (Hyal-N3) and its sulfated derivative (HyalS-N3) was analysed using a combination of quartz crystal microbalance (QCM) and cell adhesion assays. There was no significant differences in the amount of protein adsorbed onto the two polymers, however proteins were found to be more loosely bound to HyalS-N3 compared with Hyal-N3. Approximately 17% and 31% of the fibronectin interacting with Hyal-N3 and HyalS-N3 respectively was found to be irreversibly bound after rinsing with MilliQ water, SDS and urea. Proteins were exposed to the polymers before cell adhesion was monitored for a period of 2 hours in serum free conditions. Minimal cell adhesion was observed on albumin-coated materials as well as serum precoated Hyal-N3. Precoating the materials with fibronectin enhanced cell adhesion, although HyalS-N3 experienced higher levels of cell adhesion than Hyal-N3 and similar results were found for the serum precoated materials.

Milthorpe, B.K. 2008, 'Application of biomechanics to tissue engineering: a personal view', Journal of Mechanics in Medicine & Biology, vol. 8, no. 2, pp. 153-160.
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Cellular biomechanics is an area of study that is receiving more attention as time progresses. The response of cells to their mechanical environment, including biomechanical stimuli, has far-reaching ramifications for the area of tissue engineering, especially for tissues designed to withstand mechanical loading (e.g. bone, cartilage, tendons and ligaments, and arteries). The effects of mechanical stimuli on cells are only recently being examined, and the potential role of mechanical stimuli in tissue engineering is still one that is largely ignored in the design of tissue engineering scaffolds. The relationship of mechanical properties of scaffolds or of mechanical stimuli to cell behavior is complex, but vital to the development of the field. Also, understanding the complex interplay of form and environment on cells involves an increase in our knowledge of how cells react to their total environment including mechanical stimuli and material properties. In order to improve tissue engineering outcomes, a nexus must be developed between the mechanical, biochemical, and biological studies of cellular behavior, in the context of extremely complex systems

Jones, A.C., Arns, C.H., Sheppard, A.P., Hutmacher, D.W., Milthorpe, B.K. & Knackstedt, m.A. 2007, 'Assessment of bone ingrowth into porous biomaterials using MICRO-CT', Biomaterials, vol. 28, no. 15, pp. 2491-2504.
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The three-dimensional (3D) structure and architecture of biomaterial scaffolds play a critical role in bone formation as they affect the functionality of the tissue-engineered constructs. Assessment techniques for scaffold design and their efficacy in bone ingrowth studies require an ability to accurately quantify the 3D structure of the scaffold and an ability to visualize the bone regenerative processes within the scaffold structure. In this paper, a 3D micro-CT imaging and analysis study of bone ingrowth into tissue-engineered scaffold materials is described. Seven specimens are studied in this paper; a set of three specimens with a cellular structure, varying pore size and implant material, and a set of four scaffolds with two different scaffold designs investigated at early (4 weeks) and late (12 weeks) explantation times. The difficulty in accurately phase separating the multiple phases within a scaffold undergoing bone regeneration is first highlighted. A sophisticated three-phase segmentation approach is implemented to develop high-quality phase separation with minimal artifacts. A number of structural characteristics and bone ingrowth characteristics of the scaffolds are quantitatively measured on the phase separated images. Porosity, pore size distributions, pore constriction sizes, and pore topology are measured on the original pore phase of the scaffold volumes. The distribution of bone ingrowth into the scaffold pore volume is also measured. For early explanted specimens we observe that bone ingrowth occurs primarily at the periphery of the scaffold with a constant decrease in bone mineralization into the scaffold volume.

Ko, H.C., Milthorpe, B.K. & McFarland, C.D. 2007, 'Engineering thick tissues - the vascularisation problem', European Cells and Materials, vol. 14, no. 1, pp. 1-19.
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The ability to create thick tissues is a major tissue engineering challenge, requiring the development of a suitable vascular supply. Current trends are seeing the utilization of cells seeded into hybrid matrix/scaffold systems to create in vitro vascular analogues. Approaches that aim to create vasculature in vitro include the use of biological extracellular matrices such as collagen hydrogels, porous biodegradable polymeric scaffolds with macro- and micro-lumens and micro-channels, co-culture of cells, incorporation of growth factors, culture in dynamic bioreactor environments, and combinations of these. Of particular interest are those approaches that aim to create bioengineered tissues in vitro that can be readily connected to the host's vasculature following implantation in order to maintain cell viability.

Hitchcock, R., Sears, W., Gillies, R.M., Milthorpe, B.K. & Walsh, W.R. 2006, 'In vitro study of shear force on interbody implants', Journal of Spinal Disorders & Techniques, vol. 19, no. 1, pp. 32-36.
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The lordosis of the lumbar spine and body weight result in significant shear forces through the lumbosacral elise spaces. These forces result in translational motion across the disc space, which is resisted but not completely abolished by pedicle screw stabilization. It is postulated that this motion may be a factor in the development of nonunion of lumbar interbody fusions. An in vitro study of the micromotion of porcine specimens with serrated or smooth interbody spacers and subjected to shear forces under compressive preload was conducted to determinewhether the surface serrations on vertebral interbody implants significantly resist shear forces and resulting sagittal translation.

Lord, M.S., Stenzel, M., Simmons, A. & Milthorpe, B.K. 2006, 'Lysozyme Interactions with poly(HEMA)-based Hydrogel', Biomaterials, vol. 27, no. 8, pp. 1341-1345.
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Lysozyme interaction with an acrylic-based hydrogel, poly(2-hydroxyethyl methacrylate) co-methacrylic acid (P(HEMA-MAA)), was investigated using a combination of quartz crystal microbalance with dissipation (QCM-D), surface plasmon resonance (SPR) and dual polarisation interferometry (DPI). This combination of techniques demonstrated that lysozyme initially absorbed into the hydrogel matrix and displaced water from the hydrogel while subsequent lysozyme additions were adsorbed onto the surface of the hydrogel material. QCM-D, being sensitive to bound water, showed an overall decrease in mass and stiffening of the layer after lysozyme addition. SPR, a water insensitive technique, showed a net mass increase after addition of lysozyme and buffer rinses. DPI showed that the first exposure of lysozyme to P(HEMA-MAA) was consistent with lysozyme absorption while subsequent lysozyme exposures were consistent with lysozyme adsorption.

Lord, M.S., Modin, C., Foss, M., Duch, M., Simmons, A., Pedersen, F.S., Milthorpe, B.K. & Besenbacher, F. 2006, 'Monitoring cell adhesion on tantalum and oxidised polystyrene using a quartz crystal microbalance with dissipation', Biomaterials, vol. 27, no. 26, pp. 4529-4537.
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The quartz crystal microbalance with dissipation (QCM-D) (Q-Sense AB, Sweden) has been established as a useful tool for evaluating interactions between various biological and non-biological systems, and there has been increasing interest in using the QCM-D technique for cell monitoring applications. This study investigated the potential of the QCM-D to characterise the initial adhesion and spreading of cells in contact with protein precoated biocompatible surfaces. The QCM-D technique is attractive for monitoring cell adhesion and spreading as it allows in situ real-time measurements. The adhesion of NIH3T3 (EGFP) fibroblasts to tantalum (Ta) and oxidised polystyrene (PSox) surfaces precoated with serum proteins was examined using the QCM-D for a period of either 2 or 4 h. Time-lapse photography was performed at 30 min intervals to visually examine cell adhesion and spreading in order to relate cell morphology to the QCM-D response.

Lord, M.S., Stenzel, M., Simmons, A. & Milthorpe, B.K. 2006, 'The effect of charged groups on protein interactions with poly(HEMA) hydrogels', Biomaterials, vol. 27, no. 4, pp. 567-575.
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Proteins, lipids and other biomolecules interact strongly with the acrylic-based biomaterials used for contact lenses. Although hydrogels are nominally resistant to protein fouling, many studies have reported considerable amounts of protein bound to poly(2-hydroxyethylmethacrylate) (PHEMA) lenses. This study examined the binding of a series of biomolecules (tear protein analogues, mucin and cholesterol) to poly(methylmethacrylate) (PMMA) and three HEMA-based hydrogels (PHEMA, HEMA plus methacrylic acid (P(HEMA+MAA)), HEMA plus methacrylic acid plus N-vinylpyrrolidone (P(HEMA+MAA+NVP))) by use of a quartz crystal microbalance with dissipation (QCM-D) monitoring. The QCM-D estimates changes in the mass and viscous constant for the adsorbed layer through measurements of frequency and dissipation.

Lord, M.S., Cousins, B.G., Doherty, P.J., Whitelock, J.M., Simmons, A., Williams, R.L. & Milthorpe, B.K. 2006, 'The effect of silica nanoparticulate coatings on serum protein adsorption and cellular response', Biomaterials, vol. 27, no. 28, pp. 4856-4862.
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Serum protein adsorption on colloidal silica surfaces was investigated using a quartz crystal microbalance with dissipation (QCM-D) monitoring. The amount of serum proteins adsorbed on colloidal silica-coated surfaces was not significantly different from the control silica surfaces, with the exception of 21 nm colloidal silica which experienced significantly less (P<0.05) fibrinogen adsorption compared with control silica. The adhesion and proliferation of human endothelial cells (C11STH) on nano-scale colloidal silica surfaces were significantly reduced compared with control silica surfaces, suggesting that the conformation of adsorbed proteins on the colloidal silica surfaces plays a role in modulating the amount of cell binding. Fibronectin is one of the main extracellular matrix proteins involved in endothelial cell attachment to biomaterial surfaces. There was reduced binding of a monoclonal anti-fibronectin antibody, that reacted specifically with the cell-binding fragment, to fibronectin-coated colloidal silica surfaces compared with control silica surfaces. This suggests that the fibronectin adsorbed on the colloidal silica-coated surfaces was conformationally changed compared with control silica reducing the availability of the cell-binding domain of fibronectin.

Wei, M., Ruys, A.J., Swain, M.V., Milthorpe, B.K. & Sorrell, C.C. 2005, 'Hydroxyapatite-coated metals: Interfacial reactions during sintering', Journal Of Materials Science-materials In Medicine, vol. 16, pp. 101-106.
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Electrophoretic deposition (EPD) is a low cost flexible process for producing HA coatings on metal implants. Its main limitation is that it requires heating the coated implant in order to densify the HA. HA typically sinters at a temperature below 1150 degreesC, but metal implants are degraded above 1000 degreesC. Further, the metal induces the decomposition of the HA coating upon sintering. Recent developments have enabled EPD of metathesis-synthesised uncalcined HA which sinters at similar to 1000 degreesC. The effects of temperature on HA-coated Ti, T16AI4V, and 316L stainless steel were investigated for dual coatings of metathesis HA sintered at 1000 degreesC. The use of dual HA coatings (coat, sinter, coat, sinter) enabled decomposition to be confined to the "undercoat" (HA layer 1), with the surface coating decomposition free. The tensile strength of the three metals was not significantly affected by the high sintering temperatures (925 degreesC < T < 1000degreesC). XRD/SEM/EDS analyses of the interfacial zones revealed that 316L had a negligible HA:metal interfacial zone (similar to1 mum) while HA:Ti and HA:Ti6Al4V had large interfacial zones (>10 mum) comprising a TiO2 oxidation zone and a CaTiO3 reaction zone

Wei, M., Ruys, A.J., Milthorpe, B.K. & Sorrell, C.C. 2005, 'Precipitation of hydroxyapatite nanoparticles: Effects of precipitation method on electrophoretic deposition', Journal Of Materials Science-materials In Medicine, vol. 16, pp. 319-324.
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Electrophoretic deposition is a low-cost, simple, and flexible coating method for producing hydroxyapatite ( HA) coatings on metal implants with a broad range of thicknesses, from < 1 mu m to > 500 mu m. As for many other HA coating techniques, densification of electrophoretically deposited coatings involves heating the coated metal to temperatures above 1000 degrees C. Metal substrates tend to react with HA coatings at such temperatures inducing decomposition at temperatures below 1050 degrees C ( decomposition for pure HA normally occurs above 1300 degrees C). Therefore, densification of these coatings needs to be conducted at temperatures lower than 1050 degrees C, and this necessitates the use of high-surface-area HA nano-precipitates, rather than commercially available pre-calcined powders, which densify at temperatures typically higher than 1200 degrees C. HA nano-precipitates were prepared by three methods and deposited on metal substrates by electrophoresis: ( 1) the acid base method, which produced plate-like nano-particles with a 2.5: 1 aspect ratio, and severely cracked coatings; ( 2) the calcium acetate method, which produced needle-like nano-particles with a 10: 1 aspect ratio, and slightly cracked coatings; ( 3) the metathesis method, which produced rounded nano-particles with a 2: 1 aspect ratio, and high-quality crack-free coatings. The results suggested that the less equiaxed the nano-particles, the more cracked the coatings obtained by the electrophoretic deposition technique

Fornusek, C., Davis, G.M., Sinclair, P. & Milthorpe, B.K. 2004, 'Development of an Isokinetic Functional Electrical Stimulation Cycle Ergometer', Neuromodulation, vol. 7, no. 1, pp. 56-64.
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An isokinetic functional electrical stimulation leg cycle ergometer (iFES-LCE) was developed for individuals with spinal cord injury (SCI). The iFES-LCE was designed to allow cycle training over a broad range of pedalling cadences (5+60 rev/min) to promote both muscular strength and cardiorespiratory fitness. A commercially available motorized cycle ergometer was integrated with a custom built FES system, a laptop computer, and a specialized chair that restricted lateral leg movements. Sample biomechanical data were collected from an SCI subject performing FES cycling to demonstrate the iFES-LCE's performance characteristics. Calibration of the iFES-LCE system revealed a linear relationship between torque applied to the axle of the motorized ergometer and the braking motor current generated to maintain velocity. Performance data derived from iFES-LCE motor torque agreed closely with similar data collected using strain-gauge instrumented pedals (cross-correlations = 0.93+0.98). The iFES-LCE was shown to work well across a range of pedaling cadences. We conclude that the new iFES-LCE system may offer improved training potential by allowing cycling over a broad range of pedaling cadences, especially low cadence. This device also improves upon the accuracy of other ergometers by adjusting for the passive load of the legs.

Jones, A.C., Sakellariou, A., Limaye, A., Arns, C.H., Senden, T.J., Sawkins, T., Knackstedt, m.A., Rohner, D., Hutmacher, D.W., Brandwood, A. & Milthorpe, B.K. 2004, 'Investigation ogf microstructural features in regenerating bone using micro computed tomography', Journal Of Materials Science-materials In Medicine, vol. 15, pp. 529-532.
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Jones, A.C., Sheppard, A.P., Sok, R.M., Arns, C.H., Limaye, A., Averdunk, H., Brandwood, A., Sakellariou, A., Senden, T.J., Milthorpe, B.K. & Knackstedt, m.A. 2004, 'Three-dimensional analysis of cortical bone structure using X-ray micro-computed tomography', Physica A: Statistical Mechanics and its Applications, vol. 339, no. 1-2, pp. 125-130.
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We demonstrate the capability of X-ray micro-computed tomography to image the micro-structure of human cortical bone. At 5 ?m voxel size we observe the complex morphology of the Haversian network in three dimensions. The local thickness of Haversian canals is measured using a maximal sphere algorithm and found to have a bimodal signature and a mean radius of 19.2 ?m. The intra-cortical porosity due to Haversian canals is measured as 3.0%. Both results are in agreement with traditional histomorphometric measurements. We show that at higher resolutions one can resolve the spatial distribution of lacunae in cortical bone.

Jones, A.C., Milthorpe, B.K., Averdunk, H., Limaye, A., Senden, T.J., Sakellariou, A., Sheppard, A.P., Sok, R.M., Knackstedt, m.A., Brandwood, A., Rohner, D. & Hutmacher, D.W. 2004, 'Analysis of 3D bone ingrowth into polymer scaffolds via micro-computed tomography imaging', Biomaterials, vol. 25, no. 20, pp. 4947-4954.
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This paper illustrates the utility of micro-computed tomography (micro-CT) to study the process of tissue engineered bone growth. A micro-CT facility for imaging and visualising biomaterials in three dimensions (3D) is described. The facility is capable of acquiring 3D images made up of 20003 voxels on specimens up to 60 mm in extent with resolutions down to 2 ?m. This allows the 3D structure of tissue engineered materials to be imaged across three orders of magnitude of detail. The capabilities of micro-CT are demonstrated by imaging the Haversian network within human femoral cortical bone (distal diaphysis) and bone ingrowth into a porous scaffold at varying resolutions. Phase identification combined with 3D visualisation enables one to observe the complex topology of the canalicular system of the cortical bone. Imaging of the tissue engineered bone at a scale of 1 cm and resolutions of 10 ?m allows visualisation of the complex ingrowth of bone into the polymer scaffold. Further imaging at 2 ?m resolution allows observation of bone ultra-structure. These observations illustrate the benefits of tomography over traditional techniques for the characterisation of bone morphology and interconnectivity and performs a complimentary role to current histomorphometric techniques.

Nordon, R.E., Shu, A., Camacho, F. & Milthorpe, B.K. 2004, 'Hollow-Fiber Assay for Ligand-Mediated Cell Adhesion', Cytometry, vol. 57A, no. 1, pp. 39-44.
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Background The investigation of receptor-ligand interactions in the cellular context presents significant technical challenges, first, to immobilize the ligand in a manner that preserves functional properties and, second, to relate ligand properties to cell adhesion and other cellular processes. Methods Ligand-mediated cell adhesion was characterized by the development of a cellulose hollow-fiber adhesion assay in which ligand (protein A) was immobilized onto the cellulose membrane as a recombinant fusion protein containing a cellulose-binding domain affinity tag. Modules containing single cellulose hollow fibers were connected to a micro-flow system for cell deposition and detachment with fluid shear stress. The cell adhesion process that occurred inside a segment of hollow fiber was observed in real time by using an inverted microscope equipped with a CCD camera and digital frame grabber. Image analysis software was developed to count cells and record digital images. Results Cell adhesion strength was characterized by counting the number of cells that were detached by application of fluid shear stress with values that ranged from 2.3 to 185 dyne/cm2. The median shear stress of detachment of KG1a cells was directly related to the duration of membrane contact and the amount of immobilized monoclonal antibody (anti-CD34). Conclusions The hollow-fiber assay provides a general method to determine functional properties of molecular domains that interact with cell surface receptors and markers

Zheng, Q., Milthorpe, B.K. & Jones, A.S. 2004, 'Direct Neural Network Application for Automated Cell Recognition', Cytometry, vol. 57A, no. 1, pp. 1-9.
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Background Automated cell recognition from histologic images is a very complex task. Traditionally, the image is segmented by some methods chosen to suit the image type, the objects are measured, and then a classifier is used to determine cell type from the object's measurements. Different classifiers have been used with reasonable success, including neural networks working with data from morphometric analysis. Methods Image data of cells were input directly into neural networks to determine the feasibility of direct classification by using pixel intensity information. Several types of neural network and their ability to work with cells in a complex patterned background were assessed for a variety of images and cell types and for the accuracy of classification. Results Inflammatory cells from animal biomaterial implants in rabbit paravertebral muscle were imaged in histologic sections. Simple, three-layer, fully connected, back-propagation neural networks and four-layer networks with two layers of a shared-weights neural network were most successful at classifying the cells from the images, with 97% and 98% correct recognition rates, respectively. Conclusions The high accuracy recognition rate shows the potential for direct classification of visual image pixel data by neural networks.

Miao, X., Ruys, A.J. & Milthorpe, B.K. 2001, 'Hydroxyapatite-316L fibre composites prepared by vibration assisted slip casting', Journal of Materials Science, vol. 36, no. 13, pp. 3323-3332.
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To prepare hydroxyapatite (HA, or HAp)-stainless steel 316L fibre composites with up to 30 vol% 316L fibres (sim1 mm long and 50 mgrm in diameter), slip casting assisted by vibration (frequency: sim55 Hz; amplitude: sim5 mm) was carried out, followed by both cold isostatic pressing (CIPing) and hot isostatic pressing (HIPing). With the addition of around 0.5 wt% sodium carboxymethylcellulose (Na-cmc), solids loadings up to 44 vol% were obtained in calcined HA powder-derived slips, which were castable only under the vibration. The slips were concentrated and viscous so that the preferential sedimentation of the dense and large 316L fibres could be avoided. Subsequent CIPing was able to increase the relative density of the cast and dried green compacts from 46% after casting to 60% after CIPing. With the dense and uniform green compacts of the HA-316L mixtures, final HIPing at 950 ¦C resulted in HA-316L fibre composites of 99% relative density. The HA-316L fibre composites had improved fracture toughness of 3.6 ¦ 0.3 MPa.m0.5, due to the bridging effect of the ductile 316L fibres. However, the mechanical strength of the composites was limited by the presence of residual thermal stresses and circumferential microcracks. The HA-316L fibre composites were biocompatible and exhibited favourable bone-bonding characteristics.

Wei, M., Ruys, A.J., Milthorpe, B.K., Sorrell, C.C. & Evan, J.H. 2001, 'Electrophoretic Deposition of Hydroxyapatite Coatings on Metal Substrates: A Nanoparticulate Dual-Coating Approach', Journal of Sol-Gel Science and Technology, vol. 21, no. 1-2, pp. 39-48.
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Hydroxyapatite coatings can be readily deposited on metal substrates by electrophoretic deposition. However, subsequent sintering is highly problematic owing to the fact that temperatures in excess of 1100¦C are required for commercial hydroxyapatite powders to achieve high density. Such temperatures damage the metal and induce metal-catalysed decomposition of the hydroxyapatite. Furthermore, the firing shrinkage of the hydroxyapatite coating on a constraining metal substrate leads to severe cracking. The present study has overcome these problems using a novel approach: the use of aged nanoparticulate hydroxyapatite sols (lower sintering temperature) and a dual coating strategy that overcomes the cracking problem. Dual layers of uncalcined hydroxyapatite (HAp) powder were electrophoretically coated on Ti, Ti6Al4V and 316L stainless steel metal substrates, sintered at 875+1000¦C, and characterised by SEM and XRD, and interfacial shear strength measurement. Dual coatings on stainless steel had an average high bond strength (about 23 MPa), and dual coatings on titanium and titanium alloy had moderate strengths (about 14 and 11 MPa, respectively), in comparison with the measured shear strength of bone (35 MPa). SEM and XRD demonstrated that the second layer blended seamlessly with the first and filled the cracks in the first. The superior result on stainless steel is attributed to a more appropriate thermal expansion match with hydroxyapatite, the thinner oxide layer, or a combination of these factors.

Garrett, Q., Griesser, H.J., Milthorpe, B.K. & Garrett, R.W. 1999, 'Irreversible adsorption of human serum albumin to hydrogel contact lenses: a study using electron spin resonance spectroscopy', Biomaterials, vol. 20, pp. 1345-1356.
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Garrett, Q., Garrett, R.W. & Milthorpe, B.K. 1999, 'Lysozyme Sorption in Hydrogel Contact Lenses', Investigative Ophthalmology & Visual Science, vol. 40, no. 5, pp. 897-903.
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To examine the processes involved in formation of protein deposits on hydrogel contact lenses. METHODS: The adsorption and/or penetration of lysozyme on or into three types of contact lenses, etafilcon A, vifilcon A, and tefilcon, were investigated in vitro using a radiolabel-tracer technique, x-ray photoelectron spectroscopy, and laser scanning confocal microscopy. RESULTS: Binding of lysozyme to high-water-content, ionic contact lenses (etafilcon A and vifilcon A) was dominated by a penetration process. The extent of this penetration was a function of charge density of the lenses, so that there was a higher degree of penetration of lysozyme in etafilcon A than in vifilcon A lenses. In contrast, the binding of lysozyme to tefilcon lenses was a surface adsorption process. The adsorption and desorption kinetics showed similar trends to those found in human serum albumin (HSA) adsorption on lens surfaces. However, the extent of lysozyme adsorption on tefilcon is much higher than HSA adsorption, probably because of the self-association of lysozyme on the tefilcon lens surface. Furthermore, either penetration or adsorption of lysozyme involved reversible and irreversible processes and were both time dependent. CONCLUSIONS: Binding of lysozyme to hydrogel lenses involves surface adsorption or matrix penetration. These processes may be reversible or irreversible. The properties of the lens materials, such as charge density (ionicity) and porosity (water content) of the lenses, determine the type and rates of these processes.

Johnson, K.A., Rogers, G.J., Roe, S.C., Howlett, C.R., Clayton, M.K., Milthorpe, B.K. & Schindhelm, K. 1999, 'Nitrous acid pretreatment of tendon xenografts cross-linked with glutaraldehyde and sterilized with gamma irradiation', Biomaterials, vol. 20, pp. 1003-1015.
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Wei, M., Ruys, A.J., Swain, M.V., Kim, S.H., Milthorpe, B.K. & Sorrell, C.C. 1999, 'interfacial bond strength of electrophoretically deposited hydroxyapatite coating on metals', Journal Of Materials Science-materials In Medicine, vol. 10, pp. 401-409.
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Wei, M., Ruys, A.J., Milthorpe, B.K. & Sorrell, C.C. 1999, 'Solution ripening of hydroxyapatite nanoparticles: Effects on electrophoretic deposition', Journal Of Biomedical Materials Research, vol. 45, no. 1, pp. 11-19.
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Electrophoretic deposition is a low-cost, simple, and flexible coating method for producing hydroxyapatite (Hap) coatings on metal implants. However, densification requires heating the coated metal to high temperatures, which, for commercial HAp powders, generally means at least 1200¦C. At such temperatures, the metal tends to react with the HAp coating, inducing decomposition, and the strength of titanium and stainless steel implants is severely degraded. With the use of raw uncalcined nanoparticulate Hap, densification can occur at 900¦-1050¦C; however, such coatings are prone to cracking due to the high drying shrinkage. This problem was solved by precipitating nanoparticulate HAp by the metathesis process [10Ca(NO3)2 + 6NH4H2PO4 + 8NH4OH] and optimizing the 30 nm of nanoprecipitates by an Ostwald ripening approach, that is, by boiling and/or ambient aging in the mother liquor. While the as-precipitated nanoparticles produced severely cracked coatings, 2 h of boiling or 10 days of ambient aging ripened the gel-like mass into unagglomerated nanoparticles, which produced crack-free coatings. Since boiling enhanced particle size but ambient aging did not, crack elimination probably was due to the transition from the highly agglomerated gel-like state to the dispersed nanoparticulate state rather than to particle growth. Furthermore, boiling only reduced the amount of cracking whereas aging completely eliminated cracking.

Garrett, Q., Chatelier, R.C., Griesser, H.J. & Milthorpe, B.K. 1998, 'Effect of charged groups on the adsorption and penetration of proteins onto and into carboxymethylated poly(HEMA) hydrogels', Biomaterials, vol. 19, pp. 2175-2186.
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Proteins, lipids and other biomolecules interact strongly with the acrylic-based biomaterials used for contact lenses. Although hydrogels are nominally resistant to protein fouling, many studies have reported considerable amounts of protein bound to poly(2-hydroxyethylmethacrylate) (PHEMA) lenses. This study examined the binding of a series of biomolecules (tear protein analogues, mucin and cholesterol) to poly(methylmethacrylate) (PMMA) and three HEMA-based hydrogels (PHEMA, HEMA plus methacrylic acid (P(HEMA+MAA)), HEMA plus methacrylic acid plus N-vinylpyrrolidone (P(HEMA+MAA+NVP))) by use of a quartz crystal microbalance with dissipation (QCM-D) monitoring. The QCM-D estimates changes in the mass and viscous constant for the adsorbed layer through measurements of frequency and dissipation. Protein interaction with each of the test materials caused a net increase in mass of the material indicating protein binding except for lysozyme interacting with P(HEMA+MAA). A net decrease in mass was observed for lysozyme interacting with P(HEMA+MAA) which may be ascribed to lysozyme collapsing the hydrogel by expelling water. A net mass decrease was observed for cholesterol interacting with each of the hydrogel materials, while a mass increase was observed on PMMA.

Knepper, M., Milthorpe, B.K. & Moricca, S. 1998, 'Interdiffusion in short-fibre reinforced hydroxyapatite ceramics', Journal Of Materials Science-materials In Medicine, vol. 9, no. 10, pp. 589-596.
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Sintering in air and hot isostatic pressing are production methods regarded as being capable of producing fibre-reinforced hydroxyapatite ceramics for biomedical applications. These composites may have the advantage of improved mechanical properties and be suitable for applications in areas where there are significant levels of load on the material. The use of pure hydroxyapatite is restricted to those free of dynamical load. Obtaining improved mechanical strength is a question of the bond between the matrix phase and the fibre-reinforcement phase. However, a chemical bond between both phases, indicated by large diffusion zones, might lead to the dehydration of the hydroxyapatite leading to undesired tricalcium phosphate in the matrix resulting in a weakening of the mechanical and biological stability of the composites. Composites with three fibre types, alumina, 316L-stainless steel and titanium were prepared and sintered in air or hot isostatically pressed. A reaction zone was noted around the titanium and stainless steel fibres, but not around the alumina fibres. The reaction zone was larger for stainless steel than titanium. Hot isostatic pressing also reduced the reaction zone markedly compared to sintering in air.

Knepper, M., Moricca, S. & Milthorpe, B.K. 1997, 'Stability of hydroxyapatite while processing short-fibre reinforced hydroxyapatite ceramics', Biomaterials, vol. 18, no. 23, pp. 1523-1529.
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Lewis, D.D., Milthorpe, B.K. & Bellenger, C.R. 1997, 'Mechanical comparison of materials used for extra-capsular stabilisation of the stifle joint in dogs', Australian Veterinary Journal, vol. 75, pp. 890-896.

Madden, K.N., Johnson, K.A., Howlett, C.R., Milthorpe, B.K., Robins, G., Ikada, Y. & Schindhelm, K. 1997, 'Resorbable and non-resorbable augmentation devices for tenorrhaphy of xenografts in extensor tendon deficits: 12 week study', Biomaterials, vol. 18, no. 3, pp. 225-234.
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Tan, A., Milthorpe, B.K. & Huff, J.W. 1997, 'A technique for quantitation of protein deposists on rigid gas permeable contact lenses', Eye and Contact Lens: science and clinical practic, vol. 23, no. 3, pp. 177-84.

Garrett, Q. & Milthorpe, B.K. 1996, 'Human Serum Albumin Adsorption on Hydrogel Contact Lenses In Vitro', Investigative Ophthalmology & Visual Science, vol. 37, no. 13, pp. 2594-2602.
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To improve the understanding of the formation of protein deposits on hydrogel lenses. METHODS: A study of protein adsorption on three commercial hydrogel contact lenses of different materials, Etafilcon A (2-hydroxyethyl methacrylate [HEMA] polymer with sodium methacrylate and 2-ethyl-2-hydroxymethyl-1,3-propanediol trimethacrylate), tefilcon (poly[HEMA] cross-linked and copolymerized with ethylene glycol dimethacrylate), and vifilcon A (methacrylic acid polymer with ethylene glycol dimethacrylate, HEMA and N-vinyl pyrrolidone) was undertaken by using a single protein solution, human serum albumin (HSA), and a radiolabel-tracer technique. RESULTS: Static adsorption leading to multilayer adsorption was observed. Complete reversibility for adsorbed HSA on lenses did not exist. Some was tightly bound, whereas most was loosely bound and could be removed easily by rinsing in phosphate-buffered saline. Irreversible adsorption of HSA on the lenses was found to be time dependent and did not reach a maximum value even after 48 hours of adsorption. The amount of HSA adsorbed on the lenses-irreversibly as well as totally adsorbed protein- was in the order of vifilcon A > tefilcon > etafilcon A. Adsorption of HSA on the lenses increases with decreasing pH (range, 7.4 to 4) but always follows the above trend with respect to the different types of lenses. CONCLUSIONS: Irreversible binding of HSA on lenses is governed by the kinetics of protein denaturation. Electrostatic interactions may not play a major role in HSA adsorption on hydrogel lenses. Some other factors, such as hydrophobic dehydration, and special monomer units, such as N-vinyl pyrrolidone in the lens materials, may favor adsorption of HSA.

Roe, S.C., Milthorpe, B.K., Schindhelm, K. & Howlett, C.R. 1995, 'incorporation of chemically modified and radiation-sterilised cancellous bone allografts', Veterinary and Comparative Orthopaedics and Traumatology, vol. 8, pp. 163-169.

Rogers, G.J., Milthorpe, B.K., Schindhelm, K., Howlett, C.R. & Roe, S.C. 1995, 'Shielding of augmented tendon-tendon repair', Biomaterials, vol. 16, pp. 803-807.
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Ruys, A.J., Sorrell, C.C., Brandwood, A. & Milthorpe, B.K. 1995, 'Hydroxyapatite sintering characteristics: correlation with powder morphology by high-resolution microscopy', Journal of Materials Science Letters, vol. 14, no. 10, pp. 744-747.
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Hydroxyapatite Cal0(PO4)6(OH)2 (HAp) is a hydrated calcium phosphate ceramic that closely resembles the chemical composition of bone mineral and is therefore one of the small group of bonereplacement materials classed as bioactive, i.e. capable of bonding chemically with bone [1]. This group includes calcium phosphate ceramics, which, although chemically similar to HAp, are generally more prone to in vitro dissolution [2], and certain calcium phosphate glass compositions [1]. Thus HAp is a prime candidate for bone replacement applications.

Ruys, A.J., Wei, M., Sorrell, C.C., Dickson, M.R., Brandwood, A. & Milthorpe, B.K. 1995, 'Sintering effects on the strength hydroxyapatite', Biomaterials, vol. 16, no. 5, pp. 409-415.
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Ruys, A.J., Brandwood, A., Milthorpe, B.K., Dickson, M.R., Zeigler, K.A. & Sorrell, C.C. 1995, 'The effects of sintering atmosphere on the chemical compatibility of hydroxyapatite and particulate additives at 1200 C', Journal Of Materials Science-materials In Medicine, vol. 6, pp. 297-301.
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Garcia, J.N., Milthorpe, B.K., russell, D. & Johnson, K.A. 1994, 'Biomechanical study of canine spinal fracture fixation using pins or bone screws with polymethylmethacrylate', Veterinary Surgery, vol. 23, pp. 322-329.

Jones, A.S., Milthorpe, B.K. & Howlett, C.R. 1994, 'Measurement of Microtomy Induced Section Distortion and Its Correction for 3-Dimensional Histological Reconstructions', Cytometry, vol. 15, no. 2, pp. 95-105.
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The presence of microtomy induced distortion in paraffin sections is a significant hindrance to the accurate alignment of sections for three-dimensional reconstructive techniques. Measurement of section distortion in various rat tissues demonstrated distortions to be present in all sections, with over 85% of such distortions being manifest as expansions when compared to the original distances between a series of eight drilled fiducial marks. Mean percentage dimensional changes in the direction of the cutting stroke and at right angles to this direction were -0.5 ¦ 1.5% and 3.7 ¦ 1.2% for liver, 7.6 ¦ 2.4% and 9.1 ¦ 1.2% for kidney, 6.6 ¦ 2.3% and 10.5 ¦ 1.4% for lung, and 20.3 ¦ 6.6% and 8.9 ¦ 5.9% for skeletal muscle. Individual sections invariably displayed measurable distortions, with only skeletal muscle showing any consistent pattern, in the form of barrel distortion at right angles to the cutting stroke. In addition a method of distortion correction and simultaneous image alignment is presented as a means of section alignment with full distortion correction capability. This method uses a quadratic polynomial transform in a non-linear unwarping algorithm, to correct for the rotational and translational misalignment as well as for microtomy and camera aspect ratio distortions. Application of this method to a sequence of 46 serial sections demonstrated an alignment accuracy to within 2.6 ¦ 0.8 pixels

Milthorpe, B.K. 1994, 'Xenografts for tendon and ligament repair', Biomaterials, vol. 15, no. 10, pp. 745-752.
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Nordon, R.E., Milthorpe, B.K., Schindhelm, K. & Slowiaczek, P.R. 1994, 'An Experimental Model of Affinity Cell Separation', Cytometry, vol. 16, no. 1, pp. 25-33.
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Cell affinity separations are based on the selective attachment of cell pheno-type using antibody or lectins specific for cell surface markers. The major physico-chemical factors which influence ligand-mediated cell adhesion dynamics and the efficiency of cell affinity separation have been examined. Uniform cell detachment forces were generated with a parallel-plate flow cell (plate separation 100 m, surface area 3 cm2). Hydrodynamic shear stress was used to measure cell adhesion strength and to separate cells on the basis of surface affinity. Human cell lines grown in tissue culture were separated on a flat derivatised glass immunoadsorbent which formed the floor of the flow chamber. Flow-cell residence time, detachment shear stress, temperature, and ligand density were shown to influence cell attachment probability. An understanding of the physical basis of ligand-mediated cell adhesion provided a rationale for optimisation of affinity cell separation. At room temperature attachment of positive cells was rapid (<2 min) and adhesion strength was directly related to immunoadsorbent ligand density. Purity and recovery of enriched fractions were dependent on the separation shear stress and could be optimised using this parameter. Enrichment factors were greater than 100-fold, with at least 90% of positive cells recovered in enriched fractions. Enrichment purity and yields did not decline at higher loading densities (105 cells/cm2). Selective immunoadsorbent surface chemistry is a prerequisite for efficient affinity cell separation. Purity and recovery may be optimised by fractionating enriched and depleted cell populations with uniform fluid shear stress.

Ruys, A.J., Wei, M., Brandwood, A., Milthorpe, B.K. & Sorrell, C.C. 1994, 'Fracture toughness of 316L stainless steel short-fibre-reinforced hydroxyapatite', Journal of the Australian Ceramics Society, vol. 30, no. 1, pp. 12-18.

Ketaren, P.P., Batterham, E.S., White, E., Farrell, D.J. & Milthorpe, B.K. 1993, 'Phosphorus studies in pigs - 1. Available phosphorus requirements of grower/finisher pigs', British journal of Nutrition, vol. 70, pp. 249-268.
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Two experiments were conducted to determine the available P requirements of grower and grower/finisher pigs and to define the conditions for conducting a growth assay for P availability. In the first experiment, diets with four levels of calculated available P (1-4 g/kg) and four Ca:available P ratios (1.7-2.9) were used to determine the available P requirements of grower pigs. The diets were formulated by substituting the required amounts of limestone and sodium tripolyphosphate for sugar in a soya-bean meal and sugar-based diet. In addition to measuring growth responses, a range of bones were examined to determine the most suitable criteria for assessing the response to available P. There was a small quadratic response of feed intake and growth rate of the pigs to level of available P, with maximum responses occurring to approximately 3 g available P/kg (P < 0.05). There were linear depressing effects of increasing Ca: available P ratios on carcass gain and feed conversion ratio (P < 0.01) but most of these effects occurred when the ratio exceeded 2-5: 1.

Ruys, A.J., Ehsani, B.K., Milthorpe, B.K. & Sorrell, C.C. 1993, 'Effects of nonoxied additions on the decomposition and sintering of hydroxyapatite', Journal of the Australian Ceramics Society, vol. 29, pp. 65-69.

Ruys, A.J., Wei, M., Milthorpe, B.K., Brandwood, A. & Sorrell, C.C. 1993, 'Hydrothermal sintering of ZrO2 and Al2O3 Fibre-reinforced hydroxyapatite', Journal of the Australian Ceramics Society, vol. 29, pp. 51-56.

Ruys, A.J., Wei, B.K., Milthorpe, B.K., Brandwood, A. & Sorrell, C.C. 1993, 'Optimisation of fibre content in ZrO2-Fibre-Reinforced Hydroxyapatite', Journal of the Australian Ceramics Society, vol. 29, pp. 57-64.

Ruys, A.J., Milthorpe, B.K. & Sorrell, C.C. 1993, 'Short-fibre reinforced hydroxyapatite: effects of processing on thermal stability', Journal of the Australian Ceramics Society, vol. 29, pp. 39-49.

Standard, O.C., Schindhelm, K., Milthorpe, B.K. & Sorrell, C.C. 1993, 'In vitro ageing of tetragonal zirconia polycrystal (TZP) ceramics', Journal of the Australian Ceramics Society, vol. 29, pp. 54-60.

Roe, S.C., Milthorpe, B.K., True, K., Rogers, G.J. & Schindhelm, K. 1992, 'The effect of gamma irratiation on a xenograft tendon bioprosthesis', Clinical materials, vol. 9, pp. 149-154.

James, N.L., Poole-Warren, L.A., Schindhelm, K., Milthorpe, B.K., Mitchell, R.M. & Howlett, C.R. 1991, 'Comparative evaluation of treated bovine pericardium as a xenograft for hernia repair', Biomaterials, vol. 12, no. Nov, pp. 801-809.
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Milthorpe, B.K., Schindhelm, K., Howlett, C.R., Hall, P.J. & Roger, G.J. 1991, 'Treated xenografts as gliding tendon prostheses in an ovine model', Biomaterials, vol. 12, no. 6, pp. 577-583.
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A model for testing the properties of gliding tendon grafts has been developed that allows anastomoses to be evaluated separately from the mid-portion of the graft. In addition, two different graft materials may be implanted in one sheep foreleg whilst maintaining control (not operated) tendons in both the operated leg and contralateral foreleg. The model has been used to evaluate the response of xenografts made from chemically treated kangaroo tail tendon (KTT) compared with autografts. At 3 month the mid-sections of the glutaraldehyde-fixed xenografts maintained between 57 and 82% of their initial ultimate tensile strength whereas lyophilized KTT dropped to 10% and autografts retained 91% of initial strength. Sterilization by gamma-radiation of wet xenografts did not affect the material and implant properties significantly. Longer term studies are necessary to determine the resorption behaviour of the xenografts. Anastomosis strengths were found to be about the same for all grafts, at about 25% of the strength of the original tendon. Alternatives need to be investigated to improve this strength.

Schindhelm, K., Rogers, G.J., Milthorpe, B.K., Hall, P.J., Howlett, C.R., Sekel, R., Goldberg, J. & Viglione, W. 1991, 'Autograft and Leeds-Keio reconstruction of the e anterior cruciatement', Clinical Orthopaedics and Related Research, vol. 267, pp. 278-293.

James, N.L., Schindhelm, K., Slowiaczek, P.R., Milthorpe, B.K., Dudman, N.P., Johnson, G. & Steele, J.G. 1990, 'Endothelial cell seeding of small diameter vascular grafts', Artificial Organs, vol. 14, pp. 355-360.

Roe, S.C., Schindhelm, K. & Milthorpe, B.K. 1990, 'Collagen resorption and cross-linking: the effect of glutaraldehyde concentration', Artificial Organs, vol. 14, pp. 443-448.

Roger, G.J., Milthorpe, B.K., Muratore, A. & Schindhelm, K. 1990, 'Measurement of the mechanical properties of the ovine anterior cruciate ligament bone-ligament-bone complexa: basis for prosthetic evaluation', Biomaterials, vol. 11, no. March, pp. 89-96.
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McAuslan, B.R., Reilly, W., Hannan, G., Schindhelm, K., Milthorpe, B.K. & saur, B.A. 1988, 'Induction of endothelial cell migration by proline analogues and its relevance to angiogenesis', Experimental Cell Biology, vol. 176, pp. 248-257.

Milthorpe, B.K., Roger, G.J. & Schindhelm, K. 1988, 'Microcomputer-based system for tensile testing of biological materials', Medical & Biological Engineering & Computing, vol. 26, no. 2, pp. 161-166.
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The tensile testing of biological materials poses specific problems. Biological tissue may slip in the grips of the tensometer during tensile testing, and this needs to be taken into account in the calculation of strain. The inherent variability of tissue samples requires that a large number of samples be analysed to adequately characterise a given biological structure. These problems have been addressed by integrating a digital video dimensional analyser and a microcomputer into a system that can be attached to most tensometers. Features of the system are: automated data acquisition, analysis and filing; resolution of load and extension to better than 0À1 per cent of full scale; and achievable high throughput.

Bertram, C.D. & Milthorpe, B.K. 1984, 'Optical endpoint sensing in an automatic whole blood clotting timer', Medical & Biological Engineering & Computing, vol. 22, pp. 401-405.
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Most clotting time estimations are performed manually, although attempts have been made previously to automate them. The two major methods for automatically detecting the formation of the gel-like clot are mechanical (viscometric) and optical. The latter is superior in terms of accuracy of timing and freedom from artefacts but can only be performed on blood plasma. This paper describes a device which combines centrifuging to remove red cells and optical sensing of clot formation into a single operation, therepy giving activated clotting times on a par with those obtained mechanically from whole blood. The system offers the advantage over mechanical sensing that no nondisposable parts come in contact with the blood thereby eliminating e major source of timing errors. The timer works with any liquid coagulation activator, and will also time plasma clotting. The two-chambered design of the cuvette allows the activator to be kept separate from the blood until rotor startup The start of centifugal action mixes the blood and activator and starts the time. Timing is stopped auto matically when the rate of increase of optical density in the plasma, owing to fibrin formation, reaches a predetermined fevel.

Milthorpe, B.K., Bertram, G.D., Celler, B.G. & Farrell, P.C. 1984, 'Education and careers in biomedical engineering', Journal of Electrical & Electronics Engineering, Austr..., vol. 4, pp. 105-110.

Perez, D.J., Taylor, I.W., Milthorpe, B.K., McGovern, V.J. & Tattersall, M.H. 1981, 'Identification and quantitation of tumour cells in cell suspensions: a comparison of cytology and flow cytometry', British Journal Of Cancer, vol. 43, no. 4, pp. 526-531.
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FLOW-CYTOMETRIC MEASUREMENTS of cell DNA content or narrow-angle light scatter (NLS) can provide a basis for discriminating neoplastic from non-neoplastic cells by identifying differences in DNA content and cell size. Flow-cytometric analyses of many human neoplasms have been previously reported (Barlogie et at., 977, 1978) showing aneuploidy in 80-90% of non-lymphoid solid tumours and 40- 60% of non-Hodgkin's lymphomas. In leukaemias, and multiple myelomas with and aneuploid tumour clone, the percentage of neoplastic cells in marrow or blood determined by DNA content correlates well with quantitation based on histologic smears (Barlogie et al., 1977; Latreille et al., 1980; Andreeff et al., 1980) but such a comparison has not been made on cell suspensions from solid tumours.

Milthorpe, B.K. 1980, 'FMFPAK1: A program package for routine analysis of single parameter flow microfluorimetric data on a low cost mini-computer', Computer and Biomedical Research, vol. 13, pp. 417-429.
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A program package is presented for routine analysis, on a low cost minicomputer, of data obtained by flow microfluorimetry. The programs, with the aid of a graphics capability, provide calculations of various properties of cell populations: percentage of cells in various phases of the cell-cycle (G,, S, G2 + M): mean fluorescence per cell: mean narrow-angle scatter per cell, and an approximation to relative mean volume per cell from narrow-angle light scatter measurements. The system is designed to allow either direct analysis or for storage of raw data on tape files for analysis at a later time. Provision is also made for hard copy on a digital x-y plotter.

Taylor, I.W. & Milthorpe, B.K. 1980, 'An evaluation of DNA fluorochromes, staining techniques, and analysis for flow cytometry. I. Unperturbed cell populations', Journal of Histochemistry and Cytochemistry, vol. 28, no. 11, pp. 1224-1232.
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NA

Milthorpe, B.K., Nichol, L.W. & Jeffrey, P.D. 1977, 'The polymerisation pattern of Zn(II)-Insulin at pH 7.0', Biochimica et Biophysica Acta, vol. 495, pp. 195-202.

Jeffrey, P.D., Milthorpe, B.K. & Nichol, L.W. 1976, 'Polymerization Pattern of Insulin at pH 7.0', Biochemistry, vol. 15, no. 21, pp. 4660-4665.
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Nichol, L.W., Jeffrey, P.D. & Milthorpe, B.K. 1976, 'Analysis of sedimentation equilibrium results obtained with indefinitely self-associating systems using a procedure based on Laplace transformation', Journal of Physical Chemistry, vol. 80, no. 10, pp. 1071-1075.
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Equations are developed in closed form which permit the simulation of the distribution of total concentration vs. radial distance obtainable in the sedimentation equilibrium of a system of specified initial concentration undergoing indefinite self-association. Simulated distributions obtained with a variety of systems involving one or more equilibrium constants are used to test an analysis procedure which fits the distribution to a function capable of inverse Laplace transformation and leads to a specification of the relative amounts of the species in the cell as a function of their molecular weights. It is shown that such results may be related to the equilibrium concentrations of oligomeric forms at each radial distance, thereby permitting successive equilibrium constants appropriate to the indefinite self-association to be estimated.

Nichol, L.B., Jeffrey, P.D. & Milthorpe, B.K. 1976, 'The sedimentation equilibrium of heterogeneously associating systems and mixtures of non-interacting solutes: analysis without determination of molecular weight averages', Biophysical Chemistry, vol. 4, no. 3, pp. 259-267.
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Sedimentation equilibrium is first considered of a system in which a ligand of any size binds to an acceptor at p sites, the experimental result, obtained with either interference or absorption optics, being a distribution of total solute concentration as a function of radial distance. Theory illustrated by a numerical example, is presented which shows that this distribution may be analysed to give the activity of the unbound ligand as a function of total weight concentration. It is shown that this information may be used together with conservation of mass equations written in terms of the initial mixing composition to evaluate the equilibrium constant(s) relevant to the system. Correlation with composition evaluation by use of absorption optics (when possible) is also discussed. The procedure does not involve solution of simultaneous equations which are sums of exponentials nor differentiation of experimental results to obtain apparent weight-average molecular weights. It is general in that it leads to the evaluation of the activity of the species characterized by the smallest M(l-Image ?) product and, accordingly, is shown to be useful in the analysis of non-interacting as well as of interacting systems.

Milthorpe, B.K., Jeffrey, P.D. & Nichol, L.B. 1975, 'The direct analysis of sedimentation equilibrium results obtained with polymerizing systems', Biophysical Chemistry, vol. 3, no. 2, pp. 169-176.
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Theory is presented in relation to sedimentation equilibrium results obtained with polymerizing systems, which permits evaluation of the activity of the monomer as a function of total weight concentration. In contrast to established methods, the suggested procedure does not involve the solution of simultaneous equations which are sums of exponentials or the determination of weight-average molecular weights. A major advantage of the method is that it avoids errors inherent in differentiation and integration steps. An extrapolation to infinite dilution is involved, but this is to a defined limit and is uncomplicated by the existence of critical points in the relevant plot. The method is capable of detecting possible volume changes inherent on polymer formation, of treating systems where activity coefficients of solute species are functions of total concentration and of describing the system in terms of relevant equilibrium constants. These points and comparisons with existing methods of analysis are illustrated with numerical examples and with results obtained with lysozyme at pH 6.7. The lysozyme results are interpretable in terms of either a non-ideal monomer-dimer system or a monomer-dimer-trimer system

Schindhelm, K., Milthorpe, B.K. 1985, 'Artificial organs: engineering aspects', Melbourne, Australia, March 1985 in National Engineering Conference, ed E.A. Miadna, Institute of Engineers Australia, Australia, pp. 460-470.