There remains a need to develop synthetic bone substitutes with desirable architectures and properties to repair lost bone tissue. Three-dimensional printing (3DP) has been increasingly recognised as a promising technology for the fabrication of tissue-engineering scaffolds with the advantages of fully controlled geometries. However, the development of 3DP scaffolds with ideal printability, mechanical stability and resorption behaviour is still a challenge. The aim of this research was to design and develop bioceramic-based scaffold using 3DP technology. It started with the investigation of scaffold manufacture from the commercial calcium sulphate(CaS04) powder. Poly(ε-caprolactone) (PCL) coating was used to overcome the limitations of 3DP scaffolds. It was deeply infiltrated into inter-particle spaces, thus increasing the compressive properties of scaffolds to the level of cancellous bone and prolonging the complete resorption period from less than 7 days to more than 56 days. A dynamic flow system was designed to assess effects of flow condition on scaffold resorption. This research also developed a hydroxyapatite-calcium sulphate (HA-CaS04) formulation for a reliable and consistent 3DP process. A fundamental study was conducted to quantitatively assess effects of particle size on the key 3DP process parameters, such as powder bed packing and drop penetration behaviour. Based on the results, HA-CaS04 scaffolds were manufactured via 3DP followed by coated with PCL. Same characterisations were performed on the HA-CaS04 scaffolds to evaluate their compressive properties and in vitro resorption. The feasibility of printing HA-based scaffolds by incorporating different binding additives, such as maltodextrin and polyvinyl alcohol (PVOH), into HA powder was also investigated. Scaffolds with high geometrical accuracy and excellent green compressive strength were obtained when the PVOH (high viscosity) was used as the binding additive for HA.

The rat ileum normally ceases uptake of macromolecules between 18 and 21 days of age. During this period the immature vacuolated cells on the villus are replaced by a new population of mature nonpinocytosing cells from below. This is known as ileal closure. A single intraperitoneal injection of cortisone acetate to 5 day old rats induced precocious closure within 6 days. The closure process was similar to that seen during normal ileal closure. During both normal and induced closure morphometric measurements showed that there was a gradual loss of the vacuolated cell population as one passed down the villus. This was thought to be due to a gradual increase in the degree of differentiation in the cells produced by the crypt; related perhaps to the duration of the exposure of the crypt to the closure stimulus. 10 days after cortisone acetate treatment vacuolated cells were again seen on the villus. Their distribution varied from animal to animal. sometimes covering the whole villus and sometimes only the top half of the villus. One of the possible explanations for this is discussed. Administration of corticosterone, the naturally occurring corticosteroid in the rat, to young rats failed to promote any morphological changes indicative of ileal closure at the electron microscope level. Normal closure in the ferret ileum occurs between 35 and 39 days of age, by a similar process of apparent cellular replacement as that seen during normal ileal closure in the rat. Administration, to young ferrets, of cortisone acetate at many doses up to lethal ones failed to promote any morphological changes indicative of ileal closure. The possible role of the corticosteroids in the normal closure process in the mammalian intestine and the apparent species specific effect of cortisone acetate are discussed.

Three contrasting examples of bacterial host-parasite relations were studied with a view to recognising the compounds and processes responsible for the pathogenicity of the causal organisms. In the belief that only bacteria collected from only the specific disease-process in animals (i. e. An vivo) could be relied, on to have the complete armoury of virulence factors, the three bacterial species selected: Bacillus anthracis, Pasteurella Westin and the calla group, were all initially grown in vivo. An adequate supply of such organisms was obtained, and fractionation studies of the organisms and their products for substances responsible for virulence were made, possible by the use of biological assays which simulated conditions in the host. Once the important constituents of the bacteria grown in vivo had been recognised,it was often possible to alter the culture conditions in vitro to produce bacteria having the same constituents. The main findings were as follows- Anthrax virulent organisms. had-to have two factors (1) the capsule of polyglutamic acid which prevented opsonization and (2) the extracellular toxin, which was a mixture of three components, and poisoned the defence phagocytes: The massive bactersomia found in dying guinea pigs was not essential for death.

Tissue engineering scaffolds are potential candidates to develop bone grafts with autologous properties. Scaffolds mimic the bone extracellular matrix playing an active role in tissue formation. Scaffold architecture must be controlled to deliver the adequate mechanical stimuli onto cells. Rapid prototyping methods have been widely used to fabricate scaffolds with controlled regular internal microstructure. However, inaccuracies in the fabrication process could deviate the actual scaffold from its intended design leading to variations in the micromechanical cell environment and thereby final tissue properties. In this thesis, a computational inspection protocol was developed to evaluate the quality and the reproducibility of rapid prototyping scaffolds. A commercially available scaffold with three-dimensional regular microstructure was selected and none of the inspected samples replicated the intended design. In addition, variability among samples at the pore level in terms of wall shear stress and fluid velocities was found. This could potentially lead to perturbations in cell activity and affect the transport of cells during cell seeding. Scaffold cell seeding determines the initial conditions for tissue growth and the control of this process is essential towards the automatization and translation of scaffold-based therapies into clinical treatments. Micro-particle image velocimetry and cell tracking experiments were performed to characterise cell motion during hydrodynamic seeding. These experimental data were employed for the development of a computational model to predict cell transport and cell deposition onto 3D scaffolds under fluid flow. This computational model was applied to explore in vitro cell seeding experiments. It was found that cells tend to follow the fluid streamlines reducing the probability of cell interception with scaffold substrate and therefore cell adhesion. Force due to gravity and secondary flows were identified as the main mechanisms of cell deposition during cell seeding. Flow rate and geometry of the scaffold and the microfluidic chamber are key factors to control these mechanisms of cell deposition and enhance cell seeding efficiency.

Non-destructive 3D micro-computed tomography (microCT) based finite element (microFE) model is popular in estimating bone mechanical properties in recent decades. From a fundamental scientific perspective, as the primary function of the skeleton is mechanical in nature, a lot of related biological and physiological mechanisms are mechano-regulated that becomes evident at the tissue scale. In all these research it is essential to known with the best possible accuracy the displacements, stresses, and strains induced by given loads in the bone tissue. Correspondingly, verification and validation of the microFE model has become crucial in evaluating the quality of its predictions. Because of the complex geometry of cancellous bone tissue, only a few studies have investigated the local convergence behaviour of such models and post-yield behaviour has not been reported. Moreover, the validation of their prediction of local properties remains challenging. Recent technique of digital volume correlation (DVC) combined with microCT images can measure internal displacements and deformation of bone specimen and therefore is able to provide experimental data for validation. However, the strain error of this experimental method tends to be a lot higher (in the order of several thousand microstrains) for spatial resolutions of 10-20 µm, typical element size of microFE models. Strictly speaking no validation of strain is possible. Therefore, the goal of this thesis it to conduct a local convergence study of cancellous bone microFE models generated using three microCT-based tissue modelling methods (homogeneous tetrahedral model, homogeneous hexahedral model and heterogeneous hexahedral model); to validate these models’ prediction in terms of displacement using the novel DVC technique; and finally to compare the strain field predicted by three tissue modelling methods, in order to explore the effect of specific idealisations/simplifications on the prediction of strain.

Stem cells serve as important models for the in vitro study of many in vivo micro-environments. Their function relies largely on the highly ordered organisation of cells, matrices and soluble factors within defined architectures. Therefore, the ability to manipulate microscopic objects through precise in vitro approaches provides a new means to replicate these complex arrangements and give insights into factors that influence biological development. In this study, the progression of specific patterning technologies that allow precise control over cell position at the micron scale has been investigated. Initial studies demonstrated a stencilling based micro-patterning technique, which utilised aerosol deposition of a chemical agent, to induce region selectivity and define cell adhesive areas on a poor cell responsive surface. Micro-stencils acted as a mask to spatially restrict l,l'-Carbonyldi imidazole (CDI) aerosols to poly(2-hydroxyethyl methacrylate) (polyHEMA) coated surfaces, to which fibronectin was subsequently immobilised. Surface characterisation analysis revealed that a change in surface topography and a decrease in surface hydrophilicity did not negatively affect the adhesion and proliferation of cells. NIH-3T3 cells were seeded onto micro-patterns in defined medium conditions where cell proliferation, using the MTS assay, was assessed over time. The combined use of polyHEMA substrates with this functionalisation technique produced persistent patterns that were preserved for up to 96 hours. This method achieved well defined patterns of cells over macro scales with prospective uses in future tissue engineering applications. Secondly, an optical based micro-patterning technology, utilising a bespoke holographic optical tweezer (HOT) system, was used to precision build cellular micro-architectures. N1H-3T3 cells and mouse embryonic stem (mES) cells were patterned using an infrared laser. Multiple optical traps were generated by laser reflection off a spatial light modulator (SLM). The reflected light was focused through a high resolution numerical aperture objective lens to produce a defined three dimensional (3D) pattern (the hologram). PolyHEMA was used to prevent substrate stickiness prior to pattering. Opening work of this technique patterned two dimensional (2D) cell arrangements that were retained using the developed micro-stencilling method to monitor subsequent responses of cell viability, proliferation and differentiation. LIVE/DEAD® staining and proliferation assessment using the Click-iT® EdU assay demonstrated that the laser caused no obvious damaging effects on the cells. Immunocytochemical staining of Brachyury, Nestin and GATA-4 showed differentiation of mesoderm and ectoderm but no endoderm expression of small scale structures (> 4 cells). Similarly positive Oct-3/4 expression showed evidence of undifferentiated cells. The next part of the study went on to demonstrate the stabilisation of patterned cell structures within a temperature or enzyme controlled hydrogel support network to retain 3D architectures. Complex co-cultures of cells and poly(lactic-co-glycolic acid) (PLGA) polymer materials were also investigated to reproduce analogues of the cells physical environment. This manipulation tool attained single cell resolution capable of manipulating microscopic arrangements of cells with future applications within the fields of developmental and stem cell biology. It is hoped that the engineering of 3D biological structures in vitro will allow for the generation of more complex and representative cell models with greater definable and tuneable characteristics to assess stem cell fate in the future.

No description available.

The nervous system may be simulated as a network of model neurons as a means to understand the function of the brain. Complex as the mammalian nervous system is, such simulations of any significant scale are computationally and energetically expensive. SpiNNaker is a computer architecture designed to advance the feasible scale of neural tissue models using fifty thousand chips, each containing eighteen low-power processors, to model one billion neurons and one trillion synapses in real-time. This dissertation demonstrates the success of prototype hardware with detailed models of the rodent somatosensory cortex. Simulations are built from neuroanatomical data on a host computer using a simple declarative library of functions, and are executed on SpiNNaker atop an event-driven programming interface that neatly abstracts the intricate details of the machine. Comparisons with reference simulators show that SpiNNaker correctly reproduces established results, and power readings report that each chip draws just one watt during execution. A model of the whisker barrel, derived from the literature, exhibits key responses to simulated stimuli, and a model of the wider barrel cortex, comprising 10^5 neurons and 7*10^7 synapses, demonstrates real-time, massively parallel simulation across 360 processors on 23 chips. Ultimately, SpiNNaker is shown to be an effective architecture for the correct and efficient simulation of neural tissue.

L’imagerie par spectrométrie de masse est une technique sans marquage permettant la détection et la localisation de protéines à partir de coupes de tissus. Afin de répondre à des problématiques biologiques, le nombre de protéines identifiées doit être amélioré. Une stratégie consiste à réaliser une micro-jonction liquide sur des régions particulières des coupes de tissus afin d’extraire les peptides issus de la digestion in situ des protéines. Plus de 1500 protéines ont identifié sur une zone de 650µm, correspondant à environ 1900 cellules. Une corrélation entre ces données avec celles générées par MSI a augmenté le nombre de protéines localisées. Afin d’obtenir dans le même temps, la localisation et l’identification de protéines, une méthode consiste à réaliser la microdissection de l’ensemble de la coupe après l’avoir déposée sur une lame recouverte de parafilm. PAM a également été appliquée à l’étude de l'expression différentielle de protéines dans des tumeurs de prostate. Les résultats ont permis d’identifier des biomarqueurs potentiels tels que des protéines complexées avec des petits ARN nucléolaires. Enfin, la faisabilité des méthodes MS appliquées à l’étude structurale de protéines, tel que l'échange deutérium ou le pontage chimique, ont été examinés directement sur tissu. Les résultats préliminaires suggèrent qu’une étude structurale de protéines est possible afin de déterminer des changements de structures entrainés par la modification du microenvironnement. Réunis ensemble, ces méthodes MS d'analyses directes fournissent un moyen robuste d’étude de protéines dans leur état natif afin de fournir des indications sur leur rôle dans des systèmes biologiques. / Mass spectrometry-based methods for direct tissue analysis, such as MS imaging, are label-free techniques that permit the detection and localization of proteins on tissue sections. There is a need to improve the number of protein identifications in these techniques for them to comprehensively address biological questions. One strategy to obtain high protein IDs is to realize liquid microjunction on localized regions of tissue sections to extract peptides from the in situ digestion of proteins. More than 1500 proteins were identified in a 650µm spot, corresponding to about 1900 cells. Matching these IDs with those from MSI increased the number of localized proteins. In order to achieve simultaneous identification and localization of proteins, a method consisting of microdissecting entire tissue sections mounted on parafilm-covered slides was developed. Spectral counting was then used to quantify identified proteins, and the values were used to generate images. PAM was also used to examine the differential expression of proteins on prostate tumors. Results identified potential biomarkers such as proteins in complex with small nucleolar ribosomal RNAs. Lastly, the feasibility of applying MS methods of structural analysis, such as deuterium exchange and crosslinking, directly on tissue was examined. Preliminary results suggest the possibility of this approach, which could be significative by permiting the determination of protein structural changes for a given microenvironment. Taken together, these direct MS analysis methods provide a robust means of analyzing proteins in their native state and are expected to provide insights to their role in biological systems.

Histology is a technique that has any number of diagnostic uses in modern hospital laboratories. However, as a scientific method employed in the study of ancient and mummified remains, it appears to have lost its popularity.This project explores the advantages and limitations of histology as a technique for such studies. In order to do so, soft tissue and bone samples from ten early Roman Period mummies (30 BCE – 250 CE) from the Kellis 1 cemetery in the Dakhleh Oasis have been histologically examined.While this project focuses on the scientific technique of histology, and its application for the study of ancient remains, it also aims to be cross-disciplinary by incorporating scientific results from the ten mummies with the historical data and archaeological remains uncovered during excavations of the Kellis site. By bringing the results of science and Egyptology/archaeology together, it hoped that a better understanding of ancient Egyptian society could be achieved.