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(2009) Marcal, HelderThesisRegenerative biology seeks to understand the molecular mechanisms that differentiate several animals with the ability to regenerate tissues. An approach to evaluate these mechanisms is via a comparative analysis of regeneration-competent versus regeneration-deficient tissues. This thesis analyses the regenerative capacity in the axolotl blastema during the dedifferentiation phase. Currently, there is an equivocal understanding of the molecular factors expressed during the regenerative process and further analysis is warranted. Comprehensive profiles of the proteins expressed during this process are presented. Nerve derived factors such as neuregulin-1 and neurotrophin-3 were identified in the axolotl blastema during the time of dedifferentiation. Additionally, this study investigated if there are any correlation between the nerve determinants, such as neurotrophins, from the regenerating blastema and neurogenesis that occurs throughout the lifespan of mammals in the olfactory system. This was achieved by comparing the expression proteomes of the regeneration-competent axolotl and olfactory ensheathing cells. Collectively, the study proposes that there is an influence by neurotrophins on neurogenesis in the olfactory system that are comparable to the dynamic nerve signaling networks that occur in the axolotls regenerating limb. It provides a window into the complex signaling pathways during the two processes that can be clinically translated to further develop better therapeutic strategies that involve peripheral nervous tissue of the spinal cord. The current clinical outcomes usually result in limited functional restoration. One of the most promising techniques is to induce a regenerative capacity in the affected tissues using a tissue engineering strategy. The combination of factors identified may serve as candidates to augment in vivo peripheral nerve microenvironments and to achieve a regenerative capacity in human tissues. These neurotrophins should be considered for regenerative medicine applications to induce regenerative competence in human peripheral nerve tissues.
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(2009) Green, Rylie AdelleThesisThis research investigates the use of conducting polymer coatings on platinum (Pt) electrodes for use in neuroprostheses. Conducting polymers aim to provide an environment conducive to neurite outgrowth and attachment at the electrode sites, producing intimate contact between neural cells and stimulating electrodes. Conducting polymers were electropolymerised onto model Pt electrodes. Conventional polymers polypyrrole (PPy) and poly-3,4-ethylenedioxythiphene (PEDOT) doped with polystyrenesulfonate (PSS) and para-toluenesulfonate (pTS)were investigated. Improvement of material properties was assessed through the layering of polymers with multi-walled carbon nanotubes (MWNTs). The ability to incorporate cell attachment bioactivity into polymers was examined through the doping of PEDOT with anionic laminin peptides DCDPGYIGSR and DEDEDYFQRYLI. Finally, nerve growth factor (NGF), was entrapped in PEDOT during polymerisation and tested for neurite outgrowth bioactivity against the PC12 cell line. Each polymer modification was assessed for electrical performance over multiple reduction-oxidation cycles, conductivity and impedance spectroscopy, mechanical adherence and hardness, and biological response. Scanning electron microscopy was used to visualise film topography and x-ray photon spectroscopy was employed to examine chemical constitution of the polymers. For application of electrode coatings to neural prostheses, optimal bioactive conducting polymer PEDOT/pTS/NGF was deposited on electrode arrays intended for implantation. PC12s were used to assess the bioactivity of NGF functionalised PEDOT when electrode size was micronised. Flexibility of the design was tested by tailoring PEDOT bioactivity for the cloned retinal ganglion cell, RGC-5, differentiated via staurasporine. It was established that PEDOT films had superior electrical and cell growth characteristics, but only PPy was able to benefit from incorporation of MWNTs. Bioactive polymers were produced through inclusion of both laminin peptides and NGF, but the optimum film constitution was found to be PEDOT doped with pTS with NGF entrapped during electrodeposition. Application of this polymer to an implant device was confirmed through positive neurite outgrowth on vision prosthesis electrode arrays. The design was shown to be flexible when tailored for RGC-5s, with differentiation occurring on both PEDOT/pTS and PEDOT/DEDEDYFQRYLI. Conducting polymers demonstrate the potential to improve electrode-cell interactions. Future work will focus on the effect of electrical stimulation and design of bioactive polymers with improved cell attachment properties.
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(2009) Hui, Ben Bunny Chun BunThesisMathematical models are often used to describe and, in some cases predict, excitable cellular behaviour that is based on observed experimental results. With the increase of computational power, it is now possible to solve such models in a relatively short time. This, along with an increasing knowledge of cellular and subcellular processes, has led to the development of a large number of complex cellular models, capable of describing a broad range of excitable cell behaviour. But the use of complex models can also lead to problems. Most models can accurately reproduce results associated with the data on which the models are based. However, results from complicated models, with large numbers of variables and parameters, are less reliable if the model is not placed under the same physiological conditions as defined by the model author. In order to test a model's suitability and robustness over a range of physiological conditions, one needs to fit model parameters against experimental data observed under those conditions. By using the modelling standard and repository offered by CellML, model users can easily select and adapt a large number of models to set up their own applications to fit model parameters against user-supplied experimental data. However, currently there is a lack of software that can utilise CellML model for parameter fitting. In this thesis, a Java-based utility has been developed, capable of performing least square parameter optimisation for a wide range of CellML models. Using the developed software, a number of parameter fits and identifiability analyses were performed on a selected group of CellML models. It was found that most of the models were ill-formed, with larger numbers of parameters worsening model identifiability. In some cases, the usage of multiple datasets and different objective functions can improve model identifiability. Finally, the developed software was used to perform parameter optimisation against two sets of action potentials from a sinoatrial node experiment, in the absence and presence of E9031, a specific ion channel blocker.
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(2009) Fong, Nicole Wei ShiThesisHospital-acquired infections are a significant contributor to clinically-related morbidity and mortality. The majority of these infections are associated with the use of invasive medical devices, where urinary catheters account for ~36% of cases. Current preventative strategies have shown short-term (<7 days) success, however their long-term (>28 days) efficacy is unclear. This thesis explores the use of solution-cast polyurethane nanocomposite (PUNC) materials for antimicrobial drug delivery in urinary applications. It is hypothesised that the enhanced barrier properties of PUNCs, afforded by the incorporation of well-dispersed nanoinclusions, would allow for the sustained release of an antimicrobial agent. The objectives of this research were to investigate the antibacterial, mechanical and barrier properties of PUNCs incorporating various silicates modified using antimicrobials, hypothesised to also act as dispersing agents. Organically modified silicates (OMS) were prepared at 110%, 200% and 300% cationic exchange capacity (CEC) using the biocide, chlorhexidine diacetate (CHX), which was hypothesised to perform the dual functions; dispersant and antibacterial agent. Resulting OMS were incorporated at 1wt% and 5wt% loadings into a PU matrix to produce PUNCs; PEU-CHX1.1MMT, PEU-CHX2.0MMT, and PEU-CHX3.0MMT, respectively. CHX performed well as a dispersant, producing intercalated to partially exfoliated PUNCS. Antibacterial activity was dependent on OMS type and loading. PEU-CHX1.1MMT materials had poor antibacterial properties, but the addition of free CHX into the materials significantly improved their efficacy, demonstrating long-term sterility in an in vitro urinary tract (UT) model. PEU-CHX2.0MMT and PEU-CHX3.0MMT at 5wt% OMS loadings had partially exfoliated structures and excellent antibacterial activity. Cytotoxicity was evident in all materials, although to a lesser extent in the latter. Overall, intermediate OMS loadings of CHX2.0MMT would be expected to produce PUNCs with favourable antibacterial activity and cytocompatibility. PUNC drug-release profiles demonstrated sustained release compared to pristine PU, indicative of enhanced barrier properties. Their ultimate tensile properties decreased with increased OMS loading or addition of free CHX.Higher cationic-exchanged OMS caused significant reductions in strain. Young's modulus increased in response to higher %CEC OMS and loading. PUNCs show promise as antibacterial biomaterials for long-term urinary applications, where antimicrobial release and mechanical properties can be modulated through organic modification and OMS loading.
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(2009) Wong, Yan TatThesisMicroelectronic vision prostheses aim to restore visual percepts through electrical stimulation of the surviving visual pathways in the blind. Electrical stimulation has been shown to produce spots of light in the visual field. A neurostimulator that forms the basis of a vision prosthesis was designed using a high voltage CMOS process to allow it to be able to stimulate when faced with high electrode-tissue impedances. It was implemented with novel features that allow it to be scalable, and to focus charge injection, and can stimulate multiple sites simultaneously using a current source and sink at each site. To reduce electrical cross-talk between multiple stimulation sites, six-return electrodes surround each stimulating electrode, electrically guarding them from each other. The six-return electrode configuration was shown to reduce electrical cross-talk in saline bath tests compared to single-return electrode configurations. The neurostimulator was used to evoke responses from cats through electrical stimulation via intravitreal ball electrodes, corneal electrodes, and planar electrode arrays in the suprachoroidal space. Responses were measured on the visual cortex through optical imaging of intrinsic signals, and through surface electrodes. Using the planar electrode array in the suprachoroidal space, responses were elicited to biphasic, bipolar and monopolar stimuli, with each stimulating electrode coupled with either six-return electrodes, two-return electrodes, or a single-return electrode. The average charge threshold to elicit a response for biphasic, bipolar stimulation with six-return electrodes was 76.47 ± 8.76 nC (standard error of the mean). For biphasic, bipolar stimulation, the magnitude and area of cortical response with the six-return electrode configurations was on average 2.18 ± 0.19 times smaller than single-return electrode configurations, and 1.89 �� 0.19 times smaller than two-return electrode configurations (P < 0.0001). It was also found that for biphasic stimulation, a greater magnitude and area of response was elicited for monopolar stimulation compared to bipolar stimulation. This dissertation details the design and testing of a novel, scalable neurostimulator to focus charge injection. It also shows that suprachoroidal, bipolar stimulation can elicit visual responses, and that the area of cortical activation was more focused when using bipolar, biphasic stimulation, and six-return electrodes.
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(2009) Black, Ross ArthurThesisThe human vestibulo-ocular reflex (VOR) stabilizes gaze during head movement. The reflex is typically tested in a clinic or laboratory using passive rotations or artificial stimuli which measure the amount of damage the vestibular apparatus has suffered. However, during everyday activities the vestibular system is stimulated by active, self generated head movements. Head movements are often rapid and associated with the goal of achieving either gaze-fixation or re-fixation. Patients who complain of on-going symptoms will typically identify a particular position or movement that aggravates their symptoms in their everyday life. There is a need to identify objective parameters which correlate with the subjective complaints of patients whose symptoms persist after vestibular damage. In the first study, a gaze-refixation task, patients who complain of ongoing symptoms (poorly-compensated), during rapid head turns, after unilateral vestibular de-afferentation (uVD) were compared with those who did not have the same complaints (well-compensated) and normal subjects. Well- and poorly-compensated groups were sorted according to responses on a standardized questionnaire. All subjects were then located in a real-world, non-laboratory environment in which poorly-compensated subjects reported experiencing symptoms. Each subject’s head, eye and gaze displacement and velocity, head rotation frequency and blink or eye-lid closure were measured and analysed and compared between ipsi- and contra-lesional head rotations within and between subject groups. When subjects are able to generate their own active head rotations it has been suggested that a number of vestibular and extra-vestibular strategies might be employed to compensate for an impaired VOR. In subsequent studies, high resolution scleral search coils were used to identify the compensatory mechanisms used during active head rotations during a gaze-fixation task. A corrective saccade is typically observed during passive ipsilesional head rotations or “impulses” and might be potentiated during rapid, active or self-generated head rotations. The conditions which predict or contribute to the generation of the rapid, corrective eye movement were investigated. The results were compared with responses to passive head impulses of matched velocity and acceleration to determine if active head impulses could be used to identify a lesioned vestibular apparatus as is routinely clinically achieved with passive head impulses.
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(2009) Lau, Chi Bang AbeThesisKnowledge of anatomy is an elementary step towards the understanding of the human body. First used by Alphonse Bertillon as an identification system, anthropometry refers to the measurements of human individuals. In orthopaedics, comparative analysis is widely used in the understanding of morphological variance due to races, sex and pathological conditions. The characterization of bone and joint geometry has also been a foundation of modern surgical implant design. Traditional anthropometric studies rely on physical measurements by means of osteometric table. Recent advancements of 3-D imaging modalities and image processing techniques have empowered more fine-grained anthropometric characterization. The inspiration for the study is: - the understanding of anatomy originating from the clinical domain have shown to contribute to undesirable inconsistency in the image processing domain. - the difficulty of existing automated anthropometric methodology in handling pathological femur. - the tedious amount of manual and subjective work involved with the increasing amount of high resolution imaging data. The aim of the study is to: - develop a consistent and robust methodology in accurate extraction of anthropometric parameters on the femur. - increase the level of automation on the process of anthropometric parameter extraction. With the bridging of anthropometry and the image processing disciplines, a robust methodology of anthropometric parameter extraction with high level of automation was developed, implemented and tested. A dataset comprised of femoral CT scans of 19 healthy Australian, 10 healthy Japanese, 15 Japanese diagnosed with primary or secondary hip osteoarthritis and 20 adult sheep was utilized for testing. Intra-class correlation and Cronbach's α were extensively employed to evaluate the intra-rater, interrater and repeated scans consistency of the proposed methodology. High correlation values (mean > 0.95) were noted suggesting a high consistency of the methodology. All healthy and osteoarthritis human datasets were processed successfully. With the structural similarity between the sheep and human femur, the robustness was further demonstrated by accurate processing of the sheep dataset without the need of any modification of the underlying methodology. The methodology proposed is highly automated and requires very few user interactions in the parameter extraction stage.
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(2009) Estrella, Ruby PobleteThesisGlycoproteins, and their subset proteoglycans, are an important group of molecules in joint tissues, providing crucial functions such as cartilage structural integrity and lubrication at cartilage surfaces. The functionality of these glycoproteins is attributable to their oligosaccharide components, however surprisingly little is known about their fine structural details. With the use of glycoproteomic methods, this thesis presents the development and incorporation of mass spectrometric, biochemical and immunological methods to elucidate glycoprotein structures in synovial fluids, chondrocytes and synoviocytes in order to provide insight into how their structures may contribute to their functions. Initially, anion exchange chromatography was used to extract the acidic fraction containing glycoproteins and proteoglycans in arthritic synovial fluid (SF) samples, followed by proteomic analysis to identify the main glycoproteins in 1D-SDS-PAGE gels. To complement these findings, an in-gel enzymatic digest method for glycosaminoglycan (GAG) and oligosaccharide analysis was developed for analysis of glycoproteins by graphitised carbon liquid chromatography mass spectrometry (LC-MS). Further characterization of the major glycoprotein, lubricin, was pursued by investigating its interactions with the surrounding extracellular matrix (ECM) from its cellular sources and characterising the secreted lubricin with Western blot and proteomic analysis. Finally, the graphitised carbon LC-MS method was applied to analyse the overall glycosylation profiles of lubricin. The major glycoprotein found in arthritic synovial fluid was lubricin, as identified by peptide LC-MS and Western blot. Graphitised carbon LC-MS identified the major chondroitin sulfate (CS) repeat region disaccharides and linkage region oligosaccharides of aggrecan with confirmation through tandem mass spectra and Western blots using CS linkage region stub antibodies. Application of this method to lubricin led to the discovery of O-linked oligosaccharide structures which were previously undescribed for lubricin. A higher proportion of sialylated oligosaccharide structures were detected in the rheumatoid arthritis (RA) samples compared to the osteoarthritic (OA) samples, which signifies a diagnostic difference between these diseases. Sulfated oligosaccharide structures were also detected on synovial fluid lubricin, correlated with Western blot reactivity with the MECA-79 antibody, thus suggesting a role for lubricin in inflammation. Overall the results demonstrated that glycosylation structure indicates additional functional properties for the glycoproteins such as lubricin.
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(2009) Ko, Henry Chung HungThesisA limitation to engineering viable thick tissues (greater than a few hundred microns in thickness) has been the lack of vascularisation and a vascular supply. A key element in engineering such tissues is the generation of a supporting scaffold with a defined and wellcharacterized architecture. To date relatively little attention has been paid to characterization. The objective of this research was to develop well-characterized structures which will inform the rational design of the next generation of engineered thick tissues. Specifically, this research aimed to test combinations of various culturing environments, cell mono- and co-cultures, and scaffold architectures; develop improved imaging techniques and structural/spatial analytical methods to characterise porous polymer scaffolds; and use various spatial and morphological measures to quantify the relationships between scaffold geometric structure and cell distribution. Isotropic and anisotropic pore scaffolds were manufactured and then processed with nondestructive and destructive imaging methods, and characterised using image analysis methods to measure geometric parameters such as the degree of anisotropy/isotropy, porosity, and fractal parameters of pore and strut networks. Cells were introduced into scaffolds using a range of seeding methods and cultured in static and hydrodynamic environments. Quantification of the spatial cell distribution in cell-seeded scaffolds was done with first-order spatial statistics and fractal analysis. Findings comparing various destructive and non-destructive imaging methods found that cryotape cryohistology was the most accurate method for processing bare polymer scaffolds and eliminated histological artefacts common to other techniques. It was found with the various image analysis methods, surface and internal scaffold geometric architectures were strongly isotropic for porogen-fused porogen-leached scaffolds and anisotropic for TIPS scaffolds. For both isotropic and anisotropic pore scaffolds, collagen hydrogel infusion and droplet methods gave the highest cell seeding efficiencies (at 100% efficiency). The key finding in this study was that first-order spatial statistics and fractal analysis of cell distribution revealed that the geometric structure of the scaffolds had the strongest effect on spatial cell infiltration and distribution compared to the influence of culture environment or mono- and co-culture. Isotropic pore scaffolds had a higher level of cell distribution. Further work with optimizing the growth environment parameters, and utilizing collagen-infused cell-seeded scaffolds, may assist in achieving better cell growth. The work presented therefore provides the analytical basis for the rational design of tissue engineering scaffolds.
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(2009) Chen, Spencer Chin-YuThesisA successful restoration of vision should allow the blind to look, to see and to understand. The engineering of a microelectronic vision prosthesis has come a long way over the last forty years, but the understanding of how the restored form of vision would be interpreted and functionally applied to everyday living has made little progress until recent times. Prosthetic vision is not what most people think it would be; it is a visual scene composed of relatively large, isolated, spots of light so-called "phosphenes", very much like a magnified pictorial print. This thesis dissertation seeks to obtain a complete survey of the visual description of phosphenes from the human trial reports in the literature, simulate it, obtain a measure of the functional capacity of such visual perception, and explain the measured performance against design aspects of phosphene presentation, human perception, cognition and behaviour. Specifically, "visual acuity" (VA) was assessed on normally sighted subjects (N=15) administered with "simulated prosthetic vision". VA is a functional measure of vision highly correlated to many daily activities. Aggregating the results from the study with the other VA studies in prosthetic vision, it is shown that in general, the density of the phosphene field determines the affordable VA; however, design aspects relating to the phosphene field lattice (0.03 10gMAR with the hexagonal lattice as opposed to a square lattice) and image processing routines (0.15 10gMAR at optimised settings) can be further fine-tuned to improve VA performance. Significant performance improvement also arose from learning (0.13 10gMAR over ten visitations) and visual scanning adaptation (0.20 10gMAR with a circular scanning strategy). Performance improvements are likely related to various preferences and perceptual preferences of the human visual system. A rehabilitation program targeting the appropriate behavioural adaptation coupled with image processing routine optimised for image comprehension should provide a vision prosthesis recipient with the best functional experience to restored vision.