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(2010)ThesisThis study investigated retinal ganglion cell (RGC) responses to epiretinal electrical stimulation delivered by hexagonally-arranged bipolar (Hex) electrodes. In vitro experiments were performed using rabbit retinal preparations and a computational model was developed. Electrical stimulation was delivered using 50 and 125 IJm diameter platinum Hex electrodes and single-unit RGC activities were recorded differentially with extracellular tungsten microelectrodes. The majority of the responses exhibited short latencies (<5 ms) and were time-locked to the stimulus pulse. With 100 IJS/phase anodic-first biphasic pulses, threshold charge densities were 24.0 ± 11.2 and 7.7 ± 3.2 1JC/cm2 for 50 1-1m and 125 IJm diameter electrodes, respectively. The estimated chronaxie from the strength-duration relationship was 214 IJS. The short-latency responses of most cells (7/11) persisted at 200 Hz stimulus frequencies. Cell responses (7/7) were not diminished in the presence of cadmium chloride synaptic blocker. RGC responses persisted as the stimulating electrodes moved along the axonal path. Threshold profiles and response characteristics strongly suggested that RGC axons were the neural activation site. Both the model and in vitro data indicated that the lowest threshold point occurred beneath the centre active electrode and thresholds increased rapidly with lateral electrode distance across the retina, suggesting that localised tissue stimulation is achieved with Hex electrodes. Threshold profiles were dependant on the orientation of the electrodes relative to the axons. Thresholds increased up to 20 times when the electrodes were lifted 100 1Jm above the retinal surface. Low thresholds for axonal activation as well as the threshold increase with electrode displacement above the retinal surface are major concerns for epiretinal implant design. Overall, the threshold profiles predicted by the model accurately matched the experimental data obtained in this study.
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(2010)ThesisAn implantable rotary blood pump (IRBP) is a. heart assist device used to support end-stage heart failure patients, either as a bridge to transplant or permanent support. The aim of this dissertation is to investigate the interaction between an IRBP and the cardiovascular system (CVS) using carefully designed in vivo animal experiments and a mathematical model. Using data obtained from a steady flow mock loop under a wide range of pump operating points and fluid viscosities, flow and differential pressure estimate models were obtained using dimensional analysis. Linear correlations between estimated and measured pump flow over a flow range of 0.5 to 8.0 L/min resulted in a slope of 0.98 (R2 = 0.985) and an average error of 0.20±0.14 L/min (mean±standard deviation). Similarly, linear correlations between estimated and measured pump differential pressure resulted in a slope of 1.03 (R2 = 0.997) over a pressure range of 60 to 180 mmHg, with an average error of l.84±1.54 mmHg. The pump model was then modified and validated under a pulsatile flow environment. The model was shown to be able to predict the time course of the simulated haemodynamic variables with reasonable accuracy. Based on in vivo experimental data recorded in healthy pigs and dogs under a wide range of operating conditions, a lumped parameter model of heart-pump interaction was developed. Fitting of model parameters to the experimental measurements was performed to evaluate the robustness and validity of the mathematical model under the various operating conditions. It was shown from the simulation results that the model was able to reproduce the experimental data in terms of both mean values and steady state waveforms. The individual effect of key model parameters on the efficiency of rotary pump assistance was studied using variations in parameter values. Detailed discussion regarding the applicability of some of the control parameters proposed in the literature was also presented based on the experimental results and model simulations. A deadbeat controller for the control of pulsatile pump flow in the IRBP was proposed, using noninvasive measurements of pump speed and current as inputs to a dynamical model of pulsatile flow estimation. The performance of the controller was evaluated using the mathematical model. The controller was shown to be able to respond quickly to sudden perturbations in the CVS and adjust pump flow accordingly to avoid dangerous or undesirable situations.
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(2010)ThesisMast cells are multifunctional secretory cells that are involved in allergy and autoimmune diseases and predominantly were found in blood vessels near connective tissues of the skin and mucosa. These cells respond to various stimuli by releasing pro-inflammatory mediators including histamine, growth factors, and proteases. The proximity of mast cells to blood vessels together with the mediators released by these cells upon activation suggests a role of mast cell in angiogenesis. The aim of the thesis was to characterise the structure of proteoglycans (PGs) produced by model mast cells and determine whether the proteases released by activated mast cells were capable of processing extracellular matrix components, including heparan sulfate (HS) PGs, into fragments that modulate angiogenesis. Rat and human model mast cell lines, RBL-2H3 and HMC-1, were found to produce chondroitin sulfate (CS), HS and heparin. Both cell lines were found to produce and secrete serglycin, which was detected as two protein cores (11 and 13 kDa) decorated with both CS and HS. Novel finding in this thesis is that the model mast cells are capable of producing and secreting perlecan decorated with HS and CS, which was detected as both full length material (600 kDa) and fragments of 130 and 300 kDa. This suggested that perlecan may be processed by mast cell proteases. Alignment of the cleavage specificities of mast cell proteases with the N-terminal amino acid sequences of perlecan fragments showed that cathepsin S and MMPs-3 and -13 present in mast cells are potential candidate proteases that process perlecan. Releasate from activated HMC-1 cells was found to promote endothelial cell proliferation and migration to the same extent as FGF2 in an in vitro ‘scratch’ assay. Addition of anti-FGF2, anti-VEGF165 or anti-perlecan antibodies including those specific for domains I, III and V of the protein core, significantly inhibited endothelial cell migration in the presence of activated HMC-1 cell releasate. This is another novel finding of the work presented in this thesis and suggests that mast cells release growth factors and proteases to stimulate ECM remodelling, including perlecan fragmentation and the release of bound growth factors, for endothelial cell proliferation and migration, which are essential for in vivo in the context of angiogenesis in wound healing.
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(2010)ThesisA canine ventricular dataset was obtained from the modelling team at the Bioengineering Institute, University of Auckland, formatted for use with the CMISS modelling package developed by that group. In the work of this thesis, the Auckland dataset was imported into the COMSOL commercial modelling package. In doing so, the dataset was translated from a cubic Hermite representation of anatomical structure to a Bézier curve based representation. In addition, a simple monodomain model of electrical activity was solved over the ventricular geometry. The original elements of the model were either hexahedral or triangular prisms, referred to as wedges. Scale factors were calculated to convert local nodal values to global values for use with the Bézier curves. These scale factors were calculated differently in wedge elements due to their geometry. Alternative scripts were created to account for these differences. After importation into COMSOL, it was found that Hermite interpolation was unable to accurately model discontinuous bifurcations of anatomical structures, due to a continuity requirement within the Hermite scheme, leading to parallel and overlapping element edges.. Bézier curves are not subject to this restriction, and were found to be more appropriate in modelling these cases. To validate the import process from CMISS into COMSOL, a right lung lobe dataset was also successfully converted and graphically represented. The CMISS data on the canine ventricles also contains histological information on myocyte fibre orientation, which was not incorporated in the present study. It is known that this fibre orientation affects action potential propagation in the heart. Future work will need to address this area.
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(2010)ThesisUnderstanding the biomechanisms by which the cerebral cortex folds is a fundamental problem in neuroscience. The search for biomechanisms involved in cortical folding has been motivated by a desire to understand the folding patterns unique to each human brain, and the cause of neurological disorders that result in abnormal cortical folding. These include Down’s syndrome and lissencephaly, in which the brain folds are less numerous and smaller than normal folds. Current mathematical models of cortical folding do not include three dimensional geometry or measurement of cortical growth in developing brains extracted from experimental data. Motivated by the lack of experimental measurements of developing brains integrated with mathematical models of cortical folding, this thesis combines structural and Diffusion Tensor Imaging (DTI) MRI data analysis with finite element modelling to investigate the biomechanisms of cortical folding. We quantitatively measured cortical growth, the development of cortical folding and the white matter directly from MRI data acquired from fetal sheep brains at a number of key developmental stages. Novel use of the DTI MRI data has led to the characterization of diffusion anisotropy changes in developing sub-cortical white matter. A correspondence was shown between the less developed white matter fibres, as measured by decreased anisotropic diffusion, and the development of inward cortical folding. We present two biomechanical models of cortical folding which integrate 3D geometry and information taken from our MRI data analysis. The first model uses DTI measurements of white matter fibre orientation in fetal sheep brains as a cue to the tension forces that may regulate folding. In the second model, tangential cortical growth is modelled by osmotic expansion of the tissue and regulated by inhomogeneous white matter rigidity as a biomechanism of cortical folding. We demonstrated that structural and diffusion tensor MRI can be combined with finite element modelling and an explicit growth biomechanism of the cortex to create biologically meaningful models of the cortical folding process common to higher order mammals.
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(2010)ThesisThe design of scaffold materials that could be used to assist tissue regeneration has been based on the extracellular matrix (ECM), in structure and in the presentation of molecular cues. Synthetic hydrogels resemble the ECM with their structure, high water content, and elasticity, but they lack bioactivity and recognition. Therefore, the incorporation of biomolecules into synthetic hydrogels is a growing current area of research. Heparan sulfate (HS), a glycosaminoglycan present in basement membrane, has been known to bind and signal various ECM proteins. Sustained presentation of biomolecules like HS could be achieved by covalent linking to scaffolds; however it poses a challenge on the preservation and expression of the biomolecules’ activity after incorporation. Biosynthetic hydrogels derived from heparin (a model for HS) and poly(vinyl alcohol) (PVA) were formed by photopolymerisation. PVA and heparin were both functionalised with photopolymerisable methacrylate groups prior to crosslinking, and the effect of this modification on heparin was examined. PVA/heparin co-hydrogels were made with varying compositions and assessed in terms of their structure, strength and growth factor presentation. The activity of the co-hydrogels following incubation with platelet extract (PE) was also studied, to simulate responses that might occur when the hydrogels, as tissue engineered scaffolds, come in contact with blood products. Heparin remained structurally intact and biologically active following methacrylate modification and UV exposure. The addition of up to 2.5 wt% of heparin increased the hydrogel swelling capacity without compromising the strength of the resulting hydrogel network. The specific FGF-2-signalling activity of heparin in the PVA/heparin co-hydrogels was demonstrated, with results indicating that co-hydrogels may be formulated with a minimal amount of heparin (≥0.05 wt%), thus limiting any effects on structural integrity. PE treatment of the hydrogel-bound heparin diminished its anticoagulation properties but increased the FGF-2 signalling, suggesting the heparanase activity in PE cleave at the antithrombin binding site to yield fragments that can signal cell receptors. This work has demonstrated the formation of biosynthetic co-hydrogels, capable of presenting growth factors to cells, and provides a novel insight on the molecular activation of heparin-based hydrogels upon enzymatic degradation.
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(2010)ThesisWith the ever-increasing volume and complexity of mathematical biological models and experimental datasets becoming available, there is a strong need for a set of representation languages which can describe and represent these models and data in standard forms and store them in publically available repositories for universal dissemination. To help address this issue, the Modelling Markup Language (MML) computational framework was developed in this project. It consists of representation languages and application toolsets that can represent, store and solve biological temporo-spatial models. The representation languages are comprised of ModelML, responsible for maintaining relational information between different external models, along with the Field Markup Language (FML), responsible for maintaining geometric field information such as anatomical data. The MML framework also utilises the existing CellML specification to represent biological systems models. With these three representation languages, a temporo-spatial model can be created, re-used, interchanged and shared. In addition, specially-developed application toolsets provide utilities which aid in the creation and processing of the MML models. To demonstrate the capability of the MML framework, a series of simulations of cardiac electrical activity are presented, including simulations of the cardiac pacemaker (sinoatrial node) and atrial tissue activation. In the sinoatrial node simulations, tissue electrical conductivity was adjusted to observe its effect on sinoatrial node entrainment and inhibition by the atria, using several 1D, 2D, and 3D tissue geometry layouts and cellular mathematical models. Additional simulations were performed by modifying the magnitude of the hyperpolarisation-activated membrane current (if) of the underlying cellular models, to observe its effect on pacemaker activation and impulse propagation into the atria. The use of the MML framework allowed these models to be constructed rapidly through its ability to efficiently reuse and modify underlying geometric and mathematical components.
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(2010)ThesisThe ex vivo manufacture of functional organs and tissues for implantation can impact on therapeutic needs arising from an ageing population. The rapid expansion of the field of tissue engineering has arisen in response to this clinical need. In order for mesenchymal stem cells (MSC) to be useful clinically, sufficient numbers must be obtained. It was hypothesised that MSC could be isolated from a heterogeneous population of cells, and that hollow fibre bioreactor systems may be a scalable technology for expansion. In order to manufacture functional tissues, appropriate bioreactor devices must be developed to direct cellular differentiation. It was hypothesised that long-term ex vivo tissue gradients could be established. A substrate (Aldefluor) for aldehyde dehydrogenase (ALDH) activity was employed to label cells from human umbilical cord blood (hUCB) and cells isolated from rat, murine, porcine and equine origins. Growth of anchorage dependent cells was carried out in cuprophan hollow fibres coated with a novel recombinant protein, with and without extracapillary co-culture. Micro bioreactors were manufactured using the techniques of lithography from polydimethylsiloxane, and device biocompatibility was assessed using the anchorage dependent cell line NIH 3T3. While cells isolated from rat, murine, porcine and equine origins showed an ALDH bright population, MSC could not be purified from hUCB. Cellular attachment to cellulose based substrates coated with the recombinant protein occurred within 2 hours. Anchorage dependent cells could not be maintained in the hollow fibres. Stable concentration gradient profiles were generated experimentally in two micro-devices with differing geometries. In the second device, the concentration gradient was maximal in the region of flow stasis, and cells were found to remain viable inside the cell chamber below a flow rate of 4μL/min for 72 hours. Aldefluor substrate was not useful in the prospective isolation of MSC from a heterogeneous population. Coating of cellulose substrates with a novel recombinant protein was found to be necessary for cell attachment. Growth in the hollow fibres was found to be suboptimal compared to conventional methods. Cell chemotaxis and tissue morphogenesis could be studied using the second micro-device developed without the confounding effect of fluid shear stress.
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(2010)ThesisThe use of polymer nanocomposites (NCs) in industrial applications has received growing attention over the past decade due to their improved mechanical properties. However, little work has been reported which analyses the efficacy of NCs in biological applications, including drug delivery systems and implantable materials. This thesis examines the effect of the chemistry of the organic modifier (OM) on the structure and biological performance of poly(ether)urethane NCs (PUNCs) and the influence of the method of drug incorporation on interactions between drug and NC. Organically modified silicates (OMS) were prepared using OMs varying in terminal functionality and alkyl chain length. PUNCs were solvent cast containing 1 and 3wt% OMS and particle dispersion analysed using X-ray diffraction and transmission electron microscopy. Findings revealed that use of an OM with methyl terminal, dodecylamine (12CH3), resulted in superior dispersion of OMS compared with a carboxyl terminated OM, aminododecanoic acid (12COOH), of equivalent alkyl chain length. This is believed to result from increased self interaction of 12COOH compared with 12CH3. Additionally, increased alkyl chain length was shown to improve NC dispersion with a chain length of sixteen units resulting in the optimum dispersion with a partially exfoliated NC structure. Analysis of cellular interactions with the PUNCs revealed a significant difference in both fibroblast and platelet adhesion to NCs incorporating 12CH3 compared with 12COOH. Surface analysis using ToF-SIMS demonstrated the presence of 12CH3 fragments on the NC surface supporting the hypothesis that surface expressed OMs alter cellular interactions with the NC. Altering the alkyl chain length also affected cellular interaction with an alkyl chain length of twelve units or greater, substantially reducing fibroblast adhesion without affecting cell growth inhibition or viability. Incorporation of a model drug, crystal violet, into the PUNCs demonstrated a lower degree of disruption to OMS dispersion when loaded post NC fabrication compared with pre fabrication. This is believed to result from interactions between the drug and NC constituents which also impacted on drug release from the NC system. Results show PUNC properties and biological interactions can be modulated through OM variation and fabrication method, thus showing potential for use in biomedical applications.
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(2010)ThesisThis research investigates in vitro leukocyte interactions with sericin proteins from silkworm cocoons of Bombyx mori and Antherea mylitta silkworm species. Sericin from raw silk fibres has been implicated as a cause of inflammation in previous silk suture studies and these findings have hindered development of sericin as a biomaterial. Nonetheless, recent studies involving isolated sericin have suggested that it is antibacterial, UV resistant and able to absorb and release moisture easily. Sericin has also been shown to possess attractive wound healing properties and acceptable production of inflammatory mediators, causing much debate in its use as a biomaterial composite. Ultimately, sericin may be applied to the design of biosynthetic hydrogels for tissue engineering applications. Hence, there is a need to evaluate the role of sericin in the inflammatory response and determine its use as a biomaterial. The results from these in vitro studies will give a preliminary indication of the likelihood of sericin from these species to incite an inflammatory response. Isolated sericin proteins from both species were isolated and modified with methacrylate groups (sericin-MA) to photocrosslink with poly(vinyl alcohol). In vitro studies using leukocytes from whole blood samples and the monocytic cell line, U937, were conducted for investigation of neutrophil activation. Cell surface receptor expression and macrophage differentiation was evaluated by flow cytometry and fluorescence microscopy, and the release of TNF-α was measured by ELISA. Based on the findings of this research, sericin and sericin-MA of both silk species elicited negligible neutrophil activation in blood samples. However, sericin-MA appeared to trigger the early onset of macrophage differentiation and produced significant amounts of TNF-α in blood samples. In contrast, U937 studies exhibited no significant TNF-α released and no apparent impact on macrophage differentiation. Considering that blood samples contain other cofactors that may influence inflammation, unavailable in culture media, blood samples are able to express the effects of sericin methacrylation. In conclusion, this research has demonstrated that sericin does not elicit a positive response by leukocytes and therefore indicates that sericin does not cause inflammation. Further work is required to investigate the effect of sericin-MA in chronic inflammation and in vivo response to silk/PVA hydrogels using mouse models to understand better the interactions between cells and incorporated sericin. This would greatly help in the development of silk/PVA hydrogels for applications in tissue engineering.