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(2016)ThesisOsteoarthritis is a degenerative joint disease that affects millions of people worldwide. The aims of this study were (1) to quantitatively characterise the boundary and surface features of wear particles present in the synovial fluid of patients, (2) to select key numerical parameters that describe distinctive particle features and enable osteoarthritis assessment and (3) to develop a model to assess osteoarthritis conditions using comprehensive wear debris information. Discriminant analysis was used to statistically group particles based on differences in their numerical parameters. The analysis methods agreed with the clinical osteoarthritis grades in 63%, 50% and 61% of particles for no osteoarthritis, mild osteoarthritis and severe osteoarthritis, respectively. This study has revealed particle features specific to different osteoarthritis grades and provided further understanding of the cartilage degradation process through wear particle analysis – the technique that has the potential to be developed as an objective and minimally invasive method for osteoarthritis diagnosis.
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(2017)ThesisChronic wounds fail to undergo an orderly and timely reparative process. Currently, there are no effective treatments for chronic wounds. The heparan sulphate (HS) proteoglycan (HSPG), perlecan, plays an important role in healing chronic wounds through binding and signalling of growth factors. Biomimetic materials that mimic naturally occurring HS may find application in the delivery of growth factors for skin wound healing. Chitosan is a polysaccharide with a similar structure to HS, with the exception of sulphate modifications. This thesis explored the effect of chitosan-arginine (CH-Arg), a water soluble form of chitosan, modified with different levels of sulphate substitution on skin wound healing. Specifically, this thesis investigated the effect of sulphated CH-Arg on the expression of extracellular matrix (ECM) components including perlecan produced by human keratinocytes dermal fibroblasts in 2D and 3D skin models. Perlecan produced by both skin cell types was full-length perlecan decorated with HS and chondroitin sulphate (CS) chains. In addition, smaller fragments of perlecan were present in the fibroblast samples. Exposure to highly sulphated CH-Arg (HS-CH-Arg) increased the perlecan expression in both skin cell types. CH-Arg-based materials did not alter the proliferation of either skin cell type, while sulphated CH-Arg significantly (p <0.05) enhanced keratinocyte migration, demonstrating that the sulphated CH-Arg materials promoted keratinocyte migration. An in vitro skin model was established with similar expression in basement membrane components, proteoglycans and glycosaminoglycans to human adult skin, as well as a well-developed mimicked epidermis. CH-Arg-based materials significantly (p <0.05) enhanced the epidermal thickness, indicating their role in epidermal formation. In addition, sulphated CH-Arg enhanced the expression of perlecan, laminin β1, collagen type IV, syndecans-1 and -4 and HS with the exception of serglycin. Thus, CH-Arg-based materials have the potential to mimic HS and promote skin wound healing with respect to enhancing re-epithelialisation and the formation of ECM molecules.
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(2016)ThesisThe aim of visual prostheses is to return a perception of sight for blind people. From the outset, designing a visual prosthesis system that minimizes platinum dissolution remains a challenge. Recently, laser surface modification was introduced to reduce platinum dissolution. This study will investigate the performance of laser-pattern for platinum electrodes, in particular structured laser interference patterning (SLIP), both in inorganic saline and biologically relevant solution. In this study, a new platinum electrode laser fabrication technique based on enamel was proposed, which can produce more accurate electrode exposure areas and a higher degree of control on surface morphology of microelectrodes. This new method describes the advantages of laser fabrication, which requires no clean room facilities and offers short design-to-prototype time. The feasibility of fabricating implantable electrodes with this method using medical grade enamel will require additional testing. An artificial suprachoroidal interstitial fluid (ASIF) preparation was further developed as a more appropriate test electrolyte for visual prostheses in vitro experiments. This artificial fluid was prepared by analysing residual solution on the surface of electrodes which were explanted from sheep suprachoroidal space. The extent of platinum dissolution occurring on laser-patterned electrodes stimulated at clinically-relevant levels in both Dulbecco’s phosphate buffered saline (DPBS) and ASIF was also quantified. Laser surface modification on electrodes minimised platinum dissolution during electrical stimulation and enhanced charge storage capacity of electrodes both in DPBS and ASIF. Furthermore, SLIP pattern showed the great improvement on the performance of electrodes, and that improvement was not simply related to the increase of electrode surface morphology variation but potentially because of the laser surface plasmon interference effect.
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(2014)ThesisAngiogenesis relies on the coordination of endothelial cells, smooth muscle cells and the vascular extracellular matrix around cells. Perlecan is an important heparan sulfate proteoglycan, which is widely expressed in vascular tissues and essentially involved in angiogenesis. Perlecan exhibits pro- and anti-angiogenic effects differently according to the cell line from which it was derived from and various structures especially its dependent glycosaminoglycans. The aim of the thesis was to generate and characterise monoclonal antibodies against specific domains of perlecan, and investigate their functions in modulating angiogenesis. lmmunopurified human endothelial cell-derived perlecan decorated with heparin sulfate and recombinant perlecan domain V decorated with chondroitin sulfate were characterised by Enzyme linked immunosorbent assay, immunocytochemistry and Western blotting, and then used as antigens for hybridoma screening. There were 205 single clones generated by limiting dilution, while 174 clones were able to recognise endothelial perlecan. 48 clones with high viability and lg production were tested with intact perlecan and Hep Ill treated perlecan, while they showed different immunoreactive changes between two antigens. However, none of the clones reacted with recombinant perlecan domain I or domain V. Of the newly generated monoclonal antibodies (mAbs), 5 mAbs showed potential to modulate the adhesion of endothelial cells and smooth muscle cells, especially 8C8 and 4F2 reduced the adhesion of both cell types. In addition, the 5 mAbs displayed strong immunoreactivity with perlecan derived from HCAECs and HVSMCs in Western blotting and were able to detect the perlecan expressed on two types of cells in immunocytochemistry. In spreading assay, all 5 mAbs were found to inhibit the HVSMC and HCAEC spreading to different degrees. Moreover, the 5 mAbs except 5G8 showed inhibitory effects in HCAEC migration, while only 8C8, 4F7 and 4F2 were able to reduce the migration of HVSMCs. These 5 mAbs could be useful to investigate the presence and structure of perlecan as primary antibodies, since they were able to recognise the perlecan protein core. They could also become potential tools to explain the functions of different domains of perlecan in angiogenesis and developed as immunotherapeutic drugs to mediate angiogenesis in clinical treatments.
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(2011)ThesisThe performance of implantable stimulating electrodes to restore sensory perception can be enhanced by technical advances in material processing to develop stable neural interfaces. High density neural electrode arrays have been recognised as a method to stimulate a higher amount of discrete neuronal populations resulting in a greater resolution of encoded information. This thesis addressed the issues related to the performance of high density stimulating electrode arrays fabricated by a combination of laser technology and surface micromachining. A reactive ion etching process was developed using sulphur hexafluoride (SF6) and oxygen (O2) plasma to pattern microstructures in medical grade polydimethylsiloxane (PDMS), to allow the transfer of current between the Pt conductor and neural tissue. The process was optimised by investigating the effects of varying the process parameters on the etched outcome. The surface topography and etch performance were analysed by employing surface profilometry and scanning electron micrographs (SEM). The chemical modification of the PDMS structure after SF6 and O2 plasma was investigated through x-ray photoelectron spectroscopy (XPS). The surface roughness of the underlying platinum surface was measured using atomic force microscopy (AFM). The optimum process parameters were established based on a high etch rate, clean surfaces and anisotropic sidewall profiles. In vitro cytotoxicity tests consisting of direct contact and extract tests (according to ISO 10993-5), were used to predict the cytotoxicity of the surface processed by SF6 and O2 plasma. The results showed that the morphological and compositional modifications of PDMS after the SF6 and O2 plasma did not affect the viability, morphology and proliferation of mouse fibroblasts. The in-vitro electrochemical performance was analysed using cyclic voltammetry and pulse tests. The main findings were that the charge storage densities increased proportionally with the size of the electrode. The charge injection capacitance for the plasma etched Pt surfaces was less than the value obtained for the laser etched electrodes, which was attributed to a smoother surface. The effects associated with a smaller real surface area were not found to adversely affect the electrochemical stability of Pt in saline. This preliminary investigation into the process of dry etching shows positive results as a method for micro-processing PDMS for high density electrodes.
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(2014)ThesisA simplified computational model of mouse atrial cardiomyocyte electrical activity was developed, based on the HL-1 cardiomyocyte cell line. HL-1 myocytes were characterised electrically and optically: the emission spectra of di-4-ANEPPS in HL-1 cultures, the cell type and distribution, and the optical-electrical equiva- lence of the potentiometric probe were all examined. Two major cell types were determined: pacemaking and non-pacemaking, with a distribution of 70%/30% respectively. Optical mapping of the HL-1 monolayer revealed linear wavefronts and re-entrant rotor activity. Rotors were shown to be the dominant source of spontaneous activity in the HL-1 cultures. To reproduce experimentally observed electrical behaviour, a three-current generic ionic model was employed. Sharp electrode recordings of single cells were used to fit model parameters using a custom optimisation routine. An electrical cellular network model was created to replicate electrical interactions in the HL-1 mono- layer. The action potential waveshape and conduction velocity of the network model were optimised to accurately reproduce experimental data. The model was able to faithfully reproduce linear wave fronts and re-entrant rotor activity seen in the HL-1 monolayer. The radius-angle rotor relationship of the model was within one standard deviation of that observed in the HL-1 monolayer.
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(2013)ThesisExplanted extracorporeal access devices exhibit different degrees of thrombus formation as a result of different physiological factors in vivo. Numerous computational techniques have been developed to predict regions of thrombus formation based on mechanical factors. The prediction of thrombosis in these studies has been limited to isolated cases and specific conditions to validate their use. They have not been implemented for clinical applications and the impact of physiological factors has not been investigated. This thesis explored the effect of physiological factors on the likelihood of thrombus formation in an extracorporeal access device. Four physiological velocity waves (triphasic, biphasic, sharp monophasic, and blunt monophasic) were identified in the femoral artery as a result of different levels of peripheral arterial disease progression. The four waves were used to compare the effect of different pulsatile flow conditions on the likelihood of thrombus formation. This thesis also explored the effect of different geometrical design factors on the likelihood of thrombus formation to optimise its design. Computational fluid dynamics was used to simulate the flow conditions in an occlusive femoral artery with an attached extracorporeal access device. Residence time was used to predict the location and size of thrombus formation on the surface of the cavity. It was shown that the retrograde flow had a considerable impact on the likelihood of thrombus formation, which increased as the integrity of the physiological wave decreased. The predicted location of thrombus was always in the same location, but varied in size depending on the velocity wave. It was also shown that of the device design factors, the angle of the device has the greatest impact on the likelihood of thrombus formation. The computational results were compared to five explanted devices and demonstrated similar locations and sizes of thrombus formation. This thesis examined the likelihood of thrombus formation in a particular extracorporeal access device; however, the methods adapted are beneficial in a wider context and possess a clinical relevance to patient prognostics when prescribing the implantation of a medical device.
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(2012)ThesisThe identity of cells generating the first blood cells in the mammalian embryo was unknown until recently. The formation of blood from embryonic endothelium was recently observed by live cell imaging. It was hypothesized that pulsatile shear stress was responsible for induction of blood production in the developing embryo. The aims of this thesis were to develop a microfluidic chip to mimic the embryonic heart and circulation and examine the response of endothelial cell to pulsatile shear flow. The chip was manufactured in PDMS consisting of a peristaltic pump with pneumatic actuated microvalves and ‘ventricle’ connected to parallel-plate flow-cells with recirculation. In addition, cell loading, media and reagent replacement was also controlled by three parallel micro-pneumatic valves. The timing of the cardiac cycle was controlled by a microprocessor which actuated solenoid valves driving microvalve closure and ventricular contractions. The pulse rate could be varied between 50-200 cycles/min. The temporal flow profile in circulatory system was characterised by micro particle image velocimetry (Micro-PIV), and could be adjusted by varying valve duty cycle and ventricle ejection fraction. The waveform was adjusted to mimic the embryonic aortic temporal flow profile with a maximal shear stress of 35 dyne/cm2. Bovine artery endothelial cells (BAEC) were cultured and tracked by phase contrast video microscopy over a 7-day period. The microfluidic platform can mimic the micromechanical environment of endothelial cells. Furthermore, these studies provide an in vitro model system for vascular biology research, leading to a deeper understanding of vascular endothelial mechanism.
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(2011)ThesisTowards the goal of generating specific tissue from stem or progenitor cells for regenerative medicine, it will be necessary to understand the dynamics of stem and progenitor cell development and how environmental cues trigger cell migration, mitosis, apoptosis, and lineage fate. Observing the dynamic process in a continuous manner at the single-cell level will advance our knowledge of these processes. Long-term live cell imaging systems and computational methods to automatically identify and track progenitor cell migration and division will enable this study. The aims of this thesis were to develop a live cell imaging system with semi-automated software for tracking adherent cell lines and to apply this system to study cardiac stem cell development. The imaging system was benchmarked by tracking NIH3T3 cells in vitro for 4 days. Cardiac stem cells were enriched by fluorescent activated cell sorting (FACS) from the interstitial fraction of the mouse heart. These cells form colonies (c-CFU-F) which were tracked by live cell imaging. Green fluorescent protein (GFP) transgenic mice were used to report cCFU-F cells that express β-actin or platelet-derived growth factor receptor-a (PDGFR-a). These initial studies have focused on characterizing cell motility and cell cycle dynamics of cCFU-F subpopulations. The growth rate of NIH3T3 (482 cells) tracked by the live cell imaging system was similar to conventional culture methods. Lineage maps of PDGFR-α+ cells (164 maps) and β-actin + cells (352 maps) within passage 3 colonies were constructed by continuous cell tracking over a 5 day culture period. Two distinctive cell morphologies were identified; large flattened-cells with low motility and highly motile spindle-shaped cells. The probabilities of mitosis of flattened- and spindle-shaped cells were estimated for each generation using Kaplan Meier statistics. There were significant differences between the cell cycle distribution and motility for these two subpopulations. Furthermore, Cox regression analysis was used to show that cell cycle progression was related to cell size and colony size. Large flattened-cells infrequently underwent asymmetric division giving birth to a small cell and large cell with a short and long cycle time respectively. These studies have illustrated the value of lineage mapping cCFU-F, leading to a deeper understanding of cCFU-F growth dynamics.
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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.