3D bioprinting is the additive manufacture of biological materials, such as cells, and has been implemented over the last two decades to print 3D constructs which can mature into functional tissues. Although there are many bioprinting techniques, there are few that are capable of simultaneously printing two or more cell types at high resolutions, high cell densities and high initial cell viabilities. These printing characteristics are required to pattern cells in tissue-like micro-architectures and to quickly mature physiologically relevant tissue. We developed a novel 3D bioprinter and bioprinting methodology, designed to pattern cells without compromising these printing characteristics. Initially a droplet-in-oil 3D printer was created, which assembled mm-scale aqueous droplet networks composed of thousands of picolitre volume micro-compartments. These networks were programmed to show emergent tissue-like properties, mimicking nervous tissue's rapid electrical communication or self-folding in a manner similar to muscle contractions. This printing process was then adapted to pattern HEK-293T cells within droplet networks at high droplet resolution (~50-150 μm), high initial cell viability (90±4%) and high droplet cell density (~30x106 cells/mL). Capillary-like and layered sheet cell architectures were formed with two printed fluorescent cell populations. These 3D printed constructs were transferred to culture medium with minimal loss in pattern fidelity using a novel phase transfer approach. Patterned HEK-293T cells developed into dense cell organisations which showed, high cell viabilities, high cell numbers (up to ~18,000) and mitotically active cells, when cultured over 7 days. Specialised tissues were also developed, here, patterned ovine mesenchymal stem cells were differentiated, after printing, into chondrogenic progenitor cells as affirmed by Sox9 expression.

Lower limb prosthetic socket provides the interface for the transfer of loads between the ground and the stump. During daily physical activities, the stump has to endure mechanical stresses in normal (pressure) and tangential (shear) directions to the stump/socket interface, both of which may cause discomfort, pain, and a host of stump health problems. Despite the importance of the issues, there is currently no clinically-friendly sensor technology available to monitor both pressure and shear at the stump/socket interface. In this thesis, two sensor systems were developed - both capable of simultaneously measuring dynamic pressure and shear at the lower limb stump/socket interface. In particular, two thin, capacitive sensors based on flexible elastomers (3D-printed and silicone) were designed and fabricated. To acquire sensor signals, appropriate data acquisition circuitries and PC software were produced. A viscoelastic model for real-time processing of the sensor signals was developed, to improve their dynamic response. The sensor systems were characterised in research lab settings, and their performance was verified against comprehensive design requirements. The characterised sensor systems were validated at the stump/socket interface in a series of amputee walking tests, and a range of clinical factors were investigated. Pressure and shear were measured at up to three discrete locations of a trans-femoral stump. Their temporal profiles when walking on level and inclined surfaces were obtained. The results suggest that ankle flexion resistance may have an effect on both the magnitudes and the temporal profiles of pressure and shear at the stump/socket interface. The thesis delivers a tool for measuring pressure and shear at the stump/socket interface in a clinically-friendly manner. What is more, it contributes to the prosthetic field by bringing an insight into the pressure and shear at the trans-femoral stump/socket interface, thus enhancing the understanding of its biomechanics. In the future such a sensor technology could potentially aid daily monitoring of socket fit, assist prosthetists with socket fitting, support research on the stump/socket interface biomechanics, etc.

The last few decades have seen major advances in the field of materials for healthcare applications. New approaches call for the development of biomaterials with controlled chemistry, tailored structure at different length scales and adequate degradation and dissolution profiles, all of which are crucial to the success of biomaterials for tissue regenerative therapies. Calcium phosphates are widely used to promote recovery of bone tissue due to their chemically similarity to the inorganic phase of bone. And although they have had considerable success, a discussion remains about the optimum characteristics of a bone substitute. This work aimed to develop a framework to study the combined effect of surface microroughness of calcium phosphates with varying composition - Hydroxyapatite (HA) to β-Tricalcium Phosphate (β-TCP), including biphasic materials - on human mesenchymal stem cells in vitro. Furthermore, the method used to tailor such microstructural features is translatable into 3D-printed scaffolds, i.e., it was possible to fabricated materials with complex structures, while controlling surface roughness. The thermal behaviour of calcium phosphate materials with different Ca/P ratio was studied, and adequate sintering conditions were optimised. These avoid the β to α transformation in materials with TCP. A novel processing route to control microstructure was developed: ceramic materials were functionalised with a pH-responsive polymer and used to stabilise microemulsions, where the hydrophobic phase was used as a soft porogen that evaporates. Independently of the HA/β-TCP ratio used, interconnected microporosity was obtained as a result of the emulsification process. The osteogenic potential of discs with these surface properties and three different chemical compositions (pure HA, pure β-TCP and a biphasic material HA/β-TCP) was evaluated in cultures with hMSCs from three independent donors. Finally, the behaviour of particle-stabilised emulsions was optimised to be used as inks for robocasting. Rheological characterisation and extrusion flow tests clarified the impact of different processing steps in their printability. In the end, calcium phosphate scaffolds with complex shapes and controlled chemistry and surface features (resulting from the oil templating) were fabricated.

Brain-computer interfaces (BCIs) provide means for direct braincomputer interaction, based solely on the user's brain neural activity, commonly captured by Electroencephalography (EEG), and do not rely on any degree of physical movement. From a general perspective the function of BCIs is to discriminate between a limited set of mental states, which the user enters voluntarily or unconsciously. This represents a foundation for various BCI applications such as assistive technologies, including neuroprosthetics and computer control BCIs for disabled users or mental state monitoring systems aimed for emotion, fatigue or workload recognition. A commonly used type of mental tasks for BCI control is imagination of physical movement or motor imagery, which is characterized by the local power deviation occurring in the brain areas responsible for muscles involved in the executed task. This PhD manuscript is dedicated to the design of motor imagery EEG BCIs with a particular focus on signal processing and classification approaches that incorporate the background knowledge about biophysics and EEG signal generation. These aspects are considered in the EEG source reconstruction process, which estimate the cortical currents during the EEG voltage measurements from head surface. In this work it is shown that the application of the source reconstruction in a BCI signal processing scheme effectively decreases the negative effects of EEG electrode coupling providing for an increase in class separability, given that the cortical areas involved in motor imagery are anatomically segregated. Based on these observations a novel BCI feature extraction method based on source analysis and common spatial patterns (CSP) was proposed and its performance was investigated with a common motor imagery dataset and our own real-time BCI implementation. Our results show that EEG source reconstruction reduces the influence of noise and muscular artifacts, and thus the proposed approach consistently outperforms the conventional BCI sensor feature extraction methods.

Cardiovascular disease is the number one cause of death worldwide. In the treatment of such disease, vascular surgery commonly utilises grafts to replace or bypass damaged regions of the circulatory system. Currently, autograft vessels represent the gold standard for vascular bypass; however, these are of limited availability and quality. Synthetic conduits are also available, but these are of little use as small diameter vessels (< 5 mm) due to high incidence of failure through infection or thrombosis. A need exists for a better vascular graft with tissue engineering offering an attractive solution. Although the development of tissue engineered vascular grafts (TEVGs) is being explored by a number of research groups using a wide range of methods, to date, a TEVG has not yet been produced that closely matches the mechanical performance of the autograft vessels currently favoured in vascular surgery. This research aimed to develop a method of manufacturing TEVGs with mechanical properties more similar to the current gold standard vessels. These TEVGs would be biocompatible, non-immunogenic and able to grow and remodel in vivo. Additionally, the TEVG manufacturing process would also be readily adaptable to producing more complex geometric shapes than simple tubes. It was hypothesised that this may result in an improvement in the performance of the TEVGs and may also expand the possible clinical applications. Following a review of the literature, presented in Chapter 1, a synthetic polymer scaffold based tissue engineering approach was selected as the method for producing the TEVGs. This approach has been widely adopted in the field of vascular graft tissue engineering. Synthetic polymer scaffold based methods are compatible with a range of manufacturing processes; have shown success in a variety of in vivo studies of TEVGs, including human trials; and had the potential to be adapted to produce a TEVG in a variety of predefined geometries. A synthetic polymer scaffold would be developed with properties, such as elasticity, degradation rate and porosity, specifically optimised towards the development of a TEVG with mechanical properties similar to the current gold standard autografts. TEVGs would be produced in vitro by seeding cells onto the synthetic polymer scaffold and then culturing them in a bioreactor under physiologically relevant flow to encourage cell proliferation and appropriate ECM deposition. A novel photocurable form of poly(glycerol sebacate), poly(glycerol sebacate) methacrylate (PGS-M) was proposed as the material for the synthetic polymer scaffold. This material had the potential to provide the mechanical performance and degradation properties identified as requirements for the scaffold, along with being biocompatible and easy to process into various scaffold geometries. In Chapter 2, different variants of PGS-M were produced which varied in molecular weight and degree of methacrylation (DM). These were characterised using various analytical chemistry techniques. It was determined that both the molecular weight and DM could be controlled by altering the reaction conditions used in the synthesis of the polymer. The degradation of the different variants of PGS-M was examined and this revealed that the polymer was susceptible to enzymatic degradation. Increasing the DM appeared to have an inverse effect on the degradation rate. The mechanical properties of PGS-M were also assessed and found to largely depend on the DM of the polymer and not the molecular weight. A 30% DM, low molecular weight (30% Low Mw) PGS-M was selected as the most suitable variant of PGS-M for producing a scaffold for use in culturing a TEVG. In Chapter 3, the biocompatibility of 30% Low Mw PGS-M is presented. Flat surfaces of the polymer were able to support the growth and proliferation of human dermal fibroblasts, human adipose derived stem cells and human coronary artery smooth muscle cells (SMCs) for several days in culture. Additionally, growth on the PGS-M surfaces does not appear to alter the phenotype of the SMCs. It was determined that culture on PGS-M surfaces may have effects on the metabolic activities of the three cell types investigated and that these effects may be subtle and cell specific. In Chapter 4, porous scaffold structures, suitable for use in tissue engineering, were produced from 30% Low Mw PGS-M using a porogen leaching method with sucrose particles. Combining PGS-M with sucrose particles of different sizes, at different ratios, allowed variation of the scaffolds' handling properties, pore sizes, porosities and wettability. SMCs seeded onto the porous PGS-M scaffolds remained viable for 7 days in static culture and partially infiltrated the scaffold interiors. A method was then developed to produce the porous scaffolds as tubes of suitable geometry and porosity for use in the generation of TEVGs. Additionally, a method for producing porous PGS-M scaffolds in a variety of geometries was also demonstrated as a proof-of-concept. This method used a novel hybrid additive manufacturing and porogen leaching approach. A bioreactor was required for the culture of the tubular PGS-M scaffolds, once seeded with cells, to produce TEVGs. In Chapter 5, a design brief was proposed for a bioreactor capable of culturing the TEVGs under dynamic conditions and applying mechanical stimulation. This had been identified as advantageous in previous studies in TEVGs. A design process was implemented, with a number of initial ideas evaluated to determine an integrated final solution. The final bioreactor design utilised a pulsatile flow to provide mechanical stimulation to the developing TEVGs. The bioreactor was manufactured and assessed for sterility and its ability to provide mechanical stimulation to developing TEVGs. The pulsatile flow could be modulated to produce pressures and flow rates within the range of physiological blood flow which were appropriate for the culture of TEVGs. Modifications to the design were implemented, as required, to improve performance. The knowledge gained from the previous chapters was combined in Chapter 6. Porous tubular scaffolds, produced from 30% Low Mw PGS-M were seeded with human coronary artery SMCs and cultured in the bioreactor as TEVGs. The TEVGs were cultured for 7 days under dynamic and static conditions. TEVGs cultured under dynamic conditions, with mechanical stimulation produced by the pulsatile flow in the bioreactor, displayed highly variable results, but demonstrated the partial formation of blood vessel-like tissue in a small instance. TEVGs cultured under static conditions produced repeatable results, although with reduced vascular tissue formation compared to the grafts cultured under dynamic conditions. Both culture regimes produced TEVGs containing collagen and elastin and both also appeared to cause a change in the phenotype of the attached SMCs, from contractile to proliferative. Finally, Chapter 7 suggests the possible further work that may be conducted to explore the PGS-M scaffold based TEVGs, as the original aims of the research were not fully realised. Suggestions of how the bioreactor culture may be modulated and optimised are made along with ideas for generating TEVGs of varied geometries.

No description available.

Motivation: Cerebral aneurysms have presented considerable challenges to effective treatment since their first clinical observation. For some 100 years the only clinical solution was dangerous and often debilitating open-surgery in the form of a craniotomy. The safety and efficiency of cerebral aneurysm treatment was greatly improved in the early-1990s with the introduction of minimally invasive procedures such as endovascular coiling. The development in the mid-2000s of low-porosity stents, known as flow-diverters, presented a more sophisticated treatment rationale that focussed on vessel reconstruction, and addressed cases that would otherwise be problematic to treat by other endovascular means. However, current designs of flow-diverter (and all known designs in development) are limited to near-identical products that risk under-exploiting the potential benefits of the treatment method. These devices are of a cylindrical, woven construction that necessitates a fixed mesh design, which means that any variation in design focusses on device porosity alone. As such, the precise hemodynamic mechanisms, and the resulting quality of aneurysm treatment by flow-diverter, are under-developed and poorly understood. There is also concern in the clinical community that the cylindrical nature of current flow-diverter devices may lead to daughter vessel sacrifice when used to treat bifurcation aneurysms. This thesis specifically focusses on the effect of flow-diverter mesh topology on the complex hemodynamic environment within the treated aneurysm, and the resulting implications for treatment success. Methodology: In this study, a number of both novel and commercially available flow-diverter designs are virtually implanted into idealised and patient-specific aneurysm geometries. These devices are then modelled under realistic blood flow conditions with computational fluid dynamics (CFD) techniques. The devices chosen encapsulate a range of features that allow the effects of device porosity and device mesh design to be controlled for and evaluated independently. Conventional cylindrical devices are also compared to a novel, non-cylindrical device for the dedicated treatment of bifurcation aneurysms. The aneurysm hemodynamic environment both pre- and post-treatment is quantified with a number of in-silico measures, such as flow rate, velocity, pressure, and wall shear stress. Finally, a model of angiographic contrast agent transport is developed in-silico to begin to verify the complex flow patterns predicted by CFD simulations with the corresponding in-vivo behaviour seen clinically. A number of inaccuracies of previous 'virtual contrast' models, as presented in the literature, are addressed and quantified by comparison to a gold-standard in-vivo porcine model. Results: In a number of aneurysm geometries, novel flow-diverter designs are shown to out-perform current commercially available devices. The strong influence of device porosity on aneurysm inflow reduction is confirmed with a 10% decrease in device porosity correlating with around 10-20% reduction in inflow. Variation attributed to device design alone is also seen; in a number of geometries, differences in flow-diverter mesh design, at constant porosity, are shown to introduce approximately 10-30% variation in aneurysm inflow reduction. Such variation is significant and potentially very important to the development of future endovascular treatment devices. The greatest variation in inflow reduction seen across the device designs also corresponds to changes in vorticity and secondary flow effects. The flow reduction achieved by the novel bifurcation aneurysm device discussed compares favourably to conventional devices in half of the geometries simulated, achieving a flow reduction of approximately 30-70%. Across all bifurcation aneurysms studied, conventional devices produce a relatively uniform flow reduction of 50-70%. Little change in flow rates for vessels jailed by a conventional flow diverter suggest that concerns of daughter vessel sacrifice appear unfounded, at least immediately following device deployment and prior to any neointimal formation. A number of unusual flow phenomena, which are almost entirely unreported in the literature, are observed in several aneurysm geometries. In particular, the emergence of stable and unstable laminar flow patterns under stationary conditions lead to oscillatory behaviour in one aneurysm geometry. Pseudo-stable behaviour is also observed in another aneurysm geometry whereby steady state and transient simulations conducted under identical conditions converge to solutions with radically different flow patterns. A good in-silico prediction of contrast residence is possible for a pre-treatment aneurysm geometry, where mixing is prominent. Aneurysm flow patterns after device placement reveal large spatial-variation in contrast infiltration that challenge the validity of quantifying contrast residence with a spatially-averaged decay curve. The most significant source of error between aneurysm contrast decay rate predicted in-silico and the in-vivo porcine model is found to be the variation in parent vessel flow rate, and not the density and viscosity changes of the contrast-blood mixture. Conclusions: In addition to porosity, flow-diverter device design has a substantial effect on aneurysm inflow reduction and aneurysm flow pattern. When coupled with improved knowledge of aneurysm thrombosis mechanisms, appropriate changes in flow-diverter mesh design have the potential to improve the success rate of flow-diverter treatment. However, the clinical implications of these effects are currently unknown due to the lack of design variation in commercially available devices. In cases of bifurcation aneurysms, conventional flow-diverter devices have been shown to equal or outperform a novel device designed exclusively for bifurcation cases. Although no immediate complications were predicted from daughter vessel occlusion, the long-term integrity of treatment with conventional cylindrical devices is uncertain, and dedicated devices designed to preserve daughter vessel patency may become necessary. The complexity of the flow environment within a cerebral aneurysm has been reinforced, and a number of effects have been observed that demand further investigation. In particular, the role that the observed flow instabilities and the resulting changes in aneurysm wall shear stress may have in aneurysm rupture mechanisms is an emerging area of the literature. Verification of CFD simulations with in-vivo results remains a significant challenge. The virtual modelling of angiographic contrast residence currently offers the best route to such verification; although contrast transport may be integrated into current CFD simulations with relative ease, washout behaviour appears highly sensitive to parent vessel flow rates. As such, any contrast-based validation requires knowledge of patient-specific flow rates, which begins to challenge how representative flow conditions seen during angiography may be.

A polymer-based total knee replacement (TKR) system that utilises an injection moulded polyetheretherketone (PEEK) femoral component has been proposed. The current project was designed to characterise the material at the coupon level by acknowledging the issues related to the processing route as well as the in-service conditions of the proposed component. Surface characterisation (wide angle X-ray scattering (WAXS), nanoindentation, atomic force microscopy (AFM) and photographic image processing) showed that heterogeneity was introduced to the material as a result of differential cooling that occurred during injection moulding. The crystallinity level and the nanoindentation hardness were highest at the core of the sample and lowest at the surface. These were visible as variation in shading on the cross-section of the sample. Although these findings supported the presence of an amorphous surface layer, the lack of abrupt change in properties from surface to bulk meant that its thickness could only be estimated (318m to 545m) and could not be more accurately gauged. Nonetheless, the findings showed that the mechanical properties of the amorphous surface layer were lower than that of the bulk. While this could possibly be deleterious by promoting uid ingress in-service, it could also potentially be beneficial as it might provide a crack-shielding effect to the proposed TKR femoral component. Mechanical characterisation showed that the static response of the material was rate sensitive at the coupon level, but not at the nano-scale. This could be related to the difference in global and local responses, but also attributed to the difference in the mode of testing. The design and the execution of a multi parameter fatigue test programme has successfully demonstrated how the effects of test parameters on the material could be studied in a strategic manner. Failure limits were identied, where samples failed (predominantly due to cyclic softening) as opposed to running out. The fatigue life of the material was shortened by (i) increasing the stress level, (ii) increasing the frequency, (iii) suppressing cooling, and (iv) using a sinusoidal waveform instead of a waveform taken from TKR knee contact force data from the OrthoLoad database. These suggest that testing at 5Hz using a sinusoidal waveform in an ambient environment would be a suffciently effcient and robust test method for the proposed TKR femoral component, and the developed method may be used to identify appropriate characterisation methods for other novel implants.

The operational transconductance amplifier, OTA is one of the basic building blocks in many analogue circuit applications. The low power consumption is an essential parameter in modem electronic designs for many areas particularly for portable devices and biomedical applications. For biomedical applications, the low- power low-voltage OTA-C filters operating at low-frequency ranges are desired. The low-power, low-voltage operation of electronic devices is very important for applications such as hearing aids, pacemakers, and EEG. The importance of such operation is due to the need to implant these electronic circuits inside the body of the patient for long times before re-charging or replacing the batteries as for pacemakers and future hearing aids. The small size lightweight wearable EEG systems are preferable for applications ranging from epilepsy diagnosis to brain-computer interfaces. The low power consumption is achieved by operation at very small levels of current. So, in such applications the operation in the nano-ampere current range is essential to ensure power consumption of nW or few uW. Such very small currents are obtained through the operation of MOS transistors in their sub-threshold regime. The design space in such applications is restricted by their specifications which in turn based on the nature of the application. In this work, the design and implementation of OTA-C filter topologies for two bio-medical applications are made and discussed. Those applications are represented by hearing aids and EEG applications. In hearing aids, the work focused on cochlear implant and specifically on its most important stage represented by the filter. Four OTA-C filter topologies are proposed and two of them are tested experimentally. For the filter in a hearing aid system, besides its low power operation, it is required to operate with a relatively high dynamic range of 60dB and above. The dynamic range is the operation space of the filter that specified by the range of signals which can process properly. It is bounded by the maximum power signal less than its distortion overhead level to the minimum power signal more than its noise floor. The maximum signal level the filter can perform properly represents its input linear range. The challenge in CMOS OTA sub-threshold operation is the very small input linear range which makes it extremely difficult to build low-power consumed OTA-C filters with a wide dynamic range, DR. In this work, an OTA with an input linear range of ±900mV for total harmonic distortion, THD<5% is proposed using MOSFET bumping and capacitor attenuation techniques, combined for the first time. The minimum signal level the filter can distinguish from noise is still relatively small with the use of appropriate OTA architecture and using the gm/ID methodology for MOSFET sizing. So, programmable CMOS OTA-C band-pass filter topologies operating in sub-threshold region with a dynamic range of 65dB for use in bionic ears were proposed. The power consumption for the proposed filters is in nano- Watt range for their frequency range of (lOO-I Ok) Hz. Also, a 4-channel OTA-C filter bank is designed and tested. The EEG signals have small amplitudes and frequency bands ranges of uV'S and (l-40) Hz respectively. The important issue is to design filters with small noise floor with white dominant. This is achieved with the proposed OTA which is of relatively simple architecture and with operation in the deep weak-inversion region using ±1.5V supply rails. The OTA-C filter has power consumption in the pico-Watt range for 0, e, and a signals and less than 3nW for B signals. Another topology is suggested for future work.

Mast cells form an integral part of both innate and adaptive immunity; they help to orchestrate the inflammatory immune response through the release of a variety of inflammatory mediators. Adverse reaction to allergens can lead to activation of mast cells, causing degranulation and release of a range of pro-inflammatory mediators contributing to the onset of allergy. The most studied activation pathway in the adaptive immune response of mast cells is through the Immunoglobulin E (IgE) cell surface receptor FceRI. Crosslinking of FceRI leads to degranulation and de novo synthesis of mediators. Every eukaryotic cell undergoes constitutive secretion. Alongside this general process, cells such as neuronal endocrine and immune cells, including mast cells, perform regulated secretion. This enables the cell to rapidly release mediators stored in secretory granules upon stimulation by a particular extracellular ligand. Mediators released fall into two categories; pre-formed, contained within these secretory granules; monoamines such as histamine as well as many proteases, and de novo synthesized that are released through the constitutive secretory pathway, including prostaglandins, leukotrienes, cytokines and chemokines. Elucidating the mechanisms of mast cell mediator release is imperative for understanding many disease processes; however, knowledge of the precise mechanisms by which mast cell exocytosis is controlled remains elusive. The aim of this study was to identify and characterise Soluble NSF attachment protein receptor (SNARE) proteins involved in the release of inflammatory mediators in human mast cells. Using LAD 2 human mast cells and primary human lung mast cells (HLMCS), expression of a variety of syntaxins and Vesicle associated membrane proteins (VAMPs), as well as the ubiquitously expressed SNAP-23 were found. To study the roles of individual VAMPs in exocytosis a novel technique utilising pH sensitive pHluorins was developed. Using VAMPs tagged with pHluorins, the cellular distribution of VAMP-3 and VAMP-8 containing vesicles and their behaviour upon IgE stimulation in live cells was monitored. In unstimulated cells, VAMP- 3 and 8 were found to have distinct cellular distributions. Upon IgE stimulation both VAMP-3 and VAMP-8 containing vesicles translocated to the membrane and underwent membrane fusion, consistent with roles in exocytosis. However, their responses showed distinct time courses and calcium dependences. Importantly the VAMP-3 vesicle pool could be selectively targeted with a botulinum neurotoxin serotype B (BoNT)/B LC construct and in doing so inhibited the release of IL-6. The findings in this study support the notion that distinct vesicle pools, defined in part by expression of VAMP-3 and VAMP-8, regulate the release of inflammatory mediators from mast cells and that BoNTs might provide a novel means of targeting the release of chronic inflammatory mediators from mast cells for treatment of chronic inflammatory diseases.