Cellular interaction with novel biomaterials

The objective of this thesis is to report the behaviour of mammalian cells with biocompatible synthetic polymers with potential for applications to the human body. Composite hydrogel materials were tested as possible keratoprosthetic devices. It was found that surface topography is an important consideration, pores, channels and fibres exposed on the surface of the hydrogels tested can have significant effects on the extent of cell adheson and proliferation. It is recommended that the core component is fabricated out of one of the following to provide a non cell adhesive base; A8, A11, A13, A22, A23. The haptic periphery fabricated out of one of the following would provide a cell adhesive composite; A16, A30, A33, A37, A38, A42, A43, A44. The presence of vitronectin in the ocular tissue appears to lead to higher cell adhesion to the posterior surface of a contact lens when compared to the anterior surface. Group IV contact lenses adhere more cells than Group II contact lenses - this may indicate that more protein (including vitronectin) is able to adhere to the contact lens due to the Group IV contact lenses high water content and ionic hydrogel matrix. Artificial lung surfactant analogues were found to be non cytotoxic but also decreased cell proliferation when tested at higher concentrations. Poly(lysine ethyl ester adipamide) [PLETESA] had the most favourable response on cell proliferation and commercial styrene/maleic anhydride (pMA/STY sp2) the most pronounced inhibitory response. The mode of action that decreases cell proliferation appears to be through membrane destabilization. Tissue culture well plates coated with PLETESA allowed cells to adhere in a concentration dependent manner, multilaminar liposomes possibly of PLETESA were observed in solution in PLETESA coated wells. Polyhydroxybutryate (PHB) and polyhydroxyvalerate (PHV) blends that contained hydroxyapatite were found to be the most cell adhesive material of those materials tested. The blends that were most susceptible to degradation adhered the most cells in initial stages of degradation. The initial slight increase in cell adhesion may be due to the increased rugosity of the material. As the degradation continued the number of cells adhering to the samples decreased, this may indicate that the polarity was inhibitory to cell adhesion during the later stages of degradation.

A novel plasma etching and emission monitoring system (PEEMS) to assess protein and lipid spoilation for hydrogel contact lenses

The work presents a new method that combines plasma etching with extrinsic techniques to simultaneously measure matrix and surface protein and lipid deposits. The acronym for this technique is PEEMS - Plasma Etching and Emission Monitoring System. Previous work has identified the presence of proteinaceous and lipoidal deposition on the surface of contact lenses and highlighted the probability that penetration of these spoilants will occur. This technique developed here allows unambiguous identification of the depth of penetration of spoilants to be made for various material types. It is for this reason that the technique has been employed in this thesis. The technique is applied as a 'molecular' scalpel, removing known amounts of material from the target. In this case from both the anterior .and posterior surfaces of a 'soft' contact lens. The residual material is then characterised by other analytical techniques such as UV/visible .and fluorescence spectroscopy. Several studies have be.en carried out for both in vivo and in vitro spoilt materials. The analysis and identification of absorbed protein and lipid of the substrate revealed the importance of many factors in the absorption and adsorption process. The effect of the material structure, protein nature (in terms of size, shape and charge) and environment conditions were examined in order to determine the relative uptake of tear proteins. The studies were extended to real cases in order to study the. patient dependent factors and lipoidal penetration.

Tear protein interaction with hydrogel contact lenses

The design and synthesis of biomaterials covers a growing number of biomedical applications. The use of biomaterials in biological environment is associated with a number of problems, the most important of which is biocompatabUity. If the implanted biomaterial is not compatible with the environment, it will be rejected by the biological site. This may be manifested in many ways depending on the environment in which it is used. Adsorption of proteins takes place almost instantaneously when a biomaterial comes into contact with most biological fluids. The eye is a unique body site for the study of protein interactions with biomaterials, because of its ease of access and deceptive complexity of the tears. The use of contact lenses for either vision correction and cosmetic reasons or as a route for the controlled drug delivery, has significantly increased in recent years. It is relatively easy to introduce a contact lens Into the tear fluid and remove after a few minutes without surgery or trauma to the patient. A range of analytical techniques were used and developed to measure the proteins absorbed to some existing commercial contact lens materials and also to novel hydrogels synthesised within the research group. Analysis of the identity and quantity of proteins absorbed to biomaterials revealed the importance of many factors on the absorption process. The effect of biomaterial structure, protein nature in terms of size. shape and charge and pH of the environment on the absorption process were examined in order to determine the relative up-take of tear proteins. This study showed that both lysozyme and lactoferrin penetrate the lens matrix of ionic materials. Measurement of the mobility and activity of the protein deposited into the surface and within the matrix of ionic lens materials demonstrated that the mobility is pH dependent and, within the experimental errors, the biological activity of lysozyme remained unchanged after adsorption and desorption. The study on the effect of different monomers copolymerised with hydroxyethyl methacrylate (HEMA) on the protein up-take showed that monomers producing a positive charge on the copolymer can reduce the spoilation with lysozyme. The studies were extended to real cases in order to compare the patient dependent factors. The in-vivo studies showed that the spoilation is patient dependent as well as other factors. Studies on the extrinsic factors such as dye used in colour lenses showed that the addition of colourant affects protein absorption and, in one case, its effect is beneficial to the wearer as it reduces the quantity of the protein absorbed.

Chemical studies of plasma etchants used in integrated circuit manufacture

Plasma or "dry" etching is an essential process for the production of modern microelectronic circuits. However, despite intensive research, many aspects of the etch process are not fully understood. The results of studies of the plasma etching of Si and Si02 in fluorine-containing discharges, and the complementary technique of plasma polymerisation are presented in this thesis. Optical emission spectroscopy with argon actinometry was used as the principle plasma diagnostic. Statistical experimental design was used to model and compare Si and Si02 etch rates in CF4 and SF6 discharges as a function of flow, pressure and power. Etch mechanisms m both systems, including the potential reduction of Si etch rates in CF4 due to fluorocarbon polymer formation, are discussed. Si etch rates in CF4 /SF6 mixtures were successfully accounted for by the models produced. Si etch rates in CF4/C2F6 and CHF3 as a function of the addition of oxygen-containing additives (02, N20 and CO2) are shown to be consistent with a simple competition between F, 0 and CFx species for Si surface sites. For the range of conditions studied, Si02 etch rates were not dependent on F-atom concentration, but the presence of fluorine was essential in order to achieve significant etch rates. The influence of a wide range of electrode materials on the etch rate of Si and Si02 in CF4 and CF4 /02 plasmas was studied. It was found that the Si etch rate in a CF4 plasma was considerably enhanced, relative to an anodised aluminium electrode, in the presence of soda glass or sodium or potassium "doped" quartz. The effect was even more pronounced in a CF4 /02 discharge. In the latter system lead and copper electrodes also enhanced the Si etch rate. These results could not be accounted for by a corresponding rise in atomic fluorine concentration. Three possible etch enhancement mechanisms are discussed. Fluorocarbon polymer deposition was studied, both because of its relevance to etch mechanisms and its intrinsic interest, as a function of fluorocarbon source gas (CF4, C2F6, C3F8 and CHF3), process time, RF power and percentage hydrogen addition. Gas phase concentrations of F, H and CF2 were measured by optical emission spectroscopy, and the resultant polymer structure determined by X-ray photoelectron spectroscopy and infrared spectroscopy. Thermal and electrical properties were measured also. Hydrogen additions are shown to have a dominant role in determining deposition rate and polymer composition. A qualitative description of the polymer growth mechanism is presented which accounts for both changes in growth rate and structure, and leads to an empirical deposition rate model.

Some aspects of the interaction of humic substances with metal ions

Humic substances are the major organic constituents of soils and sediments. They are heterogeneous, polyfunctional, polydisperse, macromolecular and have no accurately known chemical structure. Their interactions with radionuclides are particularly important since they provide leaching mechanisms from disposal sites. The central theme to this research is the interaction of heavy metal actinide analogues with humic materials. Studies described focus on selected aspects of the characteristics and properties of humic substances. Some novel approaches to experiments and data analysis are pursued. Several humic substances are studied; all but one are humic acids, and those used most extensively were obtained commercially. Some routine characterisation techniques are applied to samples in the first instance. Humic substances are coloured, but their ultra-violet and visible absorption spectra are featureless. Yet, they fluoresce over a wide range of wavelengths. Enhanced fluorescence in the presence of luminescent europium(III) ions is explained by energy transfer from irradiated humic acid to the metal ion in a photophysical model. Nuclear magnetic resonance spectroscopy is applied to the study of humic acids and their complexes with heavy metals. Proton and carbon-13 NMR provides some structural and functionality information; Paramagnetic lanthanide ions affect these spectra. Some heavy metals are studied as NMR nuclei, but measurements are restricted by their sensitivity. A humic acid is fractionated yielding a broad molecular weight distribution. Electrophoretic mobilities and particle radii determined by Laser Doppler Electrophoretic Light Scattering are sensitive to the conditions of the supporting media, and the concentration and particle size distribution of humic substances. In potentiometric titrations of humate dispersions, the organic matter responds slowly and the mineral acid addition is buffered. Proton concentration data is modelled and a mechanism is proposed involving two key stages, both resulting in proton release after some conformational changes.

Some aspects of the microbiological properties of polymers and composite materials

The interaction of microorganisms with glass-reinforced polyester resins(GRP), both under laboratory and simulated operating conditions, has been examined following reports of severl! fungal biodeterioration. Although GRP was not previously associated with substantial microbial growth, small amounts of microbial activity would pose problems for products associated with comestible materials. The microbiology of the raw materials was investigated, two ingredients were supportive to microbial populations whilst five materials were biostatic or inhibitory in their action. Production laminate was not susceptible to microbial deterioration or inhibitory to microbes. Incorporation of zinc stearate, one of the supportive ingredients, at 300% manufacturing level or drastic undercuring produced laminate capable of supporting microbial growth but only after a non-biotic stage of degradation. Study of the long-term population dynamics of cisterns of GRP and competitive materials under conditions simulating in-service conditions, monitoring microbial numbers within the experimental vessels and comparing with the populations of the supply water, suggests that the performance of GRP cisterns is slightly superior to conventional competitive materials. An investigation of the biological performance of GRP cisterns in an isolated area of known microbiological hazard was conducted. Severe biodeterioration had been experienced with Preform GRP articles moulded using different production techniques, but substitution of current GRP articles resulted in no recurrence of the problem. All attempts to establish the fungal isolate responsible for the phenomena in cisterns under controlled conditions failed. Scanning Electron Microscopy of GRP surfaces showed that although differences exist between current and Preform laminates, these could not satisfactorily explain the differences in service behaviour. These results and the results of the British Plastics Federation Expert Working Group interlaboratory study are discussed in relation to the original report of gross fungal biodeterioration and, to the design of future testing programmes for the products of industrial concerns.

The plasma nutriding of tool and bearing steels

There is some evidence to suggest that nitriding of alloy steels, in particular high speed tool steels, under carefully controlled conditions might sharply increase rolling contact fatigue resistance. However, the subsurface shear stresses developed in aerospace bearing applications tend to occur at depths greater than the usual case depths currently produced by nitriding. Additionally, case development must be limited with certain materials due to case spalling and may not always be sufficient to achieve the current theoretical depths necessary to ensure that peak stresses occur within the case. It was the aim of' this work to establish suitable to overcome this problem by plasma nitriding. To assist this development a study has been made of prior hardening treatment, case development, residual stress and case cracking tendency. M2 in the underhardened, undertempered and fully hardened and tempered conditions all responded similarly to plasma nitriding - maximum surface hardening being achieved by plasma nitriding at 450°C. Case development varied linearly with increasing treatment temperature and also with the square root of the treatment time. Maximum surface hardness of M5O and Tl steels was achieved by plasma nitriding in 15% nitrogen/85% hydrogen and varied logarithmically with atmosphere nitrogen content. The case-cracking contact stress varied linearly with nitriding temperature for M2. Tl and M5O supported higher stresses after nitriding in low nitrogen plasma atmospheres. Unidirectional bending fatigue of M2 has been improved up to three times the strength of the fully hardened and tempered condition by plasma nitriding for 16hrs at 400°C. Fatigue strengths of Tl and M5O have been improved by up to 30% by plasma nitriding for 16hrs at 450°C in a 75% hydrogen/25% nitrogen atmosphere.

Fin spacing interaction in fluidized bed heat exchangers

The work presented in this thesis is concerned with the heat transfer performance of a single horizontal bare tube and a variety of finned tubes immersed in a shallow air fluidized bed. Results of experimental investigations with the bare tube indicate that the tube position in the bed influences its performance narticularly where fine bed materials are used. In some cases the maximum heat transfer is obtained with the tube in the particle cloud just above the dense phase fluidized bed - a phenomenon that has not been previously observed. This was attributed to the unusual particle circulation in shallow beds. The data is also presented in dimensionless correlations which may be useful for design purposes. A close approximation to the bare tube data can be obtained by using thetransient heating of a spherical robe and this provides a valuable way of accumulating a lot of data very rapidly. The experimental data on finned tubes shows that a fin spacing less than twenty times the average particle diameter can cause a significant reduction in heat transfer due to the interaction which takes place between the particles and the surface of the fins. Furthermore, evidence is provided to show that particle shape plays an important part in the interaction with spherical particles being superior to angular particles at low fin spacing/particle diameter ratio. The finned tube data is less sensitive to tube position in the bed than bare tubes and the best performance is when the tube is positioned at the distributor.A reduction in bed depth decreases the thermal performance of the finned tube but in many practical installations the reduction in pressure drop might more than comnensate for the reduced heat flux. Information is also provided on the theoretical uerformance of fins and the effect of the root contact area between the fins and the tube was investigated.

Heat transfer from molten materials to liquids

An apparatus was designed and constructed which enabled material to be melted and heated to a maximum temperature of 1000C and then flooded with a pre-heated liquid. A series of experiments to investigate the thermal interaction between molten metals (aluminium, lead and tin) and sub-cooled water were conducted. The cooling rates of the molten materials under conditions of flooding were measured with a high speed-thermocouple and recorded with a transient recorder. A simplified model for calculating heat fluxes and metal surface temperatures was developed and used. Experimental results yielded boiling heat transfer in the transition film and stable film regions of the classic boiling curve. Maximum and minimum heat fluxes were observed at nucleate boiling crisis and the Leidenfrost point respectively. Results indicate that heat transfer from molten metals to sub-cooled water is a function of temperature and coolant depth and not a direct function of the physical properties of the metals. Heat transfer in the unstable transition film boiling region suggests that boiling dynamics in this region where a stationary molten metal is under pool boiling conditions at atmospheric pressure would not initiate a fuel-coolant interaction. Low heat fluxes around the Leidenfrost point would provide efficient fuel-coolant decoupling by a stable vapour blanket to enable coarse mixing of the fuel and coolant to occur without appreciable loss of thermal energy from the fuel. The research was conducted by Gareph Boxley and was submitted for the degree of PhD at the University of Aston in Birmingham in 1980.

Activation of the neutrophil respiratory burst by plasma from periodontitis patients is mediated by pro-inflammatory cytokines

Aim: To determine the effect of periodontitis patients' plasma on the neutrophil oxidative burst and the role of albumin, immunoglobulins (Igs) and cytokines. Materials and Methods: Plasma was collected from chronic periodontitis patients (n=11) and periodontally healthy controls (n=11) and used with/without depletion of albumin and Ig or antibody neutralization of IL-8, GM-CSF or IFN-a to prime/stimulate peripheral blood neutrophils, isolated from healthy volunteers. The respiratory burst was measured by lucigenin-dependent chemiluminescence. Plasma cytokine levels were determined by ELISA. Results: Plasmas from patients were significantly more effective in both directly stimulating neutrophil superoxide production and priming for subsequent formyl-met-leu-phe (fMLP)-stimulated superoxide production than plasmas from healthy controls (p<0.05). This difference was maintained after depletion of albumin and Ig. Plasma from patients contained higher mean levels of IL-8, GM-CSF and IFN-a. Individual neutralizing antibodies against IL-8, GM-CSF or IFN-a inhibited the direct stimulatory effect of patients' plasma, whereas the ability to prime for fMLP-stimulated superoxide production was only inhibited by neutralization of IFN-a. The stimulating and priming effects of control plasma were unaffected by antibody neutralization. Conclusions: This study demonstrates that plasma cytokines may have a role in inducing the hyperactive (IL-8, GM-CSF, IFN-a) and hyper-reactive (IFN-a) neutrophil phenotype seen in periodontitis patients.

Disruption of the interaction between PMCA2 and calcineurin triggers apoptosis and enhances paclitaxel-induced cytotoxicity in breast cancer cells

Cancer is caused by defects in the signalling mechanisms that govern cell proliferation and apoptosis. It is well known that calcium-dependent signalling pathways play a critical role in cell regulation. A tight control of calcium homeostasis by transporters and channel proteins is required to assure a proper functioning of the calcium-sensitive signal transduction pathways that regulate cell growth and apoptosis. The Plasma Membrane Calcium ATPase 2 (PMCA2) has been recently identified as a negative regulator of apoptosis that can play a significant role in cancer progression by conferring cells resistance to apoptosis. We have previously reported an inhibitory interaction between PMCA2 and the calcium-activated signalling molecule calcineurin in breast cancer cells. Here we demonstrate that disruption of the PMCA2/calcineurin interaction in a variety of human breast cancer cells results in activation of the calcineurin/NFAT pathway, up-regulation in the expression of the pro-apoptotic protein Fas Ligand, and in a concomitant loss of cell viability. Reduction in cell viability is the consequence of an increase in cell apoptosis. Impairment of the PMCA2/calcineurin interaction enhances paclitaxel-mediated cytotoxicity of breast tumoral cells. Our results suggest that therapeutic modulation of the PMCA2/calcineurin interaction might have important clinical applications to improve current treatments for breast cancer patients.

Synthesis, characterisation and applications of diamond materials

This thesis presented a detailed research work on diamond materials. Chapter 1 is an overall introduction of the thesis. In the Chapter 2, the literature review on the physical, chemical, optical, mechanical, as well as other properties of diamond materials are summarised. Followed by this chapter, several advanced diamond growth and characterisation techniques used in experimental work are also introduced. Then, the successful installation and applications of chemical vapour deposition system was demonstrated in Chapter 4. Diamond growth on a variety of different substrates has been investigated such as on silicon, diamond-like carbon or silica fibres. In Chapter 5, the single crystalline diamond substrate was used as the substrate to perform femtosecond laser inscription. The results proved the potentially feasibility of this technique, which could be utilised in fabricating future biochemistry microfluidic channels on diamond substrates. In Chapter 6, the hydrogen-terminated nanodiamond powder was studied using impedance spectroscopy. Its intrinsic electrical properties and its thermal stability were presented and analysed in details. As the first PhD student within Nanoscience Research Group at Aston, my initial research work was focused on the installation and testing of the microwave plasma enhanced chemical vapour deposition system (MPECVD), which will be beneficial to all the future researchers in the group. The fundamental of the on MPECVD system will be introduced in details. After optimisation of the growth parameters, the uniform diamond deposition has been achieved with a good surface coverage and uniformity. Furthermore, one of the most significant contributions of this work is the successful pattern inscription on diamond substrates by femtosecond laser system. Previous research of femtosecond laser inscription on diamond was simple lines or dots, with little characterisation techniques were used. In my research work, the femtosecond laser has been successfully used to inscribe patterns on diamond substrate and fully characterisation techniques, e.g. by SEM, Raman, XPS, as well as AFM, have been carried out. After the femtosecond laser inscription, the depth of microfluidic channels on diamond film has been found to be 300~400 nm, with a graphitic layer thickness of 165~190 nm. Another important outcome of this work is the first time to characterise the electrical properties of hydrogenterminated nanodiamond with impedance spectroscopy. Based on the experimental evaluation and mathematic fitting, the resistance of hydrogen-terminated nanodiamond reduced to 0.25 MO, which were four orders of magnitude lower than untreated nanodiamond. Meanwhile, a theoretical equivalent circuit has been proposed to fit the results. Furthermore, the hydrogenterminated nanodiamond samples were annealed at different temperature to study its thermal stability. The XPS and FTIR results indicate that hydrogen-terminated nanodiamond will start to oxidize over 100ºC and the C-H bonds can survive up to 400ºC. This research work reports the fundamental electrical properties of hydrogen-terminated nanodiamond, which can be used in future applications in physical or chemical area.

Role of plasma in femtosecond laser pulse propagation

This paper describes physics of nonlinear ultra-short laser pulse propagation affected by plasma created by the pulse itself. Major applications are also discussed. Nonlinear propagation of the femtosecond laser pulses in gaseous and solid transparent dielectric media is a fundamental physical phenomenon in a wide range of important applications such as laser lidars, laser micro-machining (ablation) and microfabrication etc. These applications require very high intensity of the laser field, typically 1013–1015 TW/cm2. Such high intensity leads to significant ionisation and creation of electron-ion or electron-hole plasma. The presence of plasma results into significant multiphoton and plasma absorption and plasma defocusing. Consequently, the propagation effects appear extremely complex and result from competitive counteraction of the above listed effects and Kerr effect, diffraction and dispersion. The theoretical models used for consistent description of laser-plasma interaction during femtosecond laser pulse propagation are derived and discussed. It turns out that the strongly nonlinear effects such self-focusing followed by the pulse splitting are essential. These phenomena feature extremely complex dynamics of both the electromagnetic field and plasma density with different spatio-temporal structures evolving at the same time. Some numerical approaches capable to handle all these complications are also discussed. ©2006 American Institute of Physics

Through-thickness plasma modification of biodegradable and nonbiodegradable porous polymer constructs

Pure poly(lactide-co-glycolide) and polystyrene surfaces are not very suitable to support cell adhesion/ spreading owing to their hydrophobic nature and low surface energy. The interior surfaces of large porous 3D scaffolds were modified and activated using radio-frequency, low-pressure air plasma. An increase in the wettability of the surface was observed after exposure to air plasma, as indicated by the decrease in the contact angles of the wet porous system. The surface composition of the plasma-treated polymers was studied using X-ray photoelectron spectroscopy. pH-dependent zeta-potential measurements confirm the presence of an increased number of functional groups. However, the plasma-treated surfaces have a less acidic character than the original polymer surfaces as seen by a shift in their isoelectric point. Zeta-potential, as well as contact angle measurements, on 3D scaffolds confirm that plasma treatment is a useful tool to modify the surface properties throughout the interior of large scaffolds. © 2008 Wiley Periodicals, Inc.

Atmospheric plasma treatment of porous polymer constructs for tissue engineering applications

Porous 3D polymer scaffolds prepared by TIPS from PLGA (53:47) and PS are intrinsically hydrophobic which prohibits the wetting of such porous media by water. This limits the application of these materials for the fabrication of scaffolds as supports for cell adhesion/spreading. Here we demonstrate that the interior surfaces of polymer scaffolds can be effectively modified using atmospheric air plasma (AP). Polymer films (2D) were also modified as control. The surface properties of wet 2D and 3D scaffolds were characterised using zeta-potential and wettability measurements. These techniques were used as the primary screening methods to assess surface chemistry and the wettability of wet polymer constructs prior and after the surface treatment. The surfaces of the original polymers are rather hydrophobic as highlighted but contain acidic functional groups. Increased exposure to AP improved the water wetting of the treated surfaces because of the formation of a variety of oxygen and nitrogen containing functions. The morphology and pore structure was assessed using SEM and a liquid displacement test. The PLGA and PS foam samples have central regions which are open porous interconnected networks with maximum pore diameters of 49 μm for PLGA and 73 μm for PS foams. (Figure Presented) © 2007 Wiley-VCH Verlag GmbH & Co. KGaA.