Rozprawy doktorskie na temat „Biomedical applications of FBGs”

Modern day medicine is on the brink of a new age of therapy, which aims to harness the natural power of molecular biology for disease treatment. This therapy could include replacement of dysfunctional genes that cause disorders such as cystic fibrosis (Lommatzsch and Aris, 2009), or silencing the overexpression of genes that cause disorders such as cancer (Pelengaris and Khan, 2003). In both examples, the treatment of these genetic diseases lies in the delivery of synthetic nucleic acids into diseased cells, the former being called gene replacement therapy (Dobson, 2006a), and the latter being called RNA interference (RNAi) therapy (Whitehead et al., 2009). While these techniques have long been in use as genetic research tools for gene transfection or silencing in vitro, their translation for use in clinical disease treatment has yet to be achieved. The main problem facing the development of these novel therapies is the specific delivery of nucleic acids into diseased cells within the body. It is hoped that nanoparticles (NPs) can be used to overcome this problem, by acting as vehicles to transport nucleic acids through the body for specific delivery into diseased cells. This feat can be aided by the attachment of additional functional molecules such as cell penetrating peptides (CPPs), targeting peptides, additional drug types and molecules for imaging during treatment. Many different NP design strategies are currently under development. It is essential for new designs to be extensively tested for toxicity and efficiency in human cells before they can be successfully released into the clinic. As part of this effort, this PhD project has investigated two different NP design strategies for drug delivery: 1) the use of a magnetic field (MF) and a CPP to increase the delivery of iron oxide magnetic NPs (mNPs) to cells grown in tissueequivalent 3D collagen gels, and 2) gold NPs (AuNPs) for the delivery of siRNA to silence the c-myc oncogene for cancer treatment. In the first investigation, a MF and the CPP penetratin were found to increase mNP delivery to cells grown in 3D. In the second investigation, AuNPs were assessed in a range of different cell types (grown in 2D) for their performance in 4 main areas; cellular toxicity, cellular uptake, c-myc knockdown and effect on the cell cycle.

Surface topography is known to be important in biomedical applications such as scaffolds for tissue regeneration and has been shown to affect wettability and cell behaviour. Traditionally, topographical effects such as surface texturing have been generated using methods such as photolithography, soft lithography, thermal embossing, and laser/electron beam techniques. This thesis introduces a relatively new technique known as photoembossing to create surface texturing for biomedical applications. Photoembossing is used to produce surface texturing on polymer surfaces by patterned ultraviolet (UV) exposure of a photopolymer blend without an etching step or an expensive mould. After a short general introduction and a literature review, the first experimental chapters describe surface patterning of poly(methyl methacrylate) (PMMA) photopolymer substrates by photoembossing. PMMA is blended with an acrylate monomer and photoinitiator by dissolution in a volatile solvent and processed into films by wire bar coating, and fibres are produced by electrospinning. Surface texture is achieved on both films and fibres by photoembossing. Endothelial cell culture shows that the substrates are biocompatible and cells readily adhere to the surface. In tissue regeneration applications, scaffold degradation is often important to allow tissue in-growth. Thus, in subsequent studies polylactide-co-glycolide (PLGA) is used as a polymer binder. PLGA blended with a triacrylate monomer showed partial degradation after 10 weeks, with a cross-linked acrylate network remaining. Endothelial cell adhesion was even better on the PLGA photopolymer substrates compared to PMMA. Furthermore, surface texture improved cell adhesion and proliferation on the PLGA photopolymer. To obtain completely degradable substrates, thiol monomer was used in addition to the acrylate to produce ester bonds after the thiol-ene reaction, which is cleavable by hydrolysis. Accelerated degradation in sodium hydroxide (NaOH) showed complete degradation of this photopolymer system. The degradation rate of the photopolymer could be tuned by the molecular weight of the acrylate monomer, with low molecular weight monomers degrading more slowly than high molecular weight species. Furthermore, the height of the surface relief structures could be enhanced by using low-molecular-weight acrylate monomers. Endothelial cell culture revealed biocompatibility of the blend and cells were able to adhere after 24 hours of seeding. This thesis demonstrates that photoembossing is a viable technique in producing surface texture for tissue engineering applications. This surface texture can be achieved on both biocompatible and biodegradable photopolymer films and fibres.

Chitosan, a copolymer of glucosamine and N-acetyl glucosamine, is a polycationic, biocompatible and biodegradable polymer. In addition, chitosan has different functional groups that can be modified with a wide array of ligands. Because of its unique physicochemical properties, chitosan has great potential in a range of biomedical applications, including tissue engineering, non-viral gene delivery and enzyme immobilization. In our work, the primary amine groups of chitosan were utilized for chitosan modification through biotinylation using N-hydroxysuccinimide chemistry. This was followed by the addition of avidin which strongly binds to biotin. Biotinylated ligands such as polyethylene glycol (PEG) and RGD peptide sequence, or biotinylated enzymes such as trypsin, were then added to modify the surface properties of the chitosan for a variety of purposes. Modified chitosans were formulated into nano-sized particles or cast into films. Different factors affecting fabrication of chitosan particles, such as the pH of the preparation, the inclusion of polyanions, the charge ratios and the degree of deacetylation and the molecular weight of chitosan were studied. Similarly, parameters affecting the fabrication of chitosan films, such as cross-linking, were investigated for potential applications in tissue engineering and enzyme immobilization. It was found that the inclusion of dextran sulfate resulted in optimum interaction between chitosan and DNA, as shown by the high stability of these nanoparticles and their high in vitro transfection efficiencies in HEK293 cells. When applying these formulations as DNA vaccines in vivo, chitosan nanoparticles loaded with the ovalbumin antigen and the plasmid DNA encoding the same antigen resulted in the highest antibody response in C57BL/6 mice. Furthermore, engineering of the surface of chitosan nanoparticles was done by utilizing the avidin-biotin interaction for attaching PEG and RGD. The modified formulations were tested for their in vitro gene delivery properties and it was found that these ligands improved gene transfection efficiencies significantly. Chitosan nanoparticles were optimized further for enzyme immobilization purposes using sodium sulfate and glutaraldehyde as physical and chemical cross-linking agents, respectively. These particles and chitosan films were used for immobilizing trypsin utilizing several techniques. Enzyme immobilization via avidin-biotin interaction resulted in high immobilization efficiency and high enzymatic activity in different reaction conditions. Additionally, the immobilized trypsin systems were stable and amenable to be regenerated for multiple uses. Finally, glutaraldehyde cross-linked chitosan films were modified with PEG and RGD for their cell repellant and cell adhesion properties, respectively, using avidin-biotin interaction. This method was again effective in engineering chitosan surfaces for modulating cell adhesion and proliferation. In conclusion, using avidin-biotin technique to modify biotinylated chitosan surfaces is a facile method to attach a wide variety of ligands in mild reaction conditions, while preserving the functionality of these ligands.

In the past few years, significant progress in the study of scaffolds for cells grow has taken place. This research has led to the development of a wide variety of metallic, polymeric, ceramic and composite biomaterials. This thesis describes the development of a novel composite system with tunable morphological and mechanical properties, ease of production and capability to guide the biological response. The composite system was composed by polyamide 6 (PA6) and carboxyl-functionalized multi-walled carbon nanotubes (MWCNT), which were used as reinforcement agents in the polymer matrix. Electrospinning was used as the fabrication technique for the production of anisotropic networks. Physical and biological properties of the nets were evaluated focusing on the effect of the filler addition. It was observed that the production technique induced the alignment of MWCNT within the nanofiber axis and the formation of a roughness on the fiber's surface. The biological properties of MG63 and MRC5 cell lines were enhanced if compared with the neat PA6 networks due to surface modification caused by the filler addition.

In the past few years, significant progress in the study of scaffolds for cells grow has taken place. This research has led to the development of a wide variety of metallic, polymeric, ceramic and composite biomaterials. This thesis describes the development of a novel composite system with tunable morphological and mechanical properties, ease of production and capability to guide the biological response. The composite system was composed by polyamide 6 (PA6) and carboxyl-functionalized multi-walled carbon nanotubes (MWCNT), which were used as reinforcement agents in the polymer matrix. Electrospinning was used as the fabrication technique for the production of anisotropic networks. Physical and biological properties of the nets were evaluated focusing on the effect of the filler addition. It was observed that the production technique induced the alignment of MWCNT within the nanofiber axis and the formation of a roughness on the fiber's surface. The biological properties of MG63 and MRC5 cell lines were enhanced if compared with the neat PA6 networks due to surface modification caused by the filler addition.

[Truncated abstract] In the past decade, the synthesis of superparamagnetic iron oxide nanoparticles (SPIONs) has received considerable attention due to their potential applications in biomedical fields. However, success in size and shape control of the SPIONs has been mostly achieved through organic routes using large quantities of toxic or/and expensive precursors in organic reaction medium at high reaction temperature. This has limited the biomedical applications of SPIONs and therefore, development of a synthetic method under aqueous condition that is reproducible, scalable, environmentally benign, and economically feasible for industrial production is of paramount importance in order to fully realise their practical applications. Spinning Disc Processing (SDP) has been used to synthesise superparamagnetic magnetite (Fe3O4) nanoparticles at room temperature via a modified chemical precipitation method under continuous flow condition and offer a potential alternative to be applied to SPIONs production. SDP has extremely rapid mixing under plug flow conditions, effective heat and mass transfer, allowing high throughput with low wastage solvent efficiency. The synthesis process involves passing ammonia gas over a thin aqueous film of Fe2+/3+ which is introduced through a jet feed close to the centre of a rapidly rotating disc (500-2500 rpm). Synthetic parameters such as precursor concentrations, temperature, flow rate, disc speed, and surface texture influence the particle sizes. ... Magnetic silica microspheres are receiving great attention for possible applications in magnetic targeting drug delivery, bioseparation and enzyme isolation. However, the current available methods for preparation suffer from the setback of low loading of Fe3O4 nanoparticles in the silica microsphere, which result in low magnetic moment, thereby limiting their practical applications. Therefore it is of considerable importance to develop new alternative synthetic methods for fabricating magnetic silica microspheres with high magnetic nanoparticles loading. Superparamagentic Fe3O4 nanoparticles (8-10 nm diameter) and curcumin have been encapsulated in mesoporous silica in a simple multiplestep self assembly approach process with high Fe3O4 nanoparticles loading (37%). The synthesis involves loading of curcumin in the Cetyltrimethylammonium bromide (CTAB) micellar rod in the presence of superparamagnetic Fe3O4 nanoparticles via a parallel synergistic approach. The synthesised magnetic mesoporous silica composite material is stable, superparamagnetic with high saturation magnetisation before and after curcumin leaching experiment. Under physiological pH in phosphate buffer, the curcumin is slowly released over several days. These magnetic mesoporous silica are expected to have great potential as targeted drug delivery systems.

Switchable oligopeptides, able to expose of conceal biomolecules on a surface, upon the application of an electrical potential, represent a versatile tool for the development of novel devices, presenting potential biomedical applications. Recently, several studies have demonstrated the applicability of smart devices for the control of protein binding and cellular response. In this work; a detailed analysis of the steric requirements necessary to develop a mixed oligopeptide Self-Assembled Monolayer (SAM) presenting an optimum switching ability will be described. The influence of both the SAM components surface ratio and the switching unit length on the mixed SAMs switching performance will be investigated. The findings of this investigation will be used to develop, for the first time, a platform, based on electrically switchable oligopeptides, able to control the interaction between an antigen and its relative antibody. The influence of the biological medium on the oligopeptide switching ability will also be investigated. Finally, an orthogonal functionalisation strategy, will be investigated in detail, together with a new platform able to promote human sperm cells adhesion. The results of this research thesis will also represent the first building blocks towards the development of glass-gold rnicropattemed surfaces able to control the calcium signalling in human sperm cells, presenting potential applications in the improvement of in-vitro fertilisation (NF) treatments success rates.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Materials Science and Engineering, 2017.
This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.
Cataloged from student-submitted PDF version of thesis.
Includes bibliographical references (pages 170-176).
Research on biomedical applications of magnetic nanoparticles (MNPs) has increasingly sought to demonstrate noninvasive actuation of cellular processes and material responses using heat dissipated in the presence of an alternating magnetic field (AMF). By modeling the dependence of hysteresis losses on AMF amplitude and constraining AMF conditions to be physiologically suitable, it can be shown that MNPs exhibit uniquely optimal driving conditions that depend on controllable material properties such as magnetic anisotropy, magnetization, and particle volume. "Magnetothermal multiplexing," which relies on selecting materials with substantially distinct optimal AMF conditions, enables the selective heating of different kinds of collocated MNPs by applying different AMF parameters. This effect has the potential to extend the functionality of a variety of emerging techniques with mechanisms that rely on bulk or nanoscale heating of MNPs. Experimental investigations on methods for actuating deep brain stimulation, drug release, and shape memory polymer response are summarized, with discussion of the feasibility and utility of applying magnetothermal multiplexing to similar systems. The possibility of selective heating is motivated by a discussion of various models for heat dissipation by MNPs in AMFs, and then corroborated with experimental calorimetry measurements. A heuristic method for identifying materials and AMF conditions suitable for multiplexing is demonstrated on a set of iron oxide nanoparticles doped with various concentrations of cobalt. Design principles for producing AMFs with high amplitude and ranging in frequency from 15kHz to 2.5MHz are explained in detail, accompanied by a discussion of the outlook for scalability to clinically relevant dimensions. The thesis concludes with a discussion of the state of the field and the broader lessons that can be drawn from the work it describes.
by Michael G. Christiansen.
Ph. D.

Thesis: S.M. in Engineering and Management, Massachusetts Institute of Technology, System Design and Management Program, 2018.
This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.
Cataloged from student-submitted PDF version of thesis.
Includes bibliographical references (pages 77-79).
Identifying patients with aggressive cancers is a major healthcare challenge in resource-limited settings such as sub-Saharan Africa. Holographic imaging techniques have been shown to perform diagnostic screening at low cost in order to meet this clinical need, however the computational and logistical challenges involved in deploying such systems are manifold. This thesis aims to make two specific contributions to the field of point-of-care diagnostics. First, it documents the design and construction of low-cost holographic imaging hardware which can serve as a template for future research and development. Second, it presents a novel deep-learning architecture that can potentially lower the computational burden of digital holography by replacing existing image reconstruction methods. We demonstrate the effectiveness of the algorithm by reconstructing biological samples and quantifying their structural similarity relative to spatial deconvolution methods. The approaches explored in this work could enable a standalone holographic platform that is capable of efficiently performing diagnostic screening at the point of care.
by Ismail Degani.
S.M. in Engineering and Management

Measuring chemical concentration is vital for understanding normal and disease physiology involving metabolism and signalling, but monitoring chemical concentrations in living systems poses a unique challenge because of biological heterogeneity. The purpose of this work is to develop a system able to monitor chemical concentrations in primary cultured cells, and apply it to the detection of oxygen, a nutritional status marker, and nitric oxide which is a signalling molecule. Both of these electroactive species are involved in angiogenesis which is the growth of new blood vessels, and a hallmark of cancer. The approach used in this study is to grow porcine endothelial cells onto bronectin-coated gold microelectrode arrays with diameter 25 μm and perform electrochemical measurement on them. An experimental protocol is developed for measurements of dissolved oxygen and nitric oxide around cells in their normal cell-culture environment. It includes developing instrumentation like a heating platform and silver reference microelectrode; data processing for automation and normalisation; and optimising voltammetry techniques. Culture medium is found to a ect electrochemical measurements by changing double layer capacitance, reaction rate constant and di usion. The measurement system is used to detect oxygen reduction around cells, and this is used to estimate their oxygen consumption rate. Nitric oxide produced by cells is also measured, and this is used to identify an angiogenic pathway leading to nitric oxide production by endothelial cells. Variability in cell measurements is shown to originate from the biological system rather than from sensor design. A novel electroanalytical technique for determining parameters of reversible redox systems is developed by experimentally testing an analytical solution for the current response to a large-amplitude sinusoidal voltage input. The technique is used to nd estimates for double layer capacitance, half wave potential and di usion coe cients for both potassium ferrocyanide and ruthenium hexaamine.

Dans cette thèse, nous nous sommes intéressés aux systèmes intelligents pour des applicationsmédicales et cosmétiques. Ainsi, nous avons conçu et réalisé trois instruments. Le premier estdédié à la mesure de la mouillabilité de la peau. L’originalité de ce dispositif réside en sa capacité àdonner une image 3D de la goûte de la surface de la peau explorée et de donner le comportementdynamique de la goûte. Cette stratégie nous donnera la possibilité de créer de nouvelles basesde données relatives à la mouillabilité de tout le corps humain. En effet, nous disposons que desdonnées sur la mouillabilité de l’avant-bras. Le deuxième instrument intelligent concerne la mesurede la réflectance d’une surface. Ce dispositif assure une mesure de très haute résolution angulairede la BRDF et une très bonne répétabilité de la mesure. Il a été validé sur la peau pour la mesurede l’ éclat. Et enfin le troisième instrument, basé sur une méthode originale de mesure de vibrationà l’aide d’un système de stéréo-vision associée à un motif périodique. Il a été appliqué pour lamesure du mouvement thoracique et abdominal lors de la respiration. Notre principale motivationpour développer ce système fut la réduction des artefacts, dus aux mouvements d’un patient lorsd’un examen radiologique
Smart Devices have been widely used by health care and cosmetics professionals. Indeed, they helpin many aspects of clinical practice by providing an efficient way for medical diagnosis, supportingbetter clinical decision-making and improving patient outcomes. In this thesis, we have beeninterested in three applications. The first one is related to the wettability measurement, especially forthe human skin. So we propose, a held-hand device that is based on the contact angle measurementto determine skin wettability. Besides, the device allows the visualization of the liquid dropletspreading in both dynamic and static modes. Moreover, it can measure the top and the left views ofthe droplet and provides the 3D droplet and the skin explored area profiles. The second applicationpermits the skin radiance measurement. For this purpose, we propose a miniaturized device havingan original method for the BRDF measurement associated with 3D profile measurement of the areastudied. As regards the third application, it is a non-invasive method for breath measurement that usesa stereovision system and a pseudo-periodic pattern. This system allows a high-resolution threedimensionaldisplacement measurement for the recording of the thoracoabdominal wall respiratorymovement. The devices developed during this research gives us a high accuracy, a good resolutionand repeatability of measurements

Body-Centric Wireless Communication (BCWC) is a central topic in the development of healthcare and biomedical technologies. Increasing healthcare quality, in addition to the continuous miniaturisation of sensors and the advancement in wearable electronics, embedded software, digital signal processing and biomedical technologies, has led to a new era of biomedical devices and increases possibility of continuous monitoring, diagnostic and/or treatment of many diseases. However, the major difference between BCWC, particularly implantable devices, and conventional wireless systems is the radio channel over which the communication takes place. The human body is a hostile environment from a radio propagation perspective. This environment is a highly lossy and has a high effect on the antenna elements, the radio channel parameters and, hence a dramatic drop in the implanted antenna performance. This thesis focuses on how to improve the gain of implanted antennas. In order to improve the gain and performance of implanted antennas, this thesis uses a combination of experimental and electromagnetic numerical investigations. Extensive simulation and experimental investigations are carried out to study the effects of various external elements on the performance improvement of implanted antennas. The thesis also shows the design, characterisation, simulation and measurements of four different antennas to work at ISM band and seventeen different scenarios for body wireless communication. A 3- layer (skin, fat and muscle) and a liquid homogenise phantom were used for human body modelling in both simulation and measurements. The results shows that a length of printed line and a grid can be used on top of the human skin in order enhance the performance of the implanted antennas. Moreover, a ring and a hemispherical lens can be used externally in order to enhance the performance of the implanted antenna. This approach yields a significant improvement in the antenna gain and reduces the specific absorption rate (SAR) in most cases and the obtained gain varies between 2 dB and 8 dB.

Biocompatible polymers are used exhaustively within the biomedical arena, demonstrating a mechanical and chemical diversity that few other materials possess. As polymer technologies evolves to cater for new medical demands, even the most niche biomedical application becomes an achievable reality. However, the discovery of new polymers is hindered by the complexity and intricacy in which the biological milieu interacts with a new substrate, reducing the ability to predict the appropriateness of a certain polymer for a specific application. This drawback can be countered by the high-throughput evaluation of large numbers of chemically diverse polymer candidates. In this thesis, the use of polymer microarrays is invoked to address two separate medically-relevant issues: the control of inflammation, and the improvement of cancer screening. In addition, I provide details of how polymer microarray techniques and technology can be employed to expand the repertoire of biomaterials research. Mitochondrial DNA (mtDNA) is an alarm molecule that contributes to the cytokine storm observed during severe tissue injury. An application where control of this systemic inflammation is achieved through scavenging of mtDNA by a polymer was proposed. Primary screening highlighted that 166 out of the 380 polymers evaluated bound to blood cells, making them unsuitable for a blood-based application. The remaining 214 blood-compatible polymers were cross-examined for mtDNA binding. Through polymer microarray and subsequent scale-up of promising candidates, a poly(methoxyethyl methacrylate-co-di(ethylamino)ethyl acrylate-co-methoxyethyl acrylate) was found to have a remarkable ability to scavenge mtDNA. Removal of cell-free mtDNA using this polymer is proposed to remove a key trigger of systemic inflammation. Cervical cancer screening includes the cytological evaluation of patient material for developed or developing abnormalities. An application was sought that would enrich for cancerous/pre-cancerous cells and improve upon current standards for detection. Four cancerous cervical cell lines (HeLa, CaSki, SiHa, and C33a) and four precancerous cell lines (W12E, W12G, W12GPX, and W12GPXY) were interrogated to identify polymers with consistent binding that may improve routine cytological evaluation. A short-list of 24 polymers was assembled, and cells from liquid based cytology samples from healthy patient were spiked with DiI-labelled cancerous/precancerous cells and the short-listed polymers were re-evaluated for preferential binding. An enrichment of abnormal cervical cells was observed with three polymers, which could form the foundation for improved screening resources. Inkjet printing can be a useful tool in developing patterned substrates, such as polymer microarrays. A piezoelectric drop-on-demand printer was used to explore the methods in which these can be fabricated. A wettability assay using picolitre volumes was developed and used to characterise O2 plasma treatment of glass slides. Additionally, the printing of a cell-binding polymer using this approach enabled the decoration of cells with precise spatial resolution.

In my PhD polymer microarrays have been central in discovery of new materials for cardiovascular repair, cartilage tissue engineering and bacteria resistant medical devices. This has led to the work described in the following four chapters of my thesis. In the first part of my thesis polymers for the development of novel heart valve leaflets were identified. Diseased heart valves are currently replaced with the either synthetic or bioprosthetic (acellular xenografts) valve prostheses. While synthetic prosthesis have excellent durability, thromboembolic complications are frequent, requiring patients to undergo lifelong anti-coagulation therapy. On the other hand, the leaflets of bioprosthetic valves undergo structural deterioration, resulting in the patients having to undergo follow-up replacement surgeries. In order to overcome these shortcomings, the aim of this part of my PhD was to discover polymers that will enable the development of a ‘bio-synthetic’ heart valve, with the durability of synthetic valves and the biocompatibility of bioprosthetic vales. Polymers that bind valve interstitial cells (cells with a plastic fibroblast / myofibroblast phenotype that renew the extracellular matrix components of the valve leaflets) and also enable stable expression of key markers were identified. Immunohistochemistry and RNA expression analysis identified polymers for coating 3-D scaffolds, with the coated scaffolds showing excellent cell invasion, viability and maintenance of valve interstitial cell markers. To mimic the regions of the valve leaflets with differing stiffness, the response of valve interstitial cells to substrate stiffness was studied with various crosslinked gels. Thus, polymeric gels, prepared with the same chemical composition but with different Young’s modulus (covering 3 orders of magnitude) showed valve interstitial cell attachment with the cells showing differing behaviour based on the stiffness of the gels. In the second part of this thesis, polymers were identified for cartilage repair. Hyaline articular cartilage has very low potential for self-renewal, therefore cell-based therapies with autologous chondrocyte implantation are desired. Due to limited availability from biopsies, chondrocytes have to be expanded by in vitro culture; and fully defined synthetic culture substrates are essential for regulatory approvals. Using the high throughput approach I identified ‘hit’ polymers that allowed adhesion, proliferation and long-term culture of primary human chondrocytes and also chondrocytes derived from Mesenchymal stem cells. 2-D scale-up identified 2 lead polymers that supported long-term attachment and maintenance of chondrocyte markers. Since prolonged monolayer culture is known to induce loss of chondrocyte phenotype (dedifferentiation), 3D versions of the polymers were prepared and their potential for their long-term maintenance of chondrocytes via immunohistochemistry and RNA expression was demonstrated. The 3D gels were also used to encapsulate chondrocytes and their long-term maintenance of phenotype within these matrices, offers the exciting possibility of using these matrices for cartilage regeneration. The third part and fourth parts of the thesis focussed on reducing medical device associated infections. Thus polymers identified that prevented binding of a variety of bacteria including clinical isolates from infected medical devices, were used to coat two commercially available central venous catheters resulting in up to 96% reduction in bacterial binding. This non-binding was enhanced by the generation of polymeric nanocapsules containing the anti-bacterial eugenol (or its natural source clove oil). A coating consisting of eugenol nanocapsules entrapped within an interpenetrating network of the best bacteria repellent polymer, allowed slow-release of eugenol and further improved its performance.

La resposta del nostre cos als biomaterials suposa una gran obstacle per la efectivitat de múltiples teràpies basades en biomaterials. Accionats per la absorció inespecífica de biomolècules a la superfície del material, barreres com el sistema immune o les superfícies mucoses eliminen els materials del cos, evitant que arribin al seu destí i realitzin la seva funció. Els materials zwitteriònics han emergit en els últims anys com a materials antiadherents prometedors per a superar les mencionades barreres. Tot i que molts sistemes han utilitzat els materials zwitteriònics com a recobriments, les seves propietats úniques de superhidrofilicitat i versatilitat química suggereixen múltiples beneficis en utilitzar-los com a material principal. Aquí, dos sistemes diferents basats en materials zwitteriònics són presentats. En primer lloc una plataforma de alliberament de fàrmac antiadherent basat en copolímers de bloc amfifílics (CBA) és desenvolupada. Els CBAs zwitteriònics són sintetitzats i optimitzats perquè s’auto-organitzin en nanopartícules zwitteriòniques. Les propietats antiadherents d’aquestes nanopartícules es demostren, al igual que el seu potencial per a esdevenir un sistema d’alliberament de fàrmac oral. Seguidament, el sistema s’utilitza com a portador per a fàrmacs contra la malària i el càncer. Les nanopartícules mostren internalització en eritròcits infectats per Plasmòdium, i nanopartícules carregades amb curcumina demostren la seva eficàcia contra la malària in vitro. S’observa absorció oral de polímer i curcumina in vivo utilitzant un model de ratolí, indicant el potencial del sistema per a esdevenir una teràpia oral contra la malària. Quan s’optimitza el sistema per la teràpia contra el càncer, nanopartícules carregades de Paclitaxel exhibeixen activitat anti-cancerígena en models in vitro de cèl·lules canceroses. En segon lloc, microrobots zwitteriònics no-immunogènics que poden evitar el reconeixement per el sistema immune són introduïts. Es desenvolupa una fotoresistència zwitteriònica per a la microimpressió de microrobots zwitteriònics a través de la polimerització de dos fotons amb una ample funcionalització: propietats mecàniques variables, anti-bioadhesió i propietats no-immunogèniques, funcionalització per a la actuació magnètica, encapsulació de biomolècules i modificació superficial per a l’alliberament de fàrmac. Els robots invisibles eviten que els macròfags del sistema immune els detectin després d’una inspecció exhaustiva (de més de 90 hores), fet que no s’ha aconseguit fins el moment en cap sistema microrobòtic. Aquests materials zwitteriònics versàtils eliminen un dels grans obstacles en el desenvolupament de microrobots biocompatibles, i serviran com una caixa d’eines de materials no-immunogènics per a crear robots biomèdics i altres dispositius per a la bioenginyeria i per a aplicacions biomèdiques.
La respuesta de nuestro cuerpo a los biomateriales supone un gran obstáculo para la efectividad de múltiples terapias basadas en los biomateriales. Accionados por la absorción de biomoléculas en la superficie del material, barreras como el sistema inmune o las superficies mucosas eliminan los materiales del cuerpo, evitando que lleguen a su destino y realicen su función. Los materiales zwitteriónicos han emergido en los últimos años como materiales antiadherentes prometedores para superar las barreras mencionadas. Aunque muchos sistemas utilizan materiales zwitteriónicos como recubrimientos, sus propiedades únicas de superhidrofilicidad i versatilidad química sugieren múltiples beneficios en utilizarlos como material principal. Aquí, dos sistemas basados en materiales zwitteriónicos son presentados. En primer lugar, una plataforma para la liberación de fármaco antiadherente basada en copolímeros de bloque amfifílicos (CBA) es desarrollada. Los CBA zwitteriónicos son sintetizados y optimizados para que se auto-organicen en nanopartículas zwitteriónicas. Las propiedades antiadherentes de estas nanopartículas son probadas, al igual que su potencial para convertirse en un sistema oral de liberación de fármaco. Seguidamente, el sistema se utiliza como portador para fármacos animalarios y anticancerígenos. Las nanopartículas muestran internalización en eritrocitos infectados por Plasmodio, y nanopartículas cargadas con curcumina demuestran su eficacia contra la malaria in vitro. Se observa la absorción oral de polímero y curcumina in vivo utilizando un modelo de ratón, indicando el potencial del sistema para convertirse en una terapia oral contra malaria. Cuando se optimiza el sistema para la terapia contra el cáncer, las nanopartículas cargadas con Paclitaxel exhiben actividad anticancerígena en modelos in vitro de células cancerosas. En segundo lugar, microrobots zwitteriónicos no-inmunológicos que pueden evitar el reconocimiento por parte del sistema inmune son introducidos. Se desarrolla una fotoresisténcia zwitteriónica para la microimpresión de microrobots zwitteriónicos a través de la polimerización de dos fotones con una amplia funcionalización: propiedades mecánicas variables, anti-bioadhesión i propiedades no-inmunogénicas, funcionalización para la actuación magnética, encapsulación de biomoléculas i modificación superficial para la liberación de fármaco. Los robots invisibles evitan que los macrófagos del sistema inmune innato los detecten después de una inspección exhaustiva (de más de 90 horas), hecho que no se ha conseguido hasta la fecha por ningún sistema microrobótico. Estos materiales zwitteriónicos versátiles eliminan uno de los grandes obstáculos en el desarrollo de microrobots biocompatibles, y servirán como una caja de herramientas de materiales no-inmunogénicos para crear robots biomédicos y otros dispositivos para la bioingeniería y para las aplicaciones biomédicas.
Body response to biomaterials suppose a major roadblock for the effectiveness of multiple biomaterial-based therapies. Triggered by unspecific absorption of biomolecules in the material surface, barriers such as immune system or mucosal surfaces clear foreign materials from the body, preventing them to reach their target and perform their function. Zwitterionic materials have emerged in the last years as promising antifouling materials to overcome the mentioned barriers. Although many systems have used zwitterionic materials as coatings, the unique properties of superhydrophilicity and chemical versatility suggest multiple benefits of using zwitterionic polymers as bulk materials. Here, two different systems based on zwitterionic materials are presented. In first place, an antifouling drug delivery platform based on zwitterionic amphiphilic polymers (ABC) is developed. Zwitterionic ABCs are synthetized and optimized to self-assemble in zwitterionic nanoparticles. The antifouling properties of zwitterionic nanoparticles are proved, together with their potential to become an oral drug delivery system. Next, the system is used as a drug carrier for antimalarial and anticancer drugs. Nanoparticles show internalization in Plasmodium infected erythrocytes, and curcumin-loaded nanoparticles prove their antimalarial efficacy in vitro. Oral absorption of polymer and curcumin is also observed in vivo using mice model, indicating the potential of this system to become oral therapy against malaria. When optimizing the system for anticancer therapy, Paclitaxel-loaded nanoparticles exhibit anticancer activity in in vitro cancer cell models. Second, non‐immunogenic stealth zwitterionic microrobots that avoid recognition from immune cells are introduced. Zwitterionic photoresist are developed for the 3D microprinting of zwitterionic hydrogel microrobots through 2-photon polymerization with ample functionalization: tunable mechanical properties, anti-biofouling and non-immunogenic properties, functionalization for magnetic actuation, encapsulation of biomolecules, and surface functionalization for drug delivery. Stealth microrobots avoid detection by macrophage cells of the innate immune system after exhaustive inspection (> 90 h), which has not been achieved in any microrobotic platform to date. These versatile zwitterionic materials eliminate a major roadblock in the development of biocompatible microrobots, and will serve as a toolbox of non-immunogenic materials for medical microrobot and other device technologies for bioengineering and biomedical applications.

Biomedicine is an area in which computers have long been expected to play a significant role. Although many of the early claims have proved unrealistic, computers are gradually becoming accepted in the biomedical, clinical and research environment. Within these application areas, expert systems appear to have met with the most resistance, especially when applied to image interpretations. In order to improve the acceptance of computerised decision support systems it is necessary to provide the information needed to make rational judgements concerning the inferences the system has made. This entails an explanation of what inferences were made, how the inferences were made and how the results of the inferences are to be interpreted. Furthermore there must be a consistent approach to the combining of information from low level computational processes through to high level expert analyses. Until recently ad hoc formalisms were seen as the only tractable approach to reasoning under uncertainty. A review of some of these formalisms suggests that they are less than ideal for the purposes of decision making. Belief networks provide a tractable way of utilising probability theory as an inference formalism by combining the theoretical consistency of probability for inference and decision making, with the ability to use the knowledge of domain experts. The potential of belief networks in biomedical applications has already been recognised and there has been substantial research into the use of belief networks for medical diagnosis and methods for handling large, interconnected networks. In this thesis the use of belief networks is extended to include detailed image model matching to show how, in principle, feature measurement can be undertaken in a fully probabilistic way. The belief networks employed are usually cyclic and have strong influences between adjacent nodes, so new techniques for probabilistic updating based on a model of the matching process have been developed. An inference shell called FLAPNet has been implemented and used to apply the belief network formalism to the tasks of model based image matching and the incremental aggregation of disparate information for diagnosis. The domains of application are fetal ultrasound imaging and cervical screening respectively. It is argued that belief networks combine the necessary quantitative features required of a decision support system with desirable qualitative features that will lead to improved acceptability of expert systems in the biomedical domain.

La télémétrie biomédicale et l’interfaçage neuronal à base de dispositifs miniatures et autonomes sans fil constituent de nouvelles applications en émergence. Elles visent à répondre à de nombreux enjeux y compris dans les domaines de la santé, du sport et bien être, ou encore de la sécurité au travail et de la défense. Parmi les applications typiques de biotélémétrie, nous pouvons citer le monitoring de certains paramètres physiologiques : température corporelle, pression artérielle, rythme cardiaque, taux de glucose et d’anticorps, détection d’agents chimiques, etc. En ce qui concerne l’interfaçage neuronal, il permet de restaurer les informations sensorielles, d’aider à la réadaptation des amputés, des personnes atteintes de paralysie ou des patients atteints de maladies neurodégénératives. L’objectif principal de cette thèse est de contribuer au développement de dispositifs miniaturisés et communicants pour le monitoring, en continu, de variables physiologiques d’humains ainsi que d’animaux. Ces dispositifs innovants nécessitent un système de communication fiable. Plus particulièrement, il s’agit d’analyser le milieu de propagation à l’intérieur des tissus biologiques et de développer des antennes miniatures innovantes ainsi que des méthodes pour leur analyse et leur caractérisation. Le verrou majeur concerne le rendement des antennes miniatures. Les effets de forte hétérogénéité, dispersion, pertes très élevées des milieux biologiques et les contraintes de miniaturisation et d’intégration dans des dispositifs in-body limitent la portée des systèmes existants à quelques dizaines de centimètres. Tout d’abord, des outils spécifiques de modélisation et d’optimisation ont été développés en collaboration avec l’Université de Bohème de l’Ouest. Ces outils sont indispensables pour l’analyse des composants de systèmes antennaires complexes : le code Agros2D (CAO interne) utilise des méthodes entièrement adaptatives. Cette approche permet de réduire la complexité d’optimisation des antennes in-body jusqu’un seul dégrée de liberté. Puis, la limite fondamentale de rendement des antennes pour les applications in-body a été définie ; les liens entre cette limite et la taille de l’antenne, sa fréquence de fonctionnement, la polarisation et les matériaux utilisés (dont hypothétiques) ont été quantifiés pour la première fois. Ce travail fondamental a d’abord pour objectif l’optimisation des performances de l’antenne actuelle de la capsule e-Celsius de l’entreprise BodyCAP pour accroître la portée de la gélule, en prenant en compte les caractéristiques des matériaux et le milieu de propagation que constituent les tissus biologiques. Dans cette étape on inclut également la fabrication des prototypes de gélules télémétriques ainsi que leurs mesures d’impédance. L’antenne optimisée a une portée trois fois plus importante que celle actuelle tout en occupant le même volume. En utilisant ces principes de conception, nous avons développé et caractérisé une antenne à 434 MHz adaptée à une large gamme d'applications in-body. Des dimensions ultra-miniatures, une robustesse et un rendement accrus permettent de l'utiliser à la fois pour des applications des capsules à implanter et à avaler. Enfin, en développant davantage les méthodes de conception et d’optimisation, nous avons conçu une antenne double-bande. Ayant la même robustesse que son équivalent actuel mono-bande, elle présente également un rendement encore plus élevé, permettant ainsi de fonctionner au-delà de 10 m. La caractéristique double-bande permet de concevoir les dispositifs in-body rechargeables sans fil dans le corps. Les antennes proposées contribuent au développement ultérieur d'une nouvelle génération de dispositifs miniatures in-body qui impliquent une intégration complexe et dense des capteurs, de la logique et de la source d'alimentation
Emerging wireless biotelemetry using miniature implantable, ingestible or injectable (in-body) devices allows continuously monitor and yield human or animal physiological parameters while maintaining mobility and quality of life. Recent advances in microelectromechanical systems and microfluidics—along with ongoing miniaturization of electronics—have empowered numerous innovations in biotelemetry devices, creating new applications in medicine, clinical research, wellness, and defense. Among the typical applications, I can mention, for example, the monitoring of physiological variables: body temperature, blood pressure, heart rate, detection of antibodies, chemical, or biological agents. Biotelemetry devices require a reliable communication system: robust, efficient, and versatile. Improving the transmission range of miniature in-body devices remains a major challenge: for the time being, they are able to operate only up to a few meters. Among the main issues to face are low radiation efficiencies (< 0.1%), antenna impedance detuning, and strong coupling to lossy and dispersive biological tissues. Thus, the main goal of the thesis is to conduct a multi-disciplinary study on development, optimization and characterization of antennas for in-body biotelemetry devices. After state-of-the-art and the context, I start with the development on both physical and numerical approaches to account for the effect of human tissues on the antenna. I propose the methodology to achieve given electromagnetic properties at a given frequency based on the full factorial experiment and surface response optimization. In addition, I describe the spherical physical phantom for the far-field characterization along with a combination of feed decoupling techniques. I proceed by reviewing the trough-body propagation mechanisms and deriving the optimal frequency for the in-body devices. I formulate the problem using four phantoms (homogeneous and heterogeneous) and perform full-wave analysis using an in-house hp-FEM code Agros 2D. Next, I study the existing antenna used by the BodyCap Company for its e-Celsius capsule and the ways on how to improve its operating range and robustness under strict integration and material constraints. The mechanisms of antenna–body coupling are analyzed and the found solution improves the antenna IEEE gain by 11 dBi (the operating range is at least tripled). The existing matching circuit and balun are optimized too for the given application reducing its size from eleven to seven discrete elements. In the following chapters, I continue studying the decoupling of antennas from a body using specific microstrip designs and dielectric loading via capsule shell. By applying the developed approaches, a high robustness and radiation efficiency can be achieved. At first, I develop a proof-of-concept antenna that demonstrates that the perfect matching (detuning immunity) is achievable for the operation within all human tissues. Based on these results, I develop a miniature and versatile biotelemetry platform: a 17 mm x 7 mm alumina capsule containing a conformal 434 MHz antenna. The antenna is well matched to 50 Ohm within the majority of human tissues and operates with an arbitrary device circuitry. Like this, one can use it ''as is,'' applying it for a wide range of in-body applications. Then, I develop a low profile conformal dual-band antenna operating in 434 MHz and 2.45 GHz bands. Such antenna can integrate both data transmission and wireless powering functionality increasing the available space inside an in-body device and increasing its scope of applications. Finally, I present the perspective developments including in-body sensing methodology. The obtained results contributes to further development of a new generation of miniature in-body devices that involve complex and dense integration of sensors, logic, and power sources

Doutoramento em Engenharia Informática
A exigente inovação na área das aplicações biomédicas tem guiado a evolução das tecnologias de informação nas últimas décadas. Os desafios associados a uma gestão, integração, análise e interpretação eficientes dos dados provenientes das mais modernas tecnologias de hardware e software requerem um esforço concertado. Desde hardware para sequenciação de genes a registos electrónicos de paciente, passando por pesquisa de fármacos, a possibilidade de explorar com precisão os dados destes ambientes é vital para a compreensão da saúde humana. Esta tese engloba a discussão e o desenvolvimento de melhores estratégias informáticas para ultrapassar estes desafios, principalmente no contexto da composição de serviços, incluindo técnicas flexíveis de integração de dados, como warehousing ou federação, e técnicas avançadas de interoperabilidade, como serviços web ou LinkedData. A composição de serviços é apresentada como um ideal genérico, direcionado para a integração de dados e para a interoperabilidade de software. Relativamente a esta última, esta investigação debruçou-se sobre o campo da farmacovigilância, no contexto do projeto Europeu EU-ADR. As contribuições para este projeto, um novo standard de interoperabilidade e um motor de execução de workflows, sustentam a sucesso da EU-ADR Web Platform, uma plataforma para realizar estudos avançados de farmacovigilância. No contexto do projeto Europeu GEN2PHEN, esta investigação visou ultrapassar os desafios associados à integração de dados distribuídos e heterogéneos no campo do varíoma humano. Foi criada uma nova solução, WAVe - Web Analyses of the Variome, que fornece uma coleção rica de dados de variação genética através de uma interface Web inovadora e de uma API avançada. O desenvolvimento destas estratégias evidenciou duas oportunidades claras na área de software biomédico: melhorar o processo de implementação de software através do recurso a técnicas de desenvolvimento rápidas e aperfeiçoar a qualidade e disponibilidade dos dados através da adopção do paradigma de web semântica. A plataforma COEUS atravessa as fronteiras de integração e interoperabilidade, fornecendo metodologias para a aquisição e tradução flexíveis de dados, bem como uma camada de serviços interoperáveis para explorar semanticamente os dados agregados. Combinando as técnicas de desenvolvimento rápidas com a riqueza da perspectiva "Semantic Web in a box", a plataforma COEUS é uma aproximação pioneira, permitindo o desenvolvimento da próxima geração de aplicações biomédicas.
The demand for innovation in the biomedical software domain has been an information technologies evolution driver over the last decades. The challenges associated with the effective management, integration, analyses and interpretation of the wealth of life sciences information stemming from modern hardware and software technologies require concerted efforts. From gene sequencing hardware to pharmacology research up to patient electronic health records, the ability to accurately explore data from these environments is vital to further improve our understanding of human health. This thesis encloses the discussion on building better informatics strategies to address these challenges, primarily in the context of service composition, including warehousing and federation strategies for resource integration, as well as web services or LinkedData for software interoperability. Service composition is introduced as a general principle, geared towards data integration and software interoperability. Concerning the latter, this research covers the service composition requirements within the pharmacovigilance field, namely on the European EU-ADR project. The contributions to this area, the definition of a new interoperability standard and the creation of a new workflow-wrapping engine, are behind the successful construction of the EUADR Web Platform, a workspace for delivering advanced pharmacovigilance studies. In the context of the European GEN2PHEN project, this research tackles the challenges associated with the integration of heterogeneous and distributed data in the human variome field. For this matter, a new lightweight solution was created: WAVe, Web Analysis of the Variome, provides a rich collection of genetic variation data through an innovative portal and an advanced API. The development of the strategies underlying these products highlighted clear opportunities in the biomedical software field: enhancing the software implementation process with rapid application development approaches and improving the quality and availability of data with the adoption of the Semantic Web paradigm. COEUS crosses the boundaries of integration and interoperability as it provides a framework for the flexible acquisition and translation of data into a semantic knowledge base, as well as a comprehensive set of interoperability services, from REST to LinkedData, to fully exploit gathered data semantically. By combining the lightness of rapid application development strategies with the richness of its "Semantic Web in a box" approach, COEUS is a pioneering framework to enhance the development of the next generation of biomedical applications.

Doutoramento em Ciência e Engenharia de Materiais
The increased longevity of humans and the demand for a better quality of life have led to a continuous search for new implant materials. Scientific development coupled with a growing multidisciplinarity between materials science and life sciences has given rise to new approaches such as regenerative medicine and tissue engineering. The search for a material with mechanical properties close to those of human bone produced a new family of hybrid materials that take advantage of the synergy between inorganic silica (SiO4) domains, based on sol-gel bioactive glass compositions, and organic polydimethylsiloxane, PDMS ((CH3)2.SiO2)n, domains. Several studies have shown that hybrid materials based on the system PDMS-SiO2 constitute a promising group of biomaterials with several potential applications from bone tissue regeneration to brain tissue recovery, passing by bioactive coatings and drug delivery systems. The objective of the present work was to prepare hybrid materials for biomedical applications based on the PDMS-SiO2 system and to achieve a better understanding of the relationship among the sol-gel processing conditions, the chemical structures, the microstructure and the macroscopic properties. For that, different characterization techniques were used: Fourier transform infrared spectrometry, liquid and solid state nuclear magnetic resonance techniques, X-ray diffraction, small-angle X-ray scattering, smallangle neutron scattering, surface area analysis by Brunauer–Emmett–Teller method, scanning electron microscopy and transmission electron microscopy. Surface roughness and wettability were analyzed by 3D optical profilometry and by contact angle measurements respectively. Bioactivity was evaluated in vitro by immersion of the materials in Kokubos’s simulated body fluid and posterior surface analysis by different techniques as well as supernatant liquid analysis by inductively coupled plasma spectroscopy. Biocompatibility was assessed using MG63 osteoblastic cells. PDMS-SiO2-CaO materials were first prepared using nitrate as a calcium source. To avoid the presence of nitrate residues in the final product due to its potential toxicity, a heat-treatment step (above 400 °C) is required. In order to enhance the thermal stability of the materials subjected to high temperatures titanium was added to the hybrid system, and a material containing calcium, with no traces of nitrate and the preservation of a significant amount of methyl groups was successfully obtained. The difficulty in eliminating all nitrates from bulk PDMS-SiO2-CaO samples obtained by sol-gel synthesis and subsequent heat-treatment created a new goal which was the search for alternative sources of calcium. New calcium sources were evaluated in order to substitute the nitrate and calcium acetate was chosen due to its good solubility in water. Preparation solgel protocols were tested and homogeneous monolithic samples were obtained. Besides their ability to improve the bioactivity, titanium and zirconium influence the structural and microstructural features of the SiO2-TiO2 and SiO2-ZrO2 binary systems, and also of the PDMS-TiO2 and PDMS-ZrO2 systems. Detailed studies with different sol-gel conditions allowed the understanding of the roles of titanium and zirconium as additives in the PDMS-SiO2 system. It was concluded that titanium and zirconium influence the kinetics of the sol-gel process due to their different alkoxide reactivity leading to hybrid xerogels with dissimilar characteristics and morphologies. Titanium isopropoxide, less reactive than zirconium propoxide, was chosen as source of titanium, used as an additive to the system PDMS-SiO2-CaO. Two different sol-gel preparation routes were followed, using the same base composition and calcium acetate as calcium source. Different microstructures with high hydrophobicit were obtained and both proved to be biocompatible after tested with MG63 osteoblastic cells. Finally, the role of strontium (typically known in bioglasses to promote bone formation and reduce bone resorption) was studied in the PDMS-SiO2-CaOTiO2 hybrid system. A biocompatible material, tested with MG63 osteoblastic cells, was obtained with the ability to release strontium within the values reported as suitable for bone tissue regeneration.
O aumento da longevidade dos seres humanos e a procura de uma melhor qualidade de vida têm conduzido a uma pesquisa contínua de novos materiais para implantes. O desenvolvimento científico, juntamente com uma crescente multidisciplinaridade entre as ciências dos materiais e as ciências da vida deram origem a novas abordagens, como a medicina regenerativa e a engenharia de tecidos. A busca de um material com propriedades mecânicas próximas das do osso humano produziu uma nova família de materiais híbridos que tiram partido da sinergia entre os domínios inorgânicos de sílica (SiO4), com base em composições de vidros bioativos obtidos por sol-gel, e os domínios orgânicos de polidimetilsiloxano, PDMS ((CH3)2.SiO2)n. Vários estudos têm demonstrado que os materiais híbridos baseados no sistema PDMS-SiO2 constituem um grupo de biomateriais promissores com várias aplicações potenciais tais como a regeneração de tecido ósseo e a recuperação do tecido cerebral, passando por revestimentos bioativos e sistemas de libertação controlada de fármacos. O objetivo do presente trabalho foi preparar materiais híbridos para aplicações biomédicas com base no sistema PDMS-SiO2 e contribuir para uma melhor compreensão das relações entre as condições de processamento sol-gel, as estruturas químicas, a microestrutura e as propriedades macroscópicas. Para alcançar tal objetivo, foram usadas diferentes técnicas de caracterização: espectroscopia de infravermelho por transformada de Fourier, ressonância magnética nuclear no estado sólido e no estado líquido, difração de raios-X, dispersão de raios-X de baixo ângulo, dispersão de neutrões de baixo ângulo, análise da área de superfície pelo método de Brunauer–Emmett–Teller, microscopia eletrónica de varrimento e microscopia eletrónica de transmissão. A rugosidade e a molhabilidade das superfícies foram analisadas por perfilometria óptica 3D e por medidas de ângulo de contacto, respectivamente. A bioatividade in vitro foi avaliada através de testes de imersão em plasma sintético e posterior observação da superfície dos materiais e análise do líquido sobrenadante por espectrometria de emissão atômica por plasma acoplado Indutivamente. A biocompatibilidade in vitro foi avaliada usando células osteoblásticas MG63. Materiais do sistema PDMS-SiO2-CaO foram inicialmente preparados usando o nitrato como fonte de cálcio. Para eliminar os resíduos de nitrato no produto final, devido à sua potencial toxicidade, é necessária uma etapa de tratamento térmico (acima dos 400° C). A fim de aumentar a estabilidade térmica dos materiais submetidos a altas temperaturas, foi adicionado titânio ao sistema híbrido. Obteve-se assim um material híbrido contendo cálcio, sem vestígios de nitrato, mantendo-se uma quantidade significativa de grupos metilo. A dificuldade de obter amostras monolíticas de híbridos PDMS-SiO2-CaO por síntese sol-gel e posterior tratamento térmico para eliminação de nitratos, criou um novo objetivo: a procura de fontes alternativas de cálcio. Novas fontes de cálcio foram avaliadas para substituir o nitrato tendo-se escolhido o acetato de cálcio devido à sua boa solubilidade em água. Estabeleceram-se protocolos de preparação por sol-gel a partir dos quais se obtiveram amostras monolíticas homogéneas. Além de melhorar a bioatividade, o titânio e o zircónio influenciam as características estruturais e microestruturais dos sistemas binários SiO2-TiO2 e SiO2-ZrO2, bem como dos sistemas PDMS-TiO2 e PDMS-ZrO2. Neste contexto, foram estudadas diferentes condições experimentais no processo sol-gel, de modo a compreender o papel destes aditivos no sistema SiO2-PDMS. Concluiu-se que o titânio e o zircónio influenciam a cinética do processo sol-gel devido à diferente reatividade dos despectivos alcóxidos, conduzindo à obtenção de xerogéis híbridos com diferentes características e morfologias. O isopropóxido de titânio, menos reativo do que o propóxido de zircónio, foi escolhido como fonte de titânio, usado como aditivo no sistema PDMS-SiO2CaO. Dois procedimentos diferentes de preparação por sol-gel foram seguidos, utilizando a mesma composição de base e o acetato de cálcio como fonte de cálcio. Foram obtidas diferentes microestruturas muito hidrofóbicas e ambas mostraram ser biocompatíveis após serem testadas com células osteoblásticas MG63. Finalmente, foi avaliado o papel do estrôncio (conhecido nos biovidros por favorecer a formação de tecido ósseo e reduzir a sua reabsorção) no sistema híbrido PDMS-CaO-SiO2-TiO2. O material produzido revelou-se biocompatível, através de testes com células osteoblásticas MG63, e com a capacidade de libertar estrôncio dentro dos limites considerados adequados para a reparação do tecido ósseo.

Recently, the interest in implantable devices for biomedical telemetry has significantly increased. Amongst the different components of the implantable device, the antenna plays the most significant role in the wireless data transmission. However, the human body around the antenna alters its overall characteristics and absorbs most of its radiation. Therefore, this thesis is mainly focused on improving the antenna characteristics (bandwidth and radiation efficiency) to overcome the human body effect and investigating new structures that reduce the power absorption by the human body tissues. A novel antenna design methodology is developed and used to design new flexible implantable antennas of much lighter weight, larger radiation efficiency, and wider bandwidth than existing embedded antennas. These antennas work for multiple ((401-406 MHz) MedRadio, 433 MHz and 2.45 GHz ISM) bands which satisfy the requirements of low power consumption and wireless power transfer. This has been combined with thorough investigations of the antenna performance in the anatomical human body. New effective evaluation parameters such as the antenna orientation are investigated for the first time. New structures inspired by complementary and multiple split ring resonators (CSRRs and MSRRs) are designed. The structures are found to reduce the electric near field and hence the absorbed power which increases the radiated power accordingly. This new promising function of metamaterial based structures for implantable applications is investigated for the first time. The path loss (between pacemaker and glucose monitoring implantable antennas inside the anatomical body model) and (between an implantable and external antennas for a wireless power channel at 433 MHz) are estimated. Moreover, the optimum antenna type for on-in body communication is investigated. Loop antennas are found to outperform patch antennas in close proximity to the human body.

The main aim of this project was to assess auxetic (negative Poisson's ratio) materials for potential in biomedical devices. Specifically, a detailed comparative indentation study has been undertaken on auxetic and conventional foams for hip protector devices; radially-gradient one-piece foams having auxetic character have been produced for the first time and shown to have potential in artificial intervertebral disc (IVD) implant devices; and auxetic honeycomb geometries have been assessed for the stem component in hip implant devices. For the hip protector application, combined compression and heat treatment of conventional polyurethane open-cell foam was used to produce monolithic auxetic foams. The foams were characterised structurally using optical microscopy, and mechanically using mechanical testing combined with videoextensometry. Static indentation using six different indenter shapes on each of the six faces of the foam specimens has been undertaken. The key conclusion here is that the enhanced indentation resistance for the converted foam is not a consequence of increased density accompanied by the usual significant increase in foam stiffness. The enhanced indentation resistance is consistent with the auxetic effect associated with the increased density, providing a localised densification mechanism under indentation (i.e. material flows under the indenter). At higher indentation displacement the Poisson’s ratios for both the unconverted and converted foams tend towards zero. In this case, the increase in foam stiffness for the converted foams at higher strain may also contribute to the indentation enhancement at high indentation displacement. New radially-gradient foams mimicking the core-sheath structure of the natural IVD have been produced through the development of a novel thermo-mechanical manufacturing route. Foam microstructural characterisation has been undertaken using optical and scanning electron microscopy, and also micro-CT scans performed by collaborators at the University of Manchester. Detailed x-y strain mapping using combined mechanical testing and videoextensometry enabled the local and global Young's modulus and Poisson's ratio responses of these new materials to be determined. In one example, global auxetic response is achieved in a foam having a positive Poisson's ratio core and auxetic sheath. It is suggested this may be a more realistic representation of the properties of natural IVD tissue. Analytical and Finite Element (FE) models have been developed to design honeycomb geometries for the stems in new total hip replacement implants. FE models of the devices implanted within bone have been developed and the auxetic stems shown to lead to reduced stress shielding effect.

Cette thèse décrit le développement de nouvelles méthodes pour obtenir des nanoparticules fonctionnelles polyvalentes qui peuvent potentiellement être utilisées pour des applications biomédicales telles que la vectorisation de médicaments, des essais biologiques et la bio-imagerie. Les nanomatériaux sont des outils polyvalents qui ont trouvé des applications comme vecteurs de médicaments, la bio-imagerie ou les biocapteurs. En particulier, les nanoparticules de type core-shell ont attiré beaucoup d'attention en raison de leur petite taille, une relation surface/volume élevée, et une biocompatibilité. Dans ce contexte, nous proposons dans la première partie de la thèse (Chapitre 2), une nouvelle méthode pour obtenir des nanoparticules core-shell via la polymérisation radicalaire en émulsion et vivante combinées. Des particules cœurs de polystyrène de 30 à 40 nm, avec une distribution de taille étroite et portant à la surface des groupements iniferter ont été utilisés pour amorcer la polymérisation supplémentaire d'une couche de polymère. Des nanoparticules core-shell ont été préparées de cette façon. Différents types d’enveloppes : anionique, zwitterioniques, à empreintes moléculaires, thermosensibles, ont ainsi été greffées. Notre méthode est une plate-forme polyvalente permettant d'ajouter des fonctionnalités multiples soit dans le noyau et/ou l'enveloppe pour les études d'interaction cellulaire et de toxicité, ainsi que des matériaux récepteurs pour l'imagerie cellulaire. Dans la deuxième partie de la thèse (Chapitre 3), nous décrivons un procédé nouveau et polyvalent pour la modification de surface des nanoparticules de conversion ascendante (UCP). Ce sont des nanocristaux fluorescents dopés de lanthanides qui ont récemment attiré beaucoup d'attention. Leur fluorescence est excitée dans le proche infrarouge, ce qui les rend idéales comme marqueurs dans des applications biomédicales telles que les tests biologiques et la bio-imagerie, l'auto-fluorescence étant réduite par rapport à des colorants organiques et les quantum dots. Cependant, les UCP sont hydrophobes et non-compatible avec les milieux aqueux, donc une modification de leur surface est essentielle. La stratégie que nous proposons utilise l'émission UV ou visible après excitation en proche infrarouge des UCP, comme source de lumière secondaire pour la photopolymérisation localisée de couches minces hydrophiles autour les UCP. Notre méthode offre de grands avantages comme la facilité d'application et la fonctionnalisation de surface rapide pour fixer divers ligands, et fournit une plateforme pour préparer des UCP encapsulée de polymères pour des différentes applications. Des hydrogels stimuli-sensibles sont des matériaux qui changent leurs propriétés physicochimiques en réponse à des stimuli externes tels que la température, le pH ou la lumière. Ces matériaux intelligents jouent un rôle critique dans des applications biomédicales telles que la vectorisation de médicaments ou l'ingénierie tissulaire. La troisième partie de cette thèse (Chapitre 4) propose un nouveau procédé de préparation d'hydrogels photo et pH sensible. Deux composantes, l'un photosensible à base dl'acide 4-[(4-méthacryloyloxy) phénylazo] benzoïque et l'autre cationic contenant des unités 2-(diéthylamino)éthyl méthacrylate, ont été synthétisés. Leur association donne des particules monodispersées de 100 nm photo et pH sensibles. Ces nanoparticules peuvent être potentiellement utilisées pour la vectorisation de médicaments, en particulier de biomolécules telles que protéines ou siARN. En conclusion, nous avons conçu plusieurs nouvelles méthodes efficaces, polyvalentes, génériques et facilement applicables pour obtenir des nanoparticules et nanocomposites de polymères fonctionnels qui peuvent être appliqués dans de différents domaines biomédicaux comme la vectorisation de médicaments, les biocapteurs, les tests biologiques et la bio-imagerie
This thesis describes the development of novel methods to obtain versatile, functional nanoparticles that can potentially be used for biomedical applications such as drug delivery, bioassays and bioimaging. Nanomaterials are versatile tools that have found applications as drug carriers, bioimaging or biosensing. In particular, core-shell type nanoparticles have attracted much attention due to their small size, high surface to volume ratio and biocompatibility. In this regard, we propose in the first part of the thesis (Chapter 2), a novel method to obtain core-shell nanoparticles via combined radical emulsion and living polymerizations. Polystyrene core seeds of 30-40 nm, with a narrow size distribution and surface-bound iniferter moieties were used to further initiate polymerization of a polymer shell. Core-shell nanoparticles were prepared in this way. Different types of shells : anionic, zwitterionic, thermoresponsive or molecularly imprinted shells, were thus grafted. Our method is a versatile platform with the ability to add multi-functionalities in either the core for optical sensing or/and the shell for cell interaction and toxicity studies, as well as receptor materials for cell imaging. In the second part of the thesis (Chapter 3), we describe a novel and versatile method for surface modification of upconverting nanoparticles (UCPs). UCPs are lanthanide-doped fluorescent nanocrystals that have recently attracted much attention. Their fluorescence is excitated in the near infrared, which makes them ideal as labels in biomedical applications such as bioimaging and bioassays, since the autofluorescence background is minimized compared to organic dyes and quantum dots. However, UCPs are hydrophobic and non-compatible with aqueous media, therefore prior surface modification is essential. The strategy that we propose makes use oft he UV or Vis emission light of near-infrared photoexcited upconverting nanoparticles, as secondary light source for the localized photopolymerization of thin hydrophilic shells around the UCPs. Our method offers great advantages like ease of application and rapid surface functionalization for attaching various ligands and therefore can provide a platform to prepare polymeric-encapsulated UCPs for applications in bioassays, optical imaging and drug delivery. Stimuli responsive hydrogels are materials that can change their physico-chemical properties in response to external stimuli such as temperature, pH or light. These smart materials play critical roles in biomedical applications such as drug delivery or tissue engineering. The third part of the thesis (Chapter 4) proposes a novel method for obtaining photo and pH-responsive supramolecularly crosslinked hydrogels. Two building blocks, one containing photoresponsive 4-[(4-methacryloyloxy)phenylazo] benzoic acid and the other, consisting of cationic 2-(diethylamino)ethyl methacrylate units, were first synthesized. Combining the two building blocks yielded photo and pH responsive monodisperse 100-nm particles. These nanoparticles can be eventually utilized for drug delivery, especially delivery of biomolecules such as siRNAs or proteins. In conclusion, we have designed several new efficient, versatile, generic and easily applicable methods to obtain functionalized polymer nanoparticles and nanocomposites that can be applied in various biomedical domains like drug delivery, biosensing, bioassays and bioimaging

The application of mass spectrometry to verification of the structure of 3-methyluridine (m³U) isolated by HPLC from normal human urine is described. m³U has been used as an internal standard for studies of urinary nucleosides, a practice that is discouraged with the confirmation of m³U as a naturally occurring compound. Mass spectrometry has been used for the identification of 5'-deoxyxanthosine (5'-dX) a novel nucleoside in normal human urine. Initial concern over availability of a reference sample of 5'-dX prompted investigations of the structure/fragmentation relationships of the TMS deratives of 2'-, 3'-, and 5'-deoxynucleosides toward differentiation between the three deoxynucleosides. Results are presented which allow discrimination between the model compounds, deoxyanalogs of adenosine. Subsequent to the deoxynucleoside fragmentation studies, a biosynthetically produced reference sample of 5'-dX became available for direct comparison of mass spectra and chromatographic retention times which, when combined with observations from the deoxynucleoside studies established the structure of 5'-dX. In response to the large number of mass spectra produced from the GC-MS analysis of a TMS derivatized urine sample, computer software has been written to aid in spectral analysis. Examples are shown in which the software uses established fragmentation rules to assign structure to ions in the mass spectrum and suggest modifications in the sugar portion of two urinary nucleosides. The structure/fragmentation relationships of the unique antitumor drug taxol has been studied by EI, CI and FAB mass spectrometry. Information is presented showing characteristic fragmentation of the side-chain and verification of functional groups attached to the taxane ring. Studies have been conducted to determine the relationship between target temperature and matrix and sample lifetime in the source of the mass spectrometer. Results are presented showing that cooling the target permits the use of matrix materials that are too volatile at ambient temperatures thus extending the range of compounds that can be studied by mass spectrometry. A recently constructed four-sector mass spectrometer is described with a detailed discussion of instrumental capabilities. Results of experiments designed to apply these capabilities to the structural analysis of TMS nucleosides using FAB ionization are discussed with an emphasis on the fragmentation unique to 4-sector daughter ion experiments compared with conventional studies and 2-sector daughter ion results.

Acousto-electric (AE) effect comes from an interaction between electrical current and acoustic pressure generated when acoustic waves travel through a conducting material. It currently has two main application areas, ultrasound current source density imaging (UCSDI) and AE hydrophone. UCSDI can detect the current direction by modulating the dipole field with ultrasound pulse, and it is now used to form 3D imaging of dipole changing in one period of treatment, such as arrhythmia in the heart and epilepsy in the brain. As ultrasound pulse passes through electrical field, it convolutes or correlates with the inner product of the electric fields formed by the dipole and detector. The polarity of UCSDI is not determined by Doppler effect that exists in pulse echo (PE) signal, but the gradient of lead field potentials created by dipole and recording electrode, making the base-banded AE voltage positive at the anode and negative at cathode. As convolution shifts spectrum lower, the base band frequency for polarity is different from the center frequency of AE signal. The simulation uses the principles of UCSDI, and helps to understand the phenomena in the experiment. 3-D Fast Fourier Transform accelerates the computing velocity to resolve the correlation in the simulation of AE signal. Most single element hydrophones depend on a piezoelectric material that converts pressure changes to electricity. These devices, however, can be expensive, susceptible to damage at high pressure, and/or have limited bandwidth and sensitivity. An AE hydrophone requires only a conductive material and can be constructed out of common laboratory supplies to generate images of an ultrasound beam pattern consistent with more expensive hydrophones. Its sensitivity is controlled by the injected bias current, hydrophone shape, thickness and width of sensitivity zone. The design of this device needs to be the tradeoff of these parameters. Simulations were made to optimize the design with experimental validation using specifically fabricated devices composed of a resistive element of indium tin oxide (ITO).

Les propriétés des nanozéolithes, à savoir leur grande surface, leur stabilité hydrothermale et leur nature non toxique, permettent leur utilisation dans des applications prospectives, notamment la biomédecine (capteurs, administration de médicaments et de gaz) et la microbiologie (agents antibactériens). De nombreuses recherches ont été consacrées à l’étude de nouvelles applications biomédicales utilisant des matériaux zéolithiques, toutefois leur plein potentiel n’a toujours pas été pleinement dévoilé.Il est bien connu que la résistance croissante aux traitements établis de tumeurs et d’infections bactériennes par radiothérapie et antibiotiques est un problème de première importance. Par conséquent, le développement de nouvelles stratégies thérapeutiques pour résoudre ces problèmes est très démandé.L'objectif de cette recherche de doctorat est de synthétiser et de modifier post-synthétiquement des zéolithes nanométriques pour des applications biomédicales. Cela implique l'échange d'ions de zéolithe avec divers cations pour trouver celui qui convient le mieux aux applications souhaitées : le traitement antimicrobien, la réoxygénation des tissus tumoraux et l’administration de gaz.Dans cette étude, nous rapportons: (i) l'effet de la zéolithe FAU de type nanométrique modifiée au cuivre sur les bactéries de type ESKAPE (chapitre 3), (ii) l’utilisation de nanozéolithes contenant du métal comme outil d'oxygénation et de visualisation tissulaire (chapitre 4) et enfin (iii) l'utilisation de nanozéolithes FAU comme vecteur de l'oxyde nitrique et du dioxyde de carbone pour prévenir des maladies potentiellement létales (chapitre 5)
The properties of nanozeolites, namely, large surface area, hydrothermal stability and non-toxic nature, enable their utilization in forward-looking applications, including biomedicine (sensors, drug and gas delivery) and microbiology (antibacterial agents). Hence, a lot of research has been devoted to study the new biomedical applications using zeolitic materials, their full potential has still not been fully unveiled.It is well recognised that growing resistance to already established treatments of tumors and bacterial infections using radiotherapy and antibiotics is a distressing matter. Therefore, the development of new therapeutic strategies towards above issues is of great demand.The goal of this PhD research is to synthesise and post-synthetically modify nanosized zeolites for biomedical applications. This involves the ion-exchange of zeolite with various cations to find the most suitable one for desired applications in regards to antimicrobial treatment, tumour tissue reoxygenation and gas delivery.In this study, we report: (i) the effect of copper modified nanosized FAU type zeolite on ESKAPE type bacteria (Chapter 3), (ii) the metal containing nanozeolite as a tool for tissue oxygenation and visualisation using MRI (Chapter 4), and lastly (iii) the use of FAU nanozeolite as nitric oxide and carbon dioxide gas vector to prevent life threatening conditions (Chapter 5)

Objects with complex shape and functions have always attracted attention and interest. The morphological diversity and complexity of naturally occurring forms and patterns have been a motivation for humans to copy and adopt ideas from Nature to achieve functional, aesthetic and social value. Biomimetics is addressed to the design and development of new synthetic materials using strategies adopted by living organisms to produce biological materials. In particular, biomineralized tissues are often sophisticate composite materials, in which the components and the interfaces between them have been defined and optimized, and that present unusual and optimal chemical-physical, morphological and mechanical properties. Moreover, biominerals are generally produced by easily traceable raw materials, in aqueous media and at room pressure and temperature, that is through cheap process and materials. Thus, it is not surprising that the idea to mimic those strategies proper of Nature has been employed in several areas of applied sciences, such as for the preparation of liquid crystals, ceramic thin films computer switches and many other advanced materials. On this basis, this PhD thesis is focused on the investigation of the interaction of biologically active ions and molecules with calcium phosphates with the aim to develop new materials for the substitution and repair of skeletal tissue, according to the following lines: I. Modified calcium phosphates. A relevant part of this PhD thesis has been addressed to study the interaction of Strontium with calcium phosphates. It was demonstrated that strontium ion can substitute for calcium into hydroxyapatite, causing appreciable structural and morphological modifications. The detailed structural analysis carried out on the nanocrystals at different strontium content provided new insight into its interaction with the structure of hydroxyapatite. At variance with the behaviour of Sr towards HA, it was found that this ion inhibits the synthesis of octacalcium phosphate. However, it can substitute for calcium in this structure up to 15 atom %, in agreement with the increase of the cell parameters observed on increasing ion concentration. A similar behaviour was found for Magnesium ion, whereas Manganese inhibits the synthesis of octacalcium phosphate and it promotes the precipitation of dicalcium phosphate dehydrate. It was also found that Strontium affects the kinetics of the reaction of hydrolysis of α-TCP. It inhibits the conversion from α-TCP to hydroxyapatite. However, the resulting apatitic phase contains significant amounts of Sr2+ suggesting that the addition of Sr2+ to the composition of α-TCP bone cements could be successfully exploited for its local delivery in bone defects. The hydrolysis of α-TCP has been investigated also in the presence of increasing amounts of gelatin: the results indicated that this biopolymer accelerates the hydrolysis reaction and promotes the conversion of α-TCP into OCP, suggesting that its addition in the composition of calcium phosphate cements can be employed to modulate the OCP/HA ratio, and as a consequence the solubility, of the set cement. II. Deposition of modified calcium phosphates on metallic substrates. Coating with a thin film of calcium phosphates is frequently applied on the surface of metallic implants in order to combine the high mechanical strength of the metal with the excellent bioactivity of the calcium phosphates surface layers. During this PhD thesis, thank to the collaboration with prof. I.N. Mihailescu, head of the Laser-Surface-Plasma Interactions Laboratory (National Institute for Lasers, Plasma and Radiation Physics – Laser Department, Bucharest) Pulsed Laser Deposition has been successfully applied to deposit thin films of Sr substituted HA on Titanium substrates. The synthesized coatings displayed a uniform Sr distribution, a granular surface and a good degree of crystallinity which slightly decreased on increasing Sr content. The results of in vitro tests carried out on osteoblast-like and osteoclast cells suggested that the presence of Sr in HA thin films can enhance the positive effect of HA coatings on osteointegration and bone regeneration, and prevent undesirable bone resorption. The possibility to introduce an active molecule in the implant site was explored using Matrix Assisted Pulsed Laser Evaporation to deposit hydroxyapatite nanocrystals at different content of alendronate, a bisphosphonate widely employed in the treatments of pathological diseases associated to bone loss. The coatings displayed a good degree of crystallinity, and the results of in vitro tests indicated that alendronate promotes proliferation and differentiation of osteoblasts even when incorporated into hydroxyapatite. III. Synthesis of drug carriers with a delayed release modulated by a calcium phosphate coating. A core-shell system for modulated drug delivery and release has been developed through optimization of the experimental conditions to cover gelatin microspheres with a uniform layer of calcium phosphate. The kinetics of the release from uncoated and coated microspheres was investigated using aspirin as a model drug. It was shown that the presence of the calcium phosphate shell delays the release of aspirin and allows to modulate its action.

Objects with complex shape and functions have always attracted attention and interest. The morphological diversity and complexity of naturally occurring forms and patterns have been a motivation for humans to copy and adopt ideas from Nature to achieve functional, aesthetic and social value. Biomimetics is addressed to the design and development of new synthetic materials using strategies adopted by living organisms to produce biological materials. In particular, biomineralized tissues are often sophisticate composite materials, in which the components and the interfaces between them have been defined and optimized, and that present unusual and optimal chemical-physical, morphological and mechanical properties. Moreover, biominerals are generally produced by easily traceable raw materials, in aqueous media and at room pressure and temperature, that is through cheap process and materials. Thus, it is not surprising that the idea to mimic those strategies proper of Nature has been employed in several areas of applied sciences, such as for the preparation of liquid crystals, ceramic thin films computer switches and many other advanced materials. On this basis, this PhD thesis is focused on the investigation of the interaction of biologically active ions and molecules with calcium phosphates with the aim to develop new materials for the substitution and repair of skeletal tissue, according to the following lines: I. Modified calcium phosphates. A relevant part of this PhD thesis has been addressed to study the interaction of Strontium with calcium phosphates. It was demonstrated that strontium ion can substitute for calcium into hydroxyapatite, causing appreciable structural and morphological modifications. The detailed structural analysis carried out on the nanocrystals at different strontium content provided new insight into its interaction with the structure of hydroxyapatite. At variance with the behaviour of Sr towards HA, it was found that this ion inhibits the synthesis of octacalcium phosphate. However, it can substitute for calcium in this structure up to 15 atom %, in agreement with the increase of the cell parameters observed on increasing ion concentration. A similar behaviour was found for Magnesium ion, whereas Manganese inhibits the synthesis of octacalcium phosphate and it promotes the precipitation of dicalcium phosphate dehydrate. It was also found that Strontium affects the kinetics of the reaction of hydrolysis of α-TCP. It inhibits the conversion from α-TCP to hydroxyapatite. However, the resulting apatitic phase contains significant amounts of Sr2+ suggesting that the addition of Sr2+ to the composition of α-TCP bone cements could be successfully exploited for its local delivery in bone defects. The hydrolysis of α-TCP has been investigated also in the presence of increasing amounts of gelatin: the results indicated that this biopolymer accelerates the hydrolysis reaction and promotes the conversion of α-TCP into OCP, suggesting that its addition in the composition of calcium phosphate cements can be employed to modulate the OCP/HA ratio, and as a consequence the solubility, of the set cement. II. Deposition of modified calcium phosphates on metallic substrates. Coating with a thin film of calcium phosphates is frequently applied on the surface of metallic implants in order to combine the high mechanical strength of the metal with the excellent bioactivity of the calcium phosphates surface layers. During this PhD thesis, thank to the collaboration with prof. I.N. Mihailescu, head of the Laser-Surface-Plasma Interactions Laboratory (National Institute for Lasers, Plasma and Radiation Physics – Laser Department, Bucharest) Pulsed Laser Deposition has been successfully applied to deposit thin films of Sr substituted HA on Titanium substrates. The synthesized coatings displayed a uniform Sr distribution, a granular surface and a good degree of crystallinity which slightly decreased on increasing Sr content. The results of in vitro tests carried out on osteoblast-like and osteoclast cells suggested that the presence of Sr in HA thin films can enhance the positive effect of HA coatings on osteointegration and bone regeneration, and prevent undesirable bone resorption. The possibility to introduce an active molecule in the implant site was explored using Matrix Assisted Pulsed Laser Evaporation to deposit hydroxyapatite nanocrystals at different content of alendronate, a bisphosphonate widely employed in the treatments of pathological diseases associated to bone loss. The coatings displayed a good degree of crystallinity, and the results of in vitro tests indicated that alendronate promotes proliferation and differentiation of osteoblasts even when incorporated into hydroxyapatite. III. Synthesis of drug carriers with a delayed release modulated by a calcium phosphate coating. A core-shell system for modulated drug delivery and release has been developed through optimization of the experimental conditions to cover gelatin microspheres with a uniform layer of calcium phosphate. The kinetics of the release from uncoated and coated microspheres was investigated using aspirin as a model drug. It was shown that the presence of the calcium phosphate shell delays the release of aspirin and allows to modulate its action.

In this thesis, pH and potassium all-solid-state ISE based on potentiometry and bioimpedance sensors were designed, fabricated and integrated in a miniaturized array for its application in endoscopic surgery for in vivo ischemia detection inside the stomach. To achieve this goal, the developed array withstood the low pH and corrosive condition in the gastric juice of the stomach, by modifying the surface with a conductive Ag/AgCl ink containing hydrophilic and hydrophobic groups. That creates a stable and robust candidate for low pH applications. However, these sensors have to demonstrate besides stability, high sensitivity, and selectivity. For this purpose, different ionophores specific to a single ion were tested. Octadecyl isonicotinate was the one that shown better results as pH ionophore and valinomycin, bis [(benzo-15-crown-4)-4-ylmethyl] pimelate for potassium detection. All these ionophores were embedded in PVC polymer membrane containing also plasticizers such as 2-nitrophenyl octyl ether, bis (1-butylpentyl) adipate (BBPA) and liphophilic anionic additives such as potassium tetrakis (4-chlorophenyl) borate (KTpClPB). The specific compositions of membranes to detect potassium or pH were optimized for the better performance of the sensors. pH ISE sensor shows a nernstian behavior (-54,38 mV/pH) at low pH and a nearly nernstian behavior at physiological pH (-34,899 mV/pH). Bioimpedance sensor was tested and optimized in vitro with different solutions of ions concentration to mimic ischemia detection and with different kinds of tissues from different nature. For this purpose, chicken fat and breast tissues were taken as a model for mimicking non-ischemic and ischemic states respectively. The effect of electrodes insulation as well as the pressure applied on the tissue was studied. The dependence of the impedance response with different pressure applied to the sensor was overcome by applying magnetic field attachment. The sensor array was modified with ring magnets which were attracted by an external magnet, giving stable and reliable signal discarding mechanical motion. The shape and size of the sensor array were designed for being adapted to the commercially available gastroendoscopes. Round shaped cylinder of 7 mm diameter was fabricated with 12 electrodes pin of 600 µm diameter, containing 3 RE, 3 pH and 2 potassium all-solid-state sensors and 4 electrodes in a row for impedance measurements. The sensor array was successfully integrated in commercial endoscope and inserted inside the pig stomach. The blood flow of certain area of the stomach was interrupted by ligating or crossclamping vessels and organ wall. Ischemia and reperfusion steps were sensed successfully with potassium and pH sensors. These results also indicate that information about hypoxic tissue damage can be collected with this array. Ischemia was also sensed on small intestine tissue by opening the abdominal part of the body and getting the sensor array in contact with the intestine. By crossclamping of mesenteric artery by tourniquets and scissors, ischemic and reperfusion states were controlled. Results proved that ischemia and reperfusion can be monitored by our integrated sensor array. As a conclusion, a novel all-solid-state potentiometric, miniaturized, low cost and mass producible pH, potassium all-solid-state ISE and impedance sensors integrated in an array was successfully fabricated for detecting ischemia inside the stomach by means of endoscopic techniques and also on small intestine. This array was tested in vitro and vivo giving reproducible and reliable results. The developed all-solid-state pH sensors permit low pH sensing from 0.7-2.5, which is the only example in the literature that allows so low pH detection, and so make this sensor a unique device for stomach detection.
El diagnóstico médico es uno de los campos que han obtenido más ventajas de la capacidad de los electrodos selectivos de iones (ESI) para la detección de iones, ya que los cambios en la concentración de estos elementos están directamente relacionados con diferentes enfermedades. La detección de isquemia es una de las favorecidas por estos sensores. La isquemia es una disminución del suministro de sangre a un órgano y se requiere una detección rápida y precisa. Los métodos de detección in situ en el tejido de los órganos conllevan una detección temprana de la isquemia y el estómago es uno de los órganos más importantes en la detección de Ischemia. Sin embargo, el bajo pH del jugo gástrico del estómago hace difícil la fabricación de sensores de estado sólido con ESI estables y funcionales, principalmente debido a la interferencia de aniones y al problema de la adhesión entre la membrana ESI y la superficie del electrodo. En esta tesis, se han diseñado y fabricado electrodos selección de iones de pH y potasio ESI de estado sólido basados en la potenciometría y sensores de bioimpedancia y se han integrado en una matriz en miniatura para su aplicación en la cirugía endoscópica para la detección de isquemia in vivo en el interior del estómago. El conjunto de sensores se integró con éxito en endoscopio comercial y se inserto en el interior del estómago de un cerdo. El flujo de sangre de cierta área del estómago se interrumpió mediante la ligación o pinzamiento de los vasos sanguineos y la pared del órgano. Los pasos de isquemia y reperfusión fueron detectados con éxito con los sensores de potasio y de pH. Estos resultados también indican que se puede obtener información sobre el daño en el tejido hipóxico recogido con esta matriz. Los sensores de pH de sólido desarrollados permiten la detección pH bajos de 0,7 a 2,5, que es el único ejemplo en la literatura de detección de pH tan bajos con este tipo de sensores y por lo tanto hacen que sea este sensor de un dispositivo único para la detección de isquemia en el estómago.

Els cossos d’inclusió bacterians (CIs) són dipòsits proteics que apareixen normalment durant el procés de producció de proteïna recombinant. Històricament, aquests agregats han estat classificats com a sub-productes de rebuig i, per tant, descartats. No obstant, aquesta percepció ha canviat al llarg de les dues darreres dècades. Nombrosos estudis han proporcionat evidències que demostren que aquestes partícules són, de fet, estructures supramoleculars formades per contactes estereoespecífics proteïna-proteïna, morfològicament i fisicoquímicament estables, fàcils de purificar i que mostren un alt nivell de plasticitat pel que fa a activitat biològica, mida i propietats fisicoquímiques. En el present treball s’ha explorat la possibilitat d’aprofitar les propietats úniques dels CIs per desenvolupar noves aplicacions biomèdiques. En aquest sentit, ens hem centrat en l’estudi de la idoneïtat dels CIs com a nanomaterial particulat per tal de generar nanotopografies capaces de controlar la resposta de cèl·lules animals. La modificació a nivell nanomètric de superfícies per tal d’estimular una resposta cel·lular específica resulta molt prometedora pels camps de l’enginyeria tissular i la medicina regenerativa en general. En aquest treball es presenten evidències de que els CIs es poden dipositar sobre superfícies de cultiu, obtenint nanotopografies modificades amb efecte biològic. Al cultivar cèl·lules de mamífer sobre aquestes, s’aprecia un elevada capacitat d’adhesió cel·lular. A més a més, s’ha observat que el CIs són capaços d’estimular la proliferació cel·lular mitjançant una cascada de senyalització basada en la mecanotransducció. Tant l’adhesió com la proliferació cel·lulars són factors crucials per la colonització de la superfície d’implants. S’ha demostrat també que les topografies formades per CIs són capaces de dirigir cèl·lules mare mesenquimals (MSCs) indiferenciades cap a osteoblasts. És important esmentar que l’adhesió, la proliferació i la diferenciació cel·lular mediades per CIs poden ser modulades per la correcta elecció del fons genètic de la soca utilitzada per a produir els CIs, obtenint d’aquesta manera agregats fisicoquímica i morfològicament diferents. Finalment els CIs han demostrat ser capaços d’alliberar quantitats significatives de la proteïna que els forma, en una versió activa. Aquest fet ha propiciat el seu estudi com plataforma d’entrega de fàrmacs proteics, des de nanoestructures de la superfície del substrat, cap als cultius diana. Aquesta darrera aplicació seria particularment interessant en tractaments a llarg termini, ja que el CIs proporcionarien un alliberament de la proteïna sostingut. A més, aquesta capacitat d’entrega afegiria un nivell extra de complexitat i control a les aplicacions prèviament esmentades ja que combinaria l’efecte mecànic de les topografies basades en CIs amb l’activitat biològica de la proteïna formadora de l’agregat.
Bacterial Inclusion Bodies (IBs) are protein deposits usually observed in recombinant bacteria during protein production processes. These aggregates have been historically regarded as waste by-products and therefore discarded. However, in the last two decades this perception has changed. Numerous studies have provided evidence that these particles are in fact supramolecular structures formed by stereospecific cross-molecular protein-protein interactions, morphologically and mechanically stable, easy to purify and highly tunable in terms of biological activity, size and physicochemical properties,. In the present work it has been explored to exploit appealing IB properties for biomedical applications. In this regard, we have focused on the suitability of IBs as particulate protein nanomaterial to produce engineered nanotopographies able to modulate mammalian cell responses. Nanomodification of scaffolds to stimulate a specific cell response is a promising field for tissue engineering and regenerative medicine. In this regard, we have provided evidence that bacterial IBs can be effectively deposited onto cell culture surfaces generating altered nanotopographies. These IB-based topographies, when used as cell culture substrate, exhibited higher capability for cell adhesion in four distinct cell lines. Moreover, IBs were shown to be able of actively stimulating cell proliferation through mechanotransductive events, being these two activities crucial for cell colonization of implant materials. On the other hand, it has been also proved how IB-based topographies are able to direct mesenchymal stem cells (MSCs) to osteogenesis. Noteworthy, cell adhesion, cell proliferation and cell differentiation can be controlled by the proper choice of Escherichia coli producing strain genetic background, rendering physicochemically and morphologically distinct IBs. Finally, IBs have been shown capable of releasing significant amounts of their forming protein, prompting their use as a delivery platform of therapeutic protein drugs, from nanostructured surfaces to cell cultures. This last application would be particularly appealing for long term treatments, since IBs provide a sustained release of their active component. Moreover, this delivery capacity adds an extra level of complexity and control to the previous mentioned IB applications, through the combination of the mechanical effect of the IB-based topographies with the biological activity of the IB-forming protein

Thesis (Ph. D.)--University of North Carolina at Chapel Hill, 2008.
Title from electronic title page (viewed Sep. 16, 2008). " ... in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Chemistry." partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Chemistry." Discipline: Chemistry; Department/School: Chemistry.

Thesis (Ph. D.)--University of North Carolina at Chapel Hill, 2007.
Title from electronic title page (viewed Sep. 16, 2008). "... in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Chemistry Analytical Chemistry." Discipline: Chemistry; Department/School: Chemistry.

Tetraazamacrocyclic frameworks of 1,4,7,10-tetraazacylcododecane (cyclen) and 1,4,8,11-tetraazacyclotetradecane (cyclam) with functional pendent arms and porphyrins with solubilising polyethylene glycol (PEG) chains have been synthesised. The pendent arms possess different properties which tailor the metal complex towards a particular role.

Magnesium has been identified as a promising biodegradable implant material because it does not cause systemic toxicity and can reduce stress shielding. However, it corrodes too quickly in the body. Titanium, which is already used ubiquitously for implants, was chosen as the alloying element because of its proven biocompatibility and corrosion resistance in physiological environments. Thus, alloying magnesium with titanium is expected to improve the corrosion resistance of magnesium. Mg-Ti alloys with a titanium content ranging from 5 to 35 at.-% were successfully synthesized by mechanical alloying. Spark plasma sintering was identified as a processing route to consolidate the alloy powders made by ball-milling into bulk material without destroying the alloy structure. This is an important finding as this metastable Mg-Ti alloy can only be heated up to max. 200C° for a limited time without reaching the stable state of separated magnesium and titanium. The superior corrosion behavior of Mg80-Ti20 alloy in a simulated physiological environment was shown through hydrogen evolution tests, where the corrosion rate was drastically reduced compared to pure magnesium and electrochemical measurements revealed an increased potential and resistance compared to pure magnesium. Cytotoxicity tests on murine pre-osteoblastic cells in vitro confirmed that supernatants made from Mg-Ti alloy were no more cytotoxic than supernatants prepared with pure magnesium. Mg and Mg-Ti alloys can also be used to make novel polymer-metal composites, e.g., with poly(lactic-co-glycolic acid) (PLGA) to avoid the polymer’s detrimental pH drop during degradation and alter its degradation pattern. Thus, Mg-Ti alloys can be fabricated and consolidated while achieving improved corrosion resistance and maintaining cytocompatibility. This work opens up the possibility of using Mg-Ti alloys for fracture fixation implants and other biomedical applications.

Filtering is a key step in digital image processing and analysis. It is mainly used for amplification or attenuation of some frequencies depending on the nature of the application. Filtering can either be performed in the spatial domain or in a transformed domain. The selection of the filtering method, filtering domain, and the filter parameters are often driven by the properties of the underlying image. This thesis presents three different kinds of biomedical image filtering applications, where the filter parameters are automatically determined from the underlying images. Filtering can be used for image enhancement. We present a robust image dependent filtering method for intensity inhomogeneity correction of biomedical images. In the presented filtering method, the filter parameters are automatically determined from the grey-weighted distance transform of the magnitude spectrum. An evaluation shows that the filter provides an accurate estimate of intensity inhomogeneity. Filtering can also be used for analysis. The thesis presents a filtering method for heart localization and robust signal detection from video recordings of rat embryos. It presents a strategy to decouple motion artifacts produced by the non-rigid embryonic boundary from the heart. The method also filters out noise and the trend term with the help of empirical mode decomposition. Again, all the filter parameters are determined automatically based on the underlying signal. Transforming the geometry of one image to fit that of another one, so called image registration, can be seen as a filtering operation of the image geometry. To assess the progression of eye disorder, registration between temporal images is often required to determine the movement and development of the blood vessels in the eye. We present a robust method for retinal image registration. The method is based on particle swarm optimization, where the swarm searches for optimal registration parameters based on the direction of its cognitive and social components. An evaluation of the proposed method shows that the method is less susceptible to becoming trapped in local minima than previous methods. With these thesis contributions, we have augmented the filter toolbox for image analysis with methods that adjust to the data at hand.

Specific interactions of biomolecules are central to cellular processes, drug discovery and immunodiagnostics. Such biological binding events are quantifiable via thermophoresis, the directed molecule movement driven by a temperature gradient. Biomolecule thermophoresis can be induced by infrared laser heating and analyzed using fluorescence. The objective of this thesis was to enhance and optimize these all-optical measurements, regarding instrumentation, assay design and biomedical applications. In the first part, a novel measurement device and approach are presented, which cut down sample consumption 50-fold compared to established capillary thermophoresis. Instead of capillaries, analysis was performed in 10 nl-sample droplets transferred into standard 1536-well plates with a non-contact liquid handler (Labcyte). To prevent evaporation, the aqueous sample droplets were stabilized in an oil-surfactant mix. Temperature induced effects in this water-in-oil system were experimentally characterized and the results agreed with numerical simulation. The system’s applicability for biomolecular interaction analysis was confirmed with a DNA aptamer. The achieved miniaturization and the easy-to-handle multi-well plate format promote automated high-throughput screens. Besides aptamers, proteins should also be measurable very well when judging from the application depth of capillary measurements. This versatility of protein investigation via capillary thermophoresis is demonstrated in the second part. Successful experiments were not only conducted in diverse liquids including crude cell lysate, but also for binding partners with a broad range of molecular weight ratios. Affinities between protein and protein, protein and peptide, as well as protein and small molecule were determined with high accuracy. Further flexibility arises from the herein presented label free approach which utilizes protein intrinsic UV fluorescence. It is caused by aromatic amino acids with tryptophan being the major intrinsic fluorophore. This approach exempts from the need to attach a dye, which saves time and excludes labeling artifacts. The wide variety of proteins that can be analyzed with thermophoresis also includes anti-bodies. Two applications of such thermophoretic immunoassays are introduced in the third part. Firstly, the therapeutically interesting antibody MCPR3-7 was assessed. MCPR3-7 binds proteinase 3 (PR3), the major autoimmune target in granulomatosis with polyangiitis. Thermophoresis allowed to quantified MCPR3-7’s affinity and selectivity for different PR3 forms. In addition, it revealed that the antibody interferes with the complexation of PR3 and alpha-1-proteinase inhibitor (alpha-1PI). Secondly, a diagnostic autocompetition assay is described, which directly determines affinity and concentration of disease related biomarkers. It was applied for autoantibodies against the cardiac β1-adrenoceptor found in patients suffering from dilated cardiomyopathy. To detect these autoantibodies, the small peptide COR1 mimicking the adrenoceptor’s dominant epitope served as an artificial antigen. This tracer was labeled with a red-fluorescent dye, which ensured selectivity for measurements directly in untreated human blood serum. The results prove that thermophoresis is a valuable tool to characterize antibodies including those of diagnostic value and those with a therapeutic potential. Taken together, the presented innovations in assay design and the novel nl-droplet approach can be expected to considerably widen the application spectrum of thermophoresis in fundamental research, industrial drug discovery and clinical laboratory diagnostics.
Spezifische Interaktionen von Biomolekülen sind von zentraler Bedeutung für zelluläre Prozesse, die Entwicklung neuer Medikamente und die Immundiagnostik. Solche biologischen Bindungsvorgänge lassen sich mittels Thermophorese, der gerichteten Molekülbewegung entlang eines Temperaturgradienten, quantifizieren. Die Thermophorese von Biomolekülen kann durch Infrarotlaser-Heizen induziert und mittels Fluoreszenz analysiert werden. Die Weiterentwicklung dieses optischen Verfahrens bezüglich des Messinstruments, des Versuchsdesigns und der biomedizinischen Anwendungen war das Ziel der vorliegenden Dissertation. Im ersten Teil wird eine neuartige Technik vorgestellt, die den Probenverbrauch verglichen mit etablierten Kapillarmessungen um den Faktor 50 verkleinert. Statt in Kapillaren wurde in 10 nl-großen Probentropfen gemessen, die mit einem kontaktfreien Liquid-Handler (Labcyte) in eine 1536-Well-Platte übertragen wurden. Zum Schutz vor Verdunstung wurden die Tropfen in eine Öl-Tensid-Schicht transferiert. Temperaturinduzierte Effekte in diesem Wasser-in-Öl-System wurden experimentell charakterisiert, wobei die Ergebnisse durch numerischen Simulationen bestätigt wurden. Dass sich die Methode für biomolekulare Interaktionstests eignet, wurde anhand eines DNA-Aptamers belegt. Die Miniaturisierung und die einfache Handhabung der Multi-Well-Platten ermöglichen automatisierte Hochdurchsatz-Screens. Neben Aptameren sollten sich auch Proteine sehr gut untersuchen lassen, wenn man von einer ähnlichen Anwendungsbreite wie bei Kapillarmessungen ausgeht. Auf derartige Proteinuntersuchungen mittels Kapillarthermophorese wird im zweiten Teil eingegangen. Analysen wurden nicht nur in diversen Puffern und sogar in rohem Zelllysat durchgeführt, sondern auch mit unterschiedlichsten Bindungspartnern. So wurden Affinitäten zwischen Protein und Protein, Protein und Peptid, sowie Protein und niedermolekularer Verbindung mit hoher Genauigkeit bestimmt. Thermophoresetests gewinnen durch das in dieser Arbeit präsentierte, markierungsfreie Verfahren zusätzlich an Flexibilität. Es basiert auf der intrinsischen UV-Fluoreszenz von Proteinen, die auf aromatische Aminosäuren, hauptsächlich Tryptophan, zurückzuführen ist. Somit müssen Proteine nicht mehr mit Fluoreszenzfarbstoffen markiert werden, was Zeit spart und Artefakte ausschließt. Der dritte Teil behandelt die Quantifizierung von Antikörpern. Thermophoretische Immunassays wurden für zwei biomedizinische Fragestellungen eingesetzt. Zunächst wurde der aus therapeutischer Sicht interessante Antikörper MCPR3-7 untersucht. Er ist gegen Proteinase 3 (PR3) gerichtet, das Hauptepitop autoimmuner Antikörper bei der granulomatösen Polyangiitis. Mithilfe der Thermophorese wurde sowohl die Affinität von MCPR3-7 für verschiedene PR3-Formen quantifiziert, als auch gezeigt, dass der Antikörper die Komplexierung von PR3 und alpha-1-Proteinaseinhibitor (alpha-1PI) stört. Ferner wird ein diagnostisches Autokompetitionsverfahren vorgestellt, das gleichzeitig die Affinität und die Konzentration von Biomarkern in humanem Blutserum quantifiziert. Autoantikörper gegen den kardialen β1-Adrenozeptor, die mit der dilatativen Kardiomyopathie assoziiert sind, wurden mithilfe des kurzen Peptides COR1 analysiert, das das dominante Epitop nachstellt. Die Ergebnisse belegen, dass die Thermophorese ein wertvolles Werkzeug für die Antikörpercharakterisierung ist. Zusammengefasst lassen die vorgestellten Neuerungen eine umfangreiche Erweiterung des Anwendungssprektrums der Biomolekülthermophorese in der Grundlagenforschung, der industriellen Wirkstoffsuche und der klinischen Labordiagnostik erwarten.

The growing rate of diabetes and undiagnosed diabetes related diseases is becoming a worldwide major health concern. The motivation of this thesis was to make use of a technology called the ‘electronic nose’ (eNose) for diagnosing diseases. It presents a comprehensive study on metabolic and gastro-intestinal disorders, choosing diabetes as a target disease. Using eNose technology with urinary volatile organic compounds (VOCs) is attractive as it allows non-invasive monitoring of various molecular constituents in urine. Trace gases in urine are linked to metabolic reactions and diseases. Therefore, urinary volatile compounds were used for diagnosis purposes in this thesis. The literature on existing eNose technologies, their pros and cons and applications in biomedical field was thoroughly reviewed, especially in detecting headspace of urine. Since the thesis investigates urinary VOCs, it is important to discover the stability of urine samples and their VOCs in time. It was discovered that urine samples lose their stability and VOCs emission after 9 months. A comprehensive study with 137 diabetic and healthy control urine samples was done to access the capability of commercially available eNose instruments for discrimination between these two groups. Metal oxide gas sensor based commercial eNose (Fox 4000, AlphaMOS Ltd) and field asymmetric ion mobility spectrometer (Lonestar, Owlstone Ltd) were used to analyse volatiles in urinary headspace. Both technologies were able to distinguish both groups with sensitivity and specificity of more than 90%. Then the project moved onto developing a Non-dispersive infrared (NDIR) sensor system that is non-invasive, low-cost, precise, rapid, simple and patient friendly, and can be used at both hospitals and homes. NDIR gas sensing is one of the most widely used optical gas detection techniques. NDIR system was used for diagnosing diabetes and gastro related diseases from patient’s wastes. To the best of the authors’ knowledge, this is the first and only developed tuneable NDIR eNose system. The developed optical eNose is able to scan the whole infrared range between 3.1μm and 10.5 μm with step size of 20 nm. To simulate the effect of background humidity and temperature on the sensor response, a gas test rig system that includes gas mixture, VOC generator, humidity generator and gas analyser was designed to enable the user to have control of gas flow, humidity and temperature. This also helps to find out system’s sensitivity and selectivity. Finally, after evaluating the sensitivity and selectivity of optical eNose, it was tested on simple and complex odours. The results were promising in discriminating the odours. Due to insufficient sample batches received from the hospital, synthetic urine samples were purchased, and diabetic samples were artificially made. The optical eNose was able to successfully separate artificial diabetic samples from non-diabetic ones.

Ti has been widely used in biomedical fields since the 1950s because of biocompatibility, corrosion resistance and suitable mechanical properties. However, corrosion-related failures of Ti implants are observed. Ti corrosion products are reported to induce unfavourable biological responses, which may lead to failures of Ti implants. Corrosion of three grades of Ti (CP-Ti Grade2, CP-Ti Grade4 and Ti6Al4V) in simulated peri-implant environments was investigated by solution analysis, surface analysis and electrochemical tests. Lipopolysaccharide, a component of bacterial cell walls and a mediator of peri-implant inflammation, was observed to enhance Ti corrosion in slightly acidic and neutral conditions (pH 4-7) whilst it inhibited Ti dissolution in highly acidic environments (pH 2). Both albumin, an abundant protein, and H₂O₂, an important inflammation product, influenced corrosion of Ti6Al4V and the co-existence of both species considerably enhanced Ti release than either species in isolation. The β phase of Ti6Al4V was preferentially attacked in H₂O₂. The presence of an early coloniser of dental implants Streptococcus sanguinis and human neutrophils, abundant immune cells, promoted Ti release. Mechanically-assisted crevice corrosion simulation was demonstrated with development of aggressive crevice chemistry. Albumin decreased the abrasion charge of Ti6Al4V while LPS and H2O2 did not show a measurable change.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Chemical Engineering, 2014.
Cataloged from PDF version of thesis.
Includes bibliographical references (pages 153-164).
Functional polymeric thin films are often stratified with nanometer level structure and distinct purposes for each layer. These nanostructured polymeric materials are useful in a wide variety of applications including drug delivery, tissue engineering, controlling condensation and polymeric batteries; all of which will be discussed in this work. The first area of my thesis will detail the use of C₆₀ cluster-ion depth profiling X-ray Photoelectron Spectroscopy (XPS) to fundamentally understand how thin film structure and function relate. This method has the unique capability to determine the atomic composition and chemical state of polymeric thin films with <10nm nanometer depth resolution without any chemical labeling or modification. Using this technique, I probed the nanostructure of functional thin films to quantify the interlayer diffusion of the biopolymer chitosan as well as demonstrate methods to stop this diffusion. I also explored the role of interlayer diffusion in the design of hydrophobic yet antifogging 'zwitter-wettable' surfaces. Additionally, I probed the lithium triflate salt distribution in solid block copolymer battery electrolytes (PS-b-POEM) to understand the lithium-ion distribution within the POEM block. In the second area of my thesis, I show how the nanostructure of materials control the function of polymeric particles in vitro and in vivo. One example is a 'Cellular Backpack' which is a flat, anisotropic, stratified polymeric particle that is hundreds of nanometers thick and microns wide. In partnership with the Mitragotri group at UCSB, we show that cellular backpacks are phagocytosis resistant, and when attached to a cell, the cell maintains native functions. These capabilities uniquely position backpacks for cell-mediated therapeutic delivery and we show in vivo that immune cells attached to backpacks maintain their ability to home to sites of inflammation. In addition, we have designed polymeric microtubes that can control their orientation on the surface of living cells. Inspired by chemically non-uniform Janus particles, we designed tube-shaped, chemically non-uniform microparticles with cell-adhesive ligands on the ends of the tubes and a cell-resistant surface on the sides. Our results show that by altering the surface chemistry on the end versus the side, we can control the orientation of tubes on living cells. This advance opens the capability to control phagocytosis and design cellular materials from the bottom up.
by Jonathan Brian Gilbert.
Ph. D.

The field of nanotechnology is growing vastly, both as a field of research and in commercial applications. This rapid growth calls for synthesis methods which can produce high quality nanomaterials, while being scalable. This thesis describes an investigation into the use of a continuous hydrothermal reactor for the synthesis of nanomaterials, with potential use in three different biomedical applications – bone scaffolds, fluorescent biomarkers, and MRI contrast agents. The first chapter of this thesis provides an overview of nanotechnology: the advantages of nanoscale, the commercial industries which can benefit, and the predominant methods currently used to produce nanomaterials. Some advantages and drawbacks of each synthesis route are given, concluding with a description of the Nozzle reactor – the patented technology used for nanomaterial synthesis in this Thesis. Chapter 2 then focusses on the characterisation techniques used in this thesis, detailing the principles of how data is obtained, as well as highlighting the limitations of each method. With the background information in place, chapters 3, 4 and 5 describe more specific nanomaterials and how they can be applied to each of the aforementioned biomedical fields. These chapters provide the technical details of how various nanomaterials can be synthesised using the Nozzle reactor, and the structural data (crystallinity, particle size) obtained from these samples. Furthermore, the functional properties of these nanomaterials are tested and the results, along with a discussion of any trends, are presented. Finally, this thesis concludes with a summary of the results described and emphasises the key areas where further work can be conducted.

Biomedical devices are commonly used in all areas of healthcare, These devices, which range from contact lenses through to endotracheal tubes, are most often fashioned from materials which allow the device to carry out its function thoroughly, but in doing so render the device susceptible to a number of complications. Two of the most major complications are that of device infection and poor frictional behaviour at the interface of the device and human tissue. This thesis details the development and characterisation of various polymeric systems which allow the resolution of these problems. With regard to infection, well established photodynamic techniques are further developed to provide a system which can bring about effective prevention of infection for prolonged durations of time, leading to a wide range of advantages, potentiating the function of the device. Biomaterial frictional behaviour is improved in a number of ways, including the development of next generation device coatings which are more easily wetted, offer improved biocompatibility, and also offer an improved tenacity of effect. Moreover, further work in this thesis has led to the development of successful photochemical attachment pathways for the addition of such coatings to the surface of commonly used biomaterial substrates.