Drug administration through the pulmonary route is an ancient technique that evolved from inhaling the smoke of certain leaves as a medicine. The optimum droplet diameter for the pulmonary system deposition has been identified to be in the range from 2 to 3.5 μm, with potential deposition rates of up to 80% of this size range. Currently, the most used aerosol generator methods are the pressurized metered dose inhalers. However, they generally exhibit low deposition efficiency with less than 20 % of the spray reaching the target area of the lungs as most of the drug deposited in the upper airways. This is for the most part due to the droplet size polydispersity that is inherent in these systems. The droplets of the biggest diameter will deposit in the upper airways, and then the deposited medicine will be swallowed and absorbed in the gastrointestinal tract. This can produce adverse medical side effects. Electrospray (ES) or electrohydrodynamic atomization (EHDA) is a promising atomization process due to its ability to produce a spray with monodisperse droplet size. The current study will investigate the feasibility of using electrospray in a pulmonary drug delivery system. Assessments, selection and characterization of suitable biocompatible solvents that can be used as a lung obstruction relief drug were carried out. Tests to identify the electrospray setup necessary to produce droplet sizes in the appropriate range for deposition in the lungs were carried out. The study found that both stable and pulsating cone jet modes can produce the required droplet size and the pulsating mode can produce at least four times higher flow than stable cone jet mode. A low-cost image analysis technique developed for this work gave satisfactory results that could be compared to droplet size scaling laws from the literature. However, it proved to be relatively time consuming and further automation of this technique would make it more suitable for large-scale studies. The image analysis results show a correlation between the cone length, cone angle and the applied voltage. The droplet scaling laws discrepancies such as the solution flow rate exponent and the constant that is used by some scaling laws may be attributed to the droplet evaporation time which is quite short for the water/ ethanol solutions. The emitter diameter and the conductivity effect on the I(Q) power law and the sensitivity of the onset voltage (Vonset) to the liquid flow rate (Q), were demonstrated for solutions of triethylene-glycol (TEG), and for an ethanol-water mixture solution.

Les hydrogels moléculaires avec un réseau de fibres auto-assembles sont utilisés dans différents domaines dont le relargage de médicaments, les senseurs, l’ingénierie tissulaire et la nano-modélisation. Les hydrogels moléculaires à base d’acides biliaires, qui sont une classe de biocomposés d’origine naturelle, montrent une biocompatibilité améliorée et sont de bons candidats pour des applications dans le domaine biomédical. Ces hydrogels présentent une bonne bio-dégradabilité et une diversité fonctionnelle grâce aux faibles interactions supramoléculaires et aux structures chimiques précisément contrôlées. Dans cette thèse, des nouveaux hydrogels moléculaires à base des acides biliaires et leurs dérivés ont été étudiés pour mieux comprendre la relation entre la structure chimique du gélifiant et la formation de gels moléculaires. Un dimère de l'acide cholique avec un groupe diéthylènetriamine est insoluble dans l'eau. Par contre, il peut former des hydrogels grâce à un réseau tri-dimensionnel de fibres en présence de certains acides carboxyliques. L'addition d'acide carboxylique peut protoner le groupe amine secondaire et défaire les interactions intermoléculaires entre les dimères et favoriser la formation des liaisons hydrogènes acide-dimère. Seuls les acides carboxyliques faibles et hydrophiles causent la gélation des dimères. La résistance mécanique des hydrogels formés peut être modifiée par un choix judicieux d'acides. Les interactions hydrophobes et les liaisons hydrogènes entre les chaînes latérales d'acides carboxyliques peuvent améliorer les propriétés mécaniques des hydrogels. La solubilité marginale du complexe acide-dimère a été considérée comme un facteur critique pour la formation d'hydrogels. Un autre système d’hydrogélation à base d’acides biliaires a été développé par l’introduction de dioxyde de carbone (CO2) dans des solutions aqueuses de certains sels d’acides biliaires, qui donne un hydrogel composé de molécules biologiques entièrement naturelles et fournit un réservoir commode du CO2 dans l’eau. Le groupement carboxylate des sels d’acides biliaires peut être partiellement protoné dans les solutions aqueuses, ce qui amène la dissolution marginale dans l’eau et la formation d’hydrogels avec une structure fibreuse. L’aspect et les propriétés mécaniques des hydrogels dépendent de la concentration de CO2. Le bullage avec CO2 pendant une ou deux secondes génère un hydrogel transparent avec des nanofibres. Le bullage supplémentaire forme des hydrogels plus forts. Mais réduit la transparence et la force mécanique des hydrogels. D’ailleurs, les hydrogels transparents ou opaques redeviennent des solutions transparentes quand ils sont chauffés avec bullage de N2. La transition sol-gel est réversible et reproductible. La force mécanique et la transparence des hydrogels peuvent être améliorées par l’addition de sels inorganiques comme NaCl par l’effet de relargage. Toutes les composantes de ces hydrogels sont naturelles, donnant des hydrogels biocompatibles et potentiellement utiles pour des applications dans le domaine biomédical. Le dimère mentionné ci-dessus possède des propriétés d’auto-assemblage dépendamment de sa concentration. Ceci a été étudié en utilisant un sel organique de dimère/acide formique avec un rapport molaire 1/1. Le sel du dimère s’auto-assemble dans l’eau et ainsi forme des nanofibres isolées et mono-dispersées à des concentrations faibles. Les fibres enchevêtrées donnent des réseaux fibreux 3D bien dispersés de façon aléatoire à des concentrations plus élevées. Quand la concentration du sel du dimère est supérieure à la concentration critique de gélation, le réseau fibreux est assez fort pour immobiliser la solution, qui provoque la formation d’un hydrogel isotrope. L’augmentation supplémentaire de la concentration du sel du dimère peut augmenter l’anisotropie de l’hydrogel et former ainsi un hydrogel nématique. La formation de domaines ordonnés des nanofibres alignées donne ces propriétés optiques à l’hydrogel. L’agitation de systèmes aqueux du sel de dimère favorise aussi la formation de nanofibres alignées. / Molecular hydrogels are soft materials formed by the self-assembly of small molecules in aqueous solutions via supramolecular interactions. Although much effort has been made in the past several decades in the study of these hydrogels, the mechanism of their formation remains to be understood and the prediction of their formation is a challenge. The main purpose of this thesis is to develop novel molecular hydrogels derived from bile acids, which are naturally occurring biocompounds, and to find the relationship between the gelator structure and the gelation ability. Two new molecular gelation systems based on bile acids and their derivatives have been developed, which may be useful in biomedical applications. The marginal solubility of the solute in water has been found to be a prerequisite for the formation of such molecular hydrogels. The alignment of the nanofibers in the gels leads to the formation of nematic hydrogels. The first gelation system is based on a cholic acid dimer as a gelator, which has two cholic acid molecules covalently linked by a diethylenetriamine spacer. This dimer is insoluble in water, but it forms hydrogels with 3-D fibrous networks in the presence of selected carboxylic acids. The carboxylic acids protonate the dimer, making it marginally soluble in water to yield hydrogels. Only weak and hydrophilic carboxylic acids were capable of inducing the gelation of the dimer and the mechanical strength of the hydrogels could be varied by judicious choice of the acids. Hydrophobic interactions and hydrogen bonding between the side chains of carboxylic acids improve the mechanical properties of hydrogels. The marginal solubility of the acid-dimer complex is regarded to be the critical factor for the formation of hydrogels. Another hydrogelation system was developed by purging to the aqueous solutions of a series of bile salts with carbon dioxide (CO2), yielding hydrogels made of entire natural biological molecules and providing a convenient storage reservoir of CO2 in water. Bile salts are well dissolved in water, while the solubility of bile acids is limited. The carboxylate group of bile salts may be partially protonated in aqueous solutions by bubbling CO2, making them only marginally soluble in water. This forms fibrous structures. Both the appearance and mechanical properties of the hydrogels depend on the amount of CO2 purged. Bubbling CO2 initially induced the formation of transparent hydrogels with nanofibers. Continued purging with CO2 strengthened the hydrogel mechanically, while further addition of CO2 reduced the transparency and mechanical strength of the hydrogel. Both the transparent and opaque hydrogels reverted to transparent solutions when heated and bubbling N2. The sol-gel transition process was reversible and repeatable. The mechanical strength and transparency of the hydrogels could be improved by adding inorganic salts such as NaCl via a salting-out effect. All the hydrogel components are naturally biological compounds, making such hydrogels biocompatible and potentially useful in biomedical applications. The cholic acid dimer linked with a diethylenetriamine spacer was able to assemble in water and form isolated nanofibers in the presence of certain carboxylic acids at a much lower concentration than the CMC of sodium cholate. These nanofibers entangle with each other to yield well-dispersed and randomly-directed 3-D fibrous networks at higher concentrations. When the concentration of dimer salt is above the minimum gelation concentration, the fibrous network is strong enough to immobilize the solution, leading to the formation of an isotropic hydrogel. Further increase of the dimer salt concentration may transit the hydrogels to be anisotropic, thus the formation of nematic hydrogels. The formation of ordered domains of the aligned nanofibers led to anisotropic optical properties of the hydrogels. Stirring the aqueous systems of dimer salt also promoted the alignment of the nanofibers. These molecular hydrogels with ordered aggregates may be useful in applications such as cell culture and mechano-optical sensing.

Ce travail de thèse porte sur la caractérisation d’un matériau composite polyacide lactique-bioverre pour application comme dispositif de réparation osseuse. Le bioverre étant trop fragile pour être utilisé seul comme dispositif de réparation osseuse, celui-ci est associé à une matrice polymère résorbable permettant d’apporter le caractère bioactif à des matériaux pouvant être mis en forme par des procédés de plasturgie. Le matériau composite polyacide lactique-bioverre est ainsi mis en forme par injection à partir de granules élaborés par voie solvant. La caractérisation des propriétés de ce matériau composite a révélé une augmentation du module élastique avec l’ajout de charges, mais une diminution des contraintes maximales admissibles et de la déformation à la rupture. Les modifications des propriétés mécaniques ont été associées à une modification des propriétés de la matrice et notamment de sa masse moléculaire. Un autre mode d’élaboration par pressage à chaud a permis de limiter la dégradation du polymère. Une meilleure maitrise de la masse moléculaire du composite serait ainsi un moyen de contrôler sa cinétique de dégradation in vivo et ainsi d’adapter ses propriétés en fonction du cahier des charges des applications visées. Dans une seconde partie, l’effet du taux de bioverre sur le caractère bioactif du composite a été évalué par immersion dans un fluide biologique de composites chargés à 20, 30 et 50% (en masse de bioverre). Un scénario de cristallisation à la surface des différents composites a ainsi été proposé. Tous les composites se sont révélés bioactifs et d’autant plus que le taux de bioverre est élevé. Le composite chargé à 50% apparait ainsi comme le matériau le plus bioactif, mais sa vitesse de dégradation est très rapide. Ce matériau étant destiné à être implanté, une étude de biocompatibilité in vitro a été menée par culture de cellules ostéoblastiques à la surface des matériaux. Enfin la biocompatibilité du composite in vivo, son caractère biorésorbable et ostéoconducteur ont été évalués par implantation du matériau composite dans les tissus musculaires et osseux de lapins. Le caractère biocompatible, bioactif et ostéoconducteur du composite chargé à 30% en masse de bioverre en fait un candidat de choix pour les applications proposées. / The aim of this work was to evaluate polylactic acid- Bioglass® composites for bone repair applications. Bioglass being too brittle to be used alone for load bearing applications, our strategy was to incorporate bioactive Bioglass® particles into a bioresorbable polymer matrix processed by conventional manufacturing techniques. The composite were processed by injection moulding from granules prepared by a solvent route. The composites exhibit higher Young modulus but lower strength and strain to failure than polymer alone. This is attributed to a decrease of molecular weight of the polymer matrix during the different steps of the process. Another processing method (hot pressing) was used to limit the drop in molecular weight of the polymer matrix: it leads to higher mechanical properties. Therefore, a careful control of the Polymer degradation may insure better mechanical properties and a better control of the degradation rate in vivo. The bioactivity of composites with 20, 30, 50 Wt. % of Bioglass® was a assessed by immersion in simulated body fluid. All the composites are bioactive, and all the more since the Bioglass® content is large. On the other side, the degradation of composites with a Bioglass® content of 50 wt. % is very rapid. Biological evaluation was conducted in vitro and in vivo. Osteoblast cell cultures and in vivo evaluation in rabbits demonstrate that polylactic acid - Bioglass® composites are biocompatible and osteoconductive. Such composites may therefore be a good option for bone repair applications in the future.

<p>There is a strong trend in fabricating <i>miniaturized total analytical systems</i>, µTAS, for various biochemical and cell biology applications. These miniaturized systems could e.g. gain better separation performances, be faster, consume less expensive reagents and be used for studies that are difficult to access in the macro world. Disposable µTAS eliminate the risk of carry-over and can be fabricated to a low cost.</p><p>This work focused on the development of µTAS modules with the intentional use for miniaturized diagnostics. Modules for blood separation, desalting, enrichment, separation and ESI-MS detection were successfully fabricated. Surface coatings were additionally developed and evaluated for applications in µTAS with complex biological samples. The first heparin coating could be easily immobilized in a one-step-process, whereas the second heparin coating was aimed to form a hydrophilic surface that was able to draw blood or plasma samples into a microfluidic system by capillary forces. </p><p>The last mentioned heparin surface was further utilized when developing a chip-based sensor for performing CD4-count in human blood, an important marker to determine the stage of an HIV-infection.</p><p>All devices in this work were fabricated in PDMS, an elastomeric polymer with the advantage of rapid and less expensive prototyping of the microfabricated master. It was shown that PDMS could be considered as the material of choice for future commercial µTAS. The devices were intentionally produced using a low grade of fabrication complexity. It was however demonstrated that even with low complexity, it is possible to integrate several functional chip modules into a single microfluidic device.</p>

There is a strong trend in fabricating miniaturized total analytical systems, µTAS, for various biochemical and cell biology applications. These miniaturized systems could e.g. gain better separation performances, be faster, consume less expensive reagents and be used for studies that are difficult to access in the macro world. Disposable µTAS eliminate the risk of carry-over and can be fabricated to a low cost. This work focused on the development of µTAS modules with the intentional use for miniaturized diagnostics. Modules for blood separation, desalting, enrichment, separation and ESI-MS detection were successfully fabricated. Surface coatings were additionally developed and evaluated for applications in µTAS with complex biological samples. The first heparin coating could be easily immobilized in a one-step-process, whereas the second heparin coating was aimed to form a hydrophilic surface that was able to draw blood or plasma samples into a microfluidic system by capillary forces. The last mentioned heparin surface was further utilized when developing a chip-based sensor for performing CD4-count in human blood, an important marker to determine the stage of an HIV-infection. All devices in this work were fabricated in PDMS, an elastomeric polymer with the advantage of rapid and less expensive prototyping of the microfabricated master. It was shown that PDMS could be considered as the material of choice for future commercial µTAS. The devices were intentionally produced using a low grade of fabrication complexity. It was however demonstrated that even with low complexity, it is possible to integrate several functional chip modules into a single microfluidic device.

The mevalonate pathway is an important biosynthetic pathway, found in all cells of virtually all known pro- as well as eukaryotic organisms. This thesis is an investigation into the use of two drugs, originally developed for different applications, but both affecting the mevalonate pathway, in to models of fracture repair. Using two different rodent models of fracture repair, a commonly used cholesterol lowering drug (statin) and two drugs used to treat osteoporosis (bisphosphonate) were applied both systemically as well as locally in order to enhance fracture repair. Papers I and II investigate the potential of simvastatin to improve the healing of femoral fractures in mice. Papers III and IV explore the use of two bisphosphonates to improve early fixation of stainless steel screws into rat bone. The statin simvastatin lead to an increased strength of the healing cellus. The application of bisphosphonates increased early screw fixation. It seems clear that both drugs have uses in orthopaedic applications. One interesting avenue of further research would be to combine the two classes of drugs and see if we can get the benefits while at the same time diminishing the drawbacks.

This thesis is focused on the design and development of an approach, allowing the fabrication of biocompatible/biodegradable self-rolled polymer tubes, which are sensitive to stimuli at physiological conditions, can be homogenously filled with cells and are able to self-assemble into a complex 3D construct with uniaxially aligned pores. These constructs are aimed to recreate the microstructure of tissues with structural anisotropy, such as of muscles and bones. The approach consists of two steps of self-assembly. As a first step, cells are adsorbed on the top of an unfolded bilayer; triggered rolling results in a parallel encapsulation of cells inside the tubes. As a second step, the formed self-rolled tubes with encapsulated cells can be assembled in a uniaxial tubular scaffold. Three polymer systems were designed and investigated in the present work in order to allow triggered folding of the bilayer. These systems allow either reversible or irreversible tube formation. The possibility to encapsulate microobjects inside self-rolled polymer tubes was demonstrated on the example of silica particles, yeast cells and mammalian cells. At conditions when bilayer film is unfolded, particles or cells were deposited from their aqueous dispersion on the top of bilayer. An appropriate change of conditions triggers folding of the bilayer and results in encapsulation of particles or cells inside the tubes. One way swelling of an active polymer allows irreversible encapsulation of cells in a way that tubes do not unroll and cells cannot escape. It was demonstrated that encapsulated cells can proliferate and divide inside the tubes for a long period of time. Since used polymers are optically transparent, encapsulated cells can be easily observed using optical and fluorescent microscopy. Reversible swelling of an active polymer provides the possibility to release encapsulated objects. It was demonstrated that in aqueous media microtubes possessing small amount of negatively charged groups on external walls self-assemble in the presence of oppositely charged microparticles that results in a formation of 3D constructs. In obtained aggregates tubes and therefore pores were well-aligned and the orientation degree was extremely high. Moreover, the approach allows the design of porous materials with complex architectures formed by tubes of different sorts. The assembly of cell-laden microtubes results in a formation of uniaxial tubular scaffold homogeneously filled with cells. The results presented in this work demonstrate that the proposed approach is of practical interest for biotechnological applications. Self-rolled tubes can be filled with cells during their folding providing the desired homogeneity of filling. Individual tubes of different diameters could be used to investigate cell behaviour in confinement in conditions of structural anisotropy as well as to mimic blood vessels. Due to their directionality tubes could be used to guide the growth of cells that is of interest for regeneration of neuronal tissue. Reversibly foldable films allow triggered capture and release of the cells that could be implemented for controlled cell delivery. In perspective, self-assembled 3D constructs with aligned pores could be used for bottom-up engineering of the scaffolds, mimicking such tissues as cortical bone and skeletal muscle, which are characterized by repeating longitudinal units. Such constructs can be also considered as a good alternative of traditional 2D flat cell culture.

This dissertation presents an investigation of micromachined three-dimensional microelectrode arrays (3-D MEAs) targeted toward in-vitro and in-vivo biomedical applications. Current 3-D MEAs are predominantly silicon-based, fabricated in a planar fashion, and are assembled to achieve a true 3-D form: a technique that cannot be extended to micro-manufacturing. The integrated 3-D MEAs developed in this work are polymer-based and thus offer potential for large-scale, high volume manufacturing. Two different techniques are developed for microfabrication of these MEAs - laser micromachining of a conformally deposited polymer on a non-planar surface to create 3-D molds for metal electrodeposition; and metal transfer micromolding, where functional metal layers are transferred from one polymer to another during the process of micromolding thus eliminating the need for complex and non-repeatable 3-D lithography processes. In-vitro and in-vivo 3-D MEAs are microfabricated using these techniques and are packaged utilizing Printed Circuit Boards (PCB) or other low-cost manufacturing techniques. To demonstrate in-vitro applications, growth of 3-D co-cultures of neurons/astrocytes and tissue-slice electrophysiology with brain tissue of rat pups were implemented. To demonstrate in-vivo application, measurements of nerve conduction were implemented. Microelectrode impedance models, noise models and various process models were evaluated. The results confirmed biocompatibility of the polymers involved, acceptable impedance range and noise of the microelectrodes, and potential to improve upon an archaic clinical diagnostic application utilizing these 3-D MEAs.

Diss. (sammanfattning) Umeå : Umeå universitet, 2010.

Made available in DSpace on 2014-06-11T19:33:28Z (GMT). No. of bitstreams: 0 Previous issue date: 2004-08-31Bitstream added on 2014-06-13T19:44:16Z : No. of bitstreams: 1 marcaccini_am_dr_arafo.pdf: 3610599 bytes, checksum: 65fbbd0cb1b0657f1b361bf4e461a54e (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Os objetivos deste estudo foram avaliar o efeito de biomateriais associados ao PRP na formação óssea em defeitos padronizados de rádio e avaliar o efeito de PRP+RTG+enxerto ósseo autógeno para tratamento de lesões de furca grau II em cães. Para tal, em um primeiro estudo, foram confeccionados 5 defeitos de 5mm em cada rádio (direito e esquerdo) em 05 cães, constituindo assim espaços para preenchimento com os enxertos ou substitutos ósseos avaliados, totalizando 50 cavidades. Os materiais testados foram DFDBA, vidro bioativo, osso autógeno e osso mineral bovino, associados ou não ao PRP. Estes grupos foram avaliados e comparados com os grupos representados por coágulo sangüíneo e PRP. Ao final de 60 dias, foi realizada biópsia e preparo laboratorial para avaliação histológica e histomorfométrica. Os grupos que apresentaram melhores resultados foram coágulo, PRP e osso autógeno associado ou não a PRP; e a utilização do PRP não promoveu maior formação óssea em relação aos demais grupos, com exceção do vidro bioativo+PRP que apresentou os melhores resultados. No segundo estudo, foram criados cirurgicamente defeitos periodontais de furca nos quartos pré-molares mandibulares, bilateralmente em 5 cães, cronificados por um período de três meses e tratados por PRP/RTG/enxerto ósseo autógeno (grupo experimental) ou RTG/enxerto ósseo autógeno (grupo controle). Quatro meses após o tratamento, os cães foram sacrificados. Na área de furca, houve maior preenchimento ósseo e extensão linear de novo cemento, nova adaptação conjuntiva e regeneração periodontal no grupo experimental (p<0.005) e maior extensão linear de epitélio no grupo controle (p<0.005). A associação do PRP a RTG e enxerto ósseo autógeno proporcionou maior regeneração dos tecidos periodontais. Portanto, o PRP promoveu melhores resultados na formação óssea somente... . / The aims of this study were to evaluate the effect of biomaterials in association to PRP on bone formation, in padronized defects of radius, and evaluate the effect of PRP/GTR/autogenous bone graft in the treatment of Class II furcation lesions in dogs. For that, in a first study, five defects of 5mm each were produced in each radius (left and right) in 5 dogs, so creating spaces to be filled with grafts or the bone substitutes under study, totalling 50 cavities. The biomaterials tested were DFDBA, bioglass, autogenous bone and bovine mineral bone grafts, associated, or not, with PRP. These biomaterials were compared with coagulum and PRP. At the end of 60 days biopsy was done and histological laminas were prepared. Under the experimental condictions, coagulum, PRP and autogenous bone associated or not to PRP presented the best results, with more new bone formation; and no difference was observed between the groups with biomaterials, associated or not with PRP, excepting bioactive glass/PRP which showded the best results. In the second study, periodontal furcation defects were surgically produced bilaterally in the fourth mandibular premolars in 5 dogs, cronified for 3 months and treated with PRP/GTR/autogenous bone graft (experimental group) or GTR/autogenous bone graft (control group). Four months after the treatment, the dogs were sacrified. In the furcation area, more bone filling and linear extension of new cement, new conjunctive adaptation and periodontal regeneration were observed to occur in the experimental group (p<0.005) and more linear extension of epitelium in the control group (p<0.005). The association PRP/GTR/autogenous bone graft was observed to produce more regeneration of periodontal tissues. Therefore, the PRP association showed the best results in bone formation only at bioactive glass group; and... (Complete abstract, click electronic address below).