The use of nanowires, nano and micro needles in biomedical applications has markedly increased in the past years, mainly due to attractive properties such as biocompatibility and simple fabrication. Specifically, these structures have shown promise in applications including cell separation, tumor cell capture, intracellular delivery, cell therapy, cancer treatment and as cell growth scaffolds. The work proposed here aims to study two platforms for different applications: a vertical magnetic nanowire array for mesenchymal stem cell differentiation and a micro needle platform for intracellular delivery. First, a thorough evaluation of the cytotoxicity of nanowires was done in order to understand how a biological system interacts with high aspect ratio structures. Nanowires were fabricated through pulsed electrodeposition and characterized by electron microscopy, vibrating sample magnetometry and energy dispersive X-ray spectroscopy. Studies of biocompatibility, cell death, cell membrane integrity, nanowire internalization and intracellular dissolution were all performed in order to characterize the cell response. Results showed a variable biocompatibility depending on nanowire concentration and incubation time, with cell death resulting from an apoptotic pathway arising after internalization. A vertical array of nanowires was then used as a scaffold for the differentiation of human mesenchymal stem cells. Using fluorescence and electron microscopy, the interactions between the dense array of nanowires and the cells were analyzed, as well as the biocompatibility of the array and its effects on cell differentiation. A magnetic field was additionally applied on the substrate to observe a possible differentiation. Stem cells grown on this scaffold showed a cytoskeleton and focal adhesion reorganization, and later expressed the osteogenic marker osteopontin. The application of a magnetic field counteracted this outcome. Lastly, a micro needle platform was fabricated through lithography and electrodeposition, characterized using the previously mentioned techniques and then evaluated as a vector for intracellular delivery. Fluorescence and electron microscopy imaging were first performed to assess the biocompatibility, cell spreading and the interface of the cells and the needles. Intracellular delivery of a fluorescent dye was achieved via inductive heating of the needles, with the results showing a dependency of delivery and cell survivability on the exposure time.

No description available.

Polymeric biomaterials have created significant advances in the field of biomedical engineering, however, very few polymeric drug delivery devices have achieved clinical and commercial success. Thus, the motivation for this thesis was to encourage long-term success of materials through expanding the fundamental understanding of polymer properties. Poly(ethylene glycol) was specifically chosen for study as its polyether backbone provides it with many unique properties that are still not fully understood, and are not seen with other similar polymers. PEG has been shown to exhibit amphiphilic character, due to its high conformational freedom, and the ability to hydrogen-bond 2-3 water molecules for each ethylene oxide subunit, creating a very structured water shell and large hydrodynamic radius. Together, the properties formed the hypothesis for the possibility for PEG to control drug release and its environment, expanding its potential in biomedical applications. This hypothesis was investigated with PEG in three states – free PEG, conjugated and blended. Free PEG was determined to inhibit melanoma cell viability by activating apoptosis via PEG effects on the osmolality of the cell medium (Chapter 3). Novel silicone hydrogels incorporating methacrylated PEG as the sole hydrophilic component showed advantageous properties for biomedical applications across a range of formulations (such as low contact angle and protein deposition), as well as altering the release of highly hydrophilic antibiotics from the materials, presumably via PEG-drug hydrogen bonding (Chapter 4). Novel siloxane-PEG blended materials were shown to have the ability to influence drug release of hydrophilic, hydrophobic and drug salts through the structure of PEG (Chapter 5). Overall, the work within this thesis expanded understanding of the abilities and limitations of PEG based on its distinct structure, and expanded the potential for PEG in biomedical applications to more than being used as simply a hydrophilic additive. / Thesis / Doctor of Philosophy (PhD) / Polymeric biomaterials have created significant advances in the field of biomedical engineering, however, very few polymeric drug delivery devices have achieved clinical and commercial success. Thus, the motivation for this thesis was to encourage long-term success of materials through expanding the fundamental understanding of polymer properties. Poly(ethylene glycol) was specifically chosen for study due to its unique exhibition of amphiphilic character and the ability to hydrogen-bond multiple water molecules, that together suggest the possibility for PEG to control drug release and its environment. Through strategic experimental designs, greater understanding of the abilities and limitations of PEG was established and shown to be the result of the distinct structure of PEG. Specifically, two novel drug delivery systems were developed with demonstrated understanding of the structure-function relationship between polymers and drugs, and the activity of PEG as a melanoma cell viability inhibitor was discovered and found correlated to the PEG structure. Overall the work within this thesis expanded the potential for PEG in biomedical applications to more than being used as simply a hydrophilic additive.

Die Anwendung von körperfremden Materialien zur Behandlung verschiedenster Krankheitsbilder, wie zum Beispiel als Zahnersatz oder Knochenstabilisierung, ist seit Jahrtausenden fester Bestandteil in der Medizin. Während damals hauptsächlich stabile Materialien genutzt wurden, die möglichst wenig mit dem menschlichen Körper interagieren, wird heutzutage ein anderer Ansatz verfolgt. Intelligente Materialien können nicht nur passiv die Heilung unterstützen, sondern aktiv zu ihr beitragen. Ein berühmtes Beispiel hierfür ist das Formgedächtnismaterial Nitinol, das in Stents zur Behandlung verengter Arterien eingesetzt wird. Diese Arbeit beschäftigt sich mit Eisen-Palladium, einem neuen Formgedächtnismaterial, bei dem der Effekt nicht wie bei Nitinol über eine Temperaturänderung sondern durch ein äußeres Magnetfeld induziert wird. Da man somit körpertemperaturbedingte Restriktionen in biomedizinischen Anwendungen umgehen kann, birgt Eisen-Palladium ein hohes Potential für Drug-Delivery Systeme oder mikromechanische Pumpen. Da eine optimale Verträglichkeit des Materials mit seiner biologischen Umgebung absolut unabdingbar ist, untersucht diese Arbeit verschiedene Möglichkeiten, die Oberfläche zu modifizieren und somit die Adhäsion biologischer Zellen zu unterstützen. Zu diesem Zweck wurden das Peptid RGD als spezifische Zelladhäsionssequenz, ein Plasmapolymer auf L-Lysin Basis als unspezifische Beschichtung und die Nanostrukturierung der Eisen-Palladium Oberfläche durch Glanzwinkeldeposition untersucht. Die verwendeten Methoden beinhalten Immunofluoreszenztests zur Quantifizierung der fokalen Kontakte zwischen Zellen und Material, theoretische Dichtefunktionaltheorie Rechnungen, sowie Kontraktilitätsmessungen mittels eines selbst entwickelten Biegebalkenaufbaus. Somit gelingt es in dieser Arbeit, die gegenseitigen Beziehungen des Materials mit der jeweiligen Oberflächenmodifikation mit den lebenden Zellen aus verschiedenen Blickwinkeln zu analysieren. Durch eine Kombination aus experimentellen und theoretischen Methoden werden die Stärken und Schwächen der einzelnen Funktionalisierungsmethoden beleuchtet und die Bildung fokaler Kontakte für eine verbesserte Zelladhäsion wird maßgeblich verbessert.

published_or_final_version / Orthopaedics and Traumatology / Master / Master of Philosophy

Even though the role of oxidative stress in a variety of disease states is known, strategies to alleviate this oxidative stress by antioxidants have not been able to achieve clinical success. Particularly, treatment of oxidative stress by small molecule antioxidants has not received due attention because of the challenges associated with its delivery. Antioxidant polymers, where small molecule antioxidants are incorporated into the polymer backbone, are an emerging class of materials that can address some of these challenges. In this work, biodegradable polymers incorporating phenolic antioxidants in the polymer backbone were synthesized. Antioxidant polymers were then characterized for their in vitro degradation, antioxidant release and their effect on oxidative stress levels (redox state) in the cells. Trolox, a water-soluble analogue of vitamin E, was polymerized to synthesize poly(trolox ester) with 100% antioxidant content which undergoes biodegradation to release trolox. Nanoparticles of poly(trolox ester) were able to suppress oxidative stress injury induced by metal nanoparticles in an in vitro cell injury model. In another study, we polymerized polyphenolic antioxidants (e.g. curcumin, quercetin) using a modified non-free-radical polymerization poly(β-amino ester) chemistry. This synthesis scheme can be extended to all polyphenolic antioxidants and allows tuning of polymer degradation rate by choosing appropriate co-monomers from a large library of monomers available for β-amino ester chemistry. Poly(antioxidant β-amino esters) (PABAE) were synthesized and characterized for their degradation, cytotoxicity and antioxidant activity. PABAE degradation products suppressed oxidative stress levels in the cells confirming antioxidant activity of degradation products.

An overarching goal of the Gerhardt laboratory is the development of an implantable neural device that allows for long-term glutamate recordings in the hippocampus. Proper L-glutamate regulation is essential for hippocampal function, while glutamate dysregulation is implicated in many neurodegenerative diseases. Direct evidence for subregional glutamate regulation is lacking in previous in vivo studies because of limitations in the spatio-temporal resolution of conventional experimental techniques. We used novel enzyme-coated microelectrode arrays (MEAs) for rapid measurements (2Hz) of extracellular glutamate in urethane-anesthetized rats. Potassium-evoked glutamate release was highest in the cornu ammonis 1 (CA1) subregion and lowest in the cornu ammonis 3 (CA3). In the dentate gyrus (DG), evoked-glutamate release was diminished at a higher potassium concentration but demonstrated faster release kinetics. These studies are the first to show subregion specific regulation of glutamate release in the hippocampus. To allow for in vivo glutamate measurements in awake rats, we have adapted our MEAs for chronic use. Resting glutamate measurements were obtained up to six days post-implantation but recordings were unreliable at later time points. To determine the cause(s) for recording failure, a detailed investigation of MEA surface characteristics was conducted. Scanning electron microscopy and atomic force microscopy showed that PT sites have unique surface chemistry, a microwell geometry and nanometer-sized features, all of which appear to be favorable for high sensitivity recordings. Accordingly, studies were initiated to improve enzyme coatings using a computer-controlled microprinting system (Microfab Technologies, Plano, TX). Preliminary testing showed that microprinting allowed greater control over the coating process and produced MEAs that met our performance criteria. Our final studies investigated the effects of chronic MEA implantation. Immunohistochemical analysis showed that the MEA produced minimal damage in the hippocampus at all time points from 1 day to 6 months. Additionally, tissue attachment to the MEA surface was minimal. Taken together with previous electrophysiology data supporting that MEAs are functional up to six months, these studies established that our chronic MEAs technology is capable of maintaining a brain-device interface that is both functional and biocompatible for extended periods of time.

Les procédés de dépôt en phase vapeur sont particulièrement performants pour la synthèse de revêtements à propriétés contrôlées. Plus spécifiquement, ce travail de recherche porte sur l'élaboration de revêtements biocompatibles, sur alliage titane TiAl6V4, obtenus par pulvérisation magnétron en conditions réactives. Dans un premier temps, nous avons décrit les procédés de mise en forme des implants commerciaux pour pouvoir les reproduire au niveau du laboratoire. Dans un second, nous avons focalisé notre étude sur l'effet de la structure cristallographique de films de zircone sur leur caractère biocompatible à partir de cultures cellulaires de fibroblastes. Les résultats ainsi obtenus démontrent très nettement des différences de comportement entre des films de zircone monoclinique, quadratique ou cubique. Dans l'objectif d'apporter des éléments d'information permettant de discuter de ces effets, d'autres séries de revêtements céramiques ont été élaborées comme par exemple des oxydes de titane ou de zirconium amorphes, de l'oxyde d'yttrium, de l'oxyde d'aluminium ou encore du carbone amorphe. Les cultures cellulaires pratiquées sur ces échantillons ont permis de démontrer le caractère biocompatible de l'oxyde d'yttrium excluant ainsi tout effet nocif de cet élément dans les zircones dopées / The vapor deposition processes are particularly successful for the synthesis of coatings with tuneable properties. More specifically, this research deals with the development of biocompatible coatings on titanium alloys TA6V obtained by magnetron sputtering in reactive conditions. At first, we described the processes to design the commercial medical implants to be able to reproduce them within the laboratory. In the second, we focused our study on the effect of the crystallographic structure of zirconia-based coatings on their biocompatible character from cell cultures of fibroblasts. The results obtained demonstrate a significant variation of cell behavior for the three the zirconia structures: monoclinic, tetragonal or cubic. In the purpose to bring relevant information that allow discussing these effects, another series of ceramic coatings were developed as for instance amorphous oxides of titanium or zirconium, yttrium oxide,, aluminum oxide or amorphous carbon. The cell response on these samples demonstrates to the biocompatible properties of the yttrium oxide, excluding any harmful effect of this element in the doped zirconia

Les travaux menés au cours de cette thèse portent sur la microfabrication d'interfaces neuro-électroniques pour des applications en neurosciences. Nous avons choisi de nos focaliser sur la réhabilitation fonctionnelle motrice et sensorielle en développant différentes matrices de micro-électrodes (MEA) respectivement, des sondes neuronales rigides et des implants rétiniens souples. Selon les applications visées, deux types de substrats ont été utilisés pour concevoir ces MEA. Pour des analyses ou expériences in-vitro, les MEA (sondes neuronales) ont plutôt été réalisées sur des substrats rigides tels que le silicium ou le verre, tandis que pour les expériences in-vivo, les MEA (implants rétiniens) ont été réalisées sur des substrats souples tels que des polymères biocompatibles (polyimide ou parylène). Ces MEA ont été fabriquées avec différents matériaux d'électrodes (diamant dopé, platine, platine noir et or) qui ont également été testés afin de déterminer leur capacité en enregistrement et/ou stimulation. De plus, à l'aide de travaux de modélisation numérique, nous avons validé le concept d'une géométrie tridimensionnelle avec grille de masse permettant une stimulation plus focale des cellules. Cette thèse a ainsi contribué à stabiliser différents procédés de fabrication pour obtenir des MEA plus reproductibles ainsi que pour améliorer leur rendement. Elle a également permis d'établir un suivi et un protocole expérimental pour assurer une traçabilité des MEA et contrôler leur performances à toutes les étapes : depuis leur fabrication au moyen de techniques électrochimiques (CV, EIS) jusqu'aux expériences biologiques in-vitro et in-vivo / The work lead during this thesis deals with microfabrication of neuro-electronic interfaces for neuroscience applications. We have chosen to focus on motor and sensory function rehabilitations by developing Micro-Electrode Arrays (MEA) respectively, rigid neural probes and flexible retinal implants. According to the targeted applications, two types of substrates have been used to achieve these MEA. For analysis or in vitro experiments, neural probes MEA have been realized on rigid substrates such as silicon or glass whereas for in-vivo experiments, retinal implants MEA have been realized on flexible substrates such as biocompatible polymers (polyimide or parylene). These MEA were made with different electrode materials (boron doped diamond, platinum, black platinum and gold) which have been tested to determine their capability in recording and/or stimulation. Moreover, with numerical modelling work, we have validated a tridimensional geometry concept with a ground grid which permits a more local stimulation of cells. This thesis has contributed to stabilize different fabrication processes to obtain more repeatable MEA and also to improve their yield. It also allowed the set-up of a follow-up and an experimental protocol to insure MEA traceability and to monitor their performances at each step since their fabrication through means of electrochemical techniques (CV, EIS) to in vitro and in-vivo biological experiments

L'objectif de ce travail est d'évaluer le rôle du facteur tissulaire (FT) dans l'initiation de la coagulation d'un circuit d'hémodialyse. A partir de l'analyse des membranes d'hémodialyse, nous avons observé que les leucocytes et, majoritairement les polynucléaires neutrophiles (PNN) adhéraient aux membranes hémocompatibles (de type AN69ST). Ces cellules expriment un FT fonctionnel. Nous avons développé différents modèles d'étude de l'expression du FT par les PNN. A partir de PNN humains obtenus chez des sujets sains, nous montrons que les PNN expriment le FT après stimulation par le TNF. L'IL-8, chemokine chimioattractante des PNN augmentent, par un effet de priming, l'expression de FT en réponse au TNF. L'inhibition de l'adhésion par un anticorps dirigé contre les β2-intégrines, induit une diminution de l'expression de FT en réponse au TNF. L'inhibition de la voie de signalisation MEK1/2, la p38 MAPK, et des radicaux libres oxygénés, inhibe également cette expression. A partir de PNN provenant de péritonites secondaires à une dialyse péritonéale, nous avons mis en évidence une forte expression de FT par ces PNN (ARNm et protéine). Le FT possède un fort potentiel pro-coagulant. Ce modèle physiopathologique est la conséquence d'une migration et d'une activation inflammatoire comparable au modèle que nous avons développé in-vitro. Dans l'objectif de faire la preuve de ce nouveau concept, nous avons évalué un facteur VII humain recombinant inactivé (FVIIai) dans un modèle d'hémodialyse chez le mouton. Nos résultats sont en faveur d'un effet anticoagulant du circuit d'hémodialyse, sans effet anticoagulant mesurable chez l'animal. / The objective of this study is to analyse the role of Tissue Factor, the unique physiological trigger on thrombin generation. Analysing haemodialysis membranes, we found that leukocytes, mainly polymorphonuclear neutrophils (PMN) adhere to hemocompatible (AN69ST) membranes. These cells express a functional TF. We next showed that human PMN obtained from healthy subjects expressed TF in response to TNF. IL-8, a major chemokine involved in PMN chemoattraction primed TNF-induced TF by PMN. TF expression was down regulated when 2 integrins were blocked by a potent antibody. The inhibition of MEK1/2, p38 MAPK and free radicals reduced TF expression. We observed, that PMN obtained from patients experiencing peritonitis, expressed high levels of TF (mRNA and protein). Functional assays measuring Xa generation and kinetics of thrombinn generation (thrombinography) indicate the stong procoagulant potential of these cells. This physiopathological model is close to our in vitro model as it results from PMN migration and inflammatoty activation. For proof of concept, we evaluated the effect of an inactivated human recombinant factor VIIa ( FVIIai) in a sheep model of hemodialysis. Our results show that FVIIai limits haemodialysis circuit coagulation without any measurable systemic anticoagulant effect