Hyaluronic acid (HA) is one of the primary chemical building blocks of the extracellular matrix and thus is an attractive material for biomedical applications. FDA approved HA-based materials are available as dermal fillers, joint viscosupplements, vitreous substitutes, and abdominal adhesion barriers. The engineering of new HA-based materials and applications is an active area of research. Here we develop several new types of HA-based hydrogels with unique and useful properties. To address the challenge of delivering hydrophobic drugs from hydrophilic hydrogel matrices we have grafted HA hydrogels with [Beta]-cyclodextrin to create hydrogels capable of binding poorly water soluble drugs. To create HA hydrogels with unique anisotropic swelling behavior we have developed a dual-crosslinking technique in which a super-swelling chemically crosslinked hydrogel is patterned with low-swelling photocrosslinked domains. When this dual-crosslinked hydrogel is swelled it contorts into a new shape because of differential swelling among photopatterned regions. To address the challenge of creating hydrogel scaffolds with biomimetic branched porosity we have invented a "crystal templating" technique. This technique grows dendritic crystals throughout a biopolymer solution, crosslinks the biopolymer around the crystals, and washes the crystals away to yield a hydrogel with a dendritic macroporous network. Lastly, we invented a method for patterning a substrate with a microarray of hydrogel compartments. A microarray of living cells is obtained when cells are seeded on the hydrogel patterned substrate. This method addresses the need for an inexpensive, simple method for obtaining living cell microarrays that does not require clean room labs and lithographic expertise. Each of these new materials were based on hyaluronic acid hydrogels but the methods are generalizable to hydrogels of other polymers too. In conclusion, the novel methods in this dissertation are a significant contribution to the engineering of HA-based materials. / text

Η παρούσα διδακτορική διατριβή πραγματεύεται την κατασκευή και τον έλεγχο ενός καινοτόμου τεχνητού χεριού, για προσθετικές κυρίως εφαρμογές, κάνοντας χρήση βιομιμητικών ενεργοποιητών και πιο συγκεκριμένα ειδικά κατεργασμένων λεπτών κυλινδρικών αγωγών από μορφομνήμονα μεταλλικά κράματα νικελίου-τιτανίου. Εκμεταλλευόμενοι τα συγκριτικά πλεονεκτήματα των ενεργοποιητών αυτών έναντι των αντίστοιχων συμβατικών, αναπτύσσεται μια πλήρως λειτουργική συσκευή με μικρό μέγεθος και βάρος, ανθρωπομορφική εμφάνιση, αθόρυβη λειτουργία και χαμηλό κόστος κατασκευής και συντήρησης, ικανή να εκπληρώσει σε μεγάλο βαθμό τις απαιτήσεις των ατόμων με αναπηρία στα άνω άκρα. Για τη φυσική υλοποίηση του σκελετού του τεχνητού αυτού χεριού χρησιμοποιείται η τεχνολογία της ταχείας προτυποποίησης. Καθένα από τα πέντε δάκτυλά του ελέγχεται ανεξάρτητα μέσω ενός υπο-ενεργοποιούμενου μηχανισμού κίνησης με τεχνητούς τένοντες. Για τον έλεγχο θέσης κάθε δακτύλου, αναπτύσσεται και εφαρμόζεται μία νέα μέθοδος ελέγχου βασισμένη στην έμφυτη δυνατότητα ανάδρασης θέσης των προαναφερθέντων ενεργοποιητών μέσω μέτρησης της ηλεκτρικής τους αντίστασης. Επιπλέον, αναπτύσσεται κατάλληλος αλγόριθμος για τον σχηματισμό διαφόρων θέσεων και συλλήψεων του τεχνητού χεριού. Για τη βελτίωση του ελέγχου, το χέρι εξοπλίζεται με αισθητήρες αφής στα ακροδάκτυλα, καθώς και με τη δυνατότητα οδήγησης συσκευών οπτικής και απτικής ανάδρασης. Όλα τα ηλεκτρονικά κυκλώματα που είναι απαραίτητα για την οδήγηση των ενεργοποιητών και τον έλεγχο του χεριού αναπτύσσονται και ενσωματώνονται στο εσωτερικό του φυσικού πρωτοτύπου. Με τη βοήθεια ειδικού προγραμματιστικού πακέτου, σχεδιάζεται μία γραφική διεπαφή ελέγχου μέσω της οποίας μελετάται και αξιολογείται η δυνατότητα του αναπτυχθέντος χεριού σε πειράματα σύλληψης διαφόρων αντικειμένων. Τέλος, προτείνονται διάφορες τεχνικές ελέγχου του χεριού από τους χρήστες του, ενώ αναπτύσσεται και κατάλληλος αλγόριθμος ελέγχου βασισμένος στη χρήση ηλεκτρομυογραφικών σημάτων. / This doctoral thesis presents the development and control of an innovative artificial hand, mostly for use in prosthetic applications, utilizing biomimetic actuators, and, more specifically, specially processed thin cylindrical wires made of shape memory nickel-titanium alloys. By exploiting the comparative advantages of these actuators over the conventional ones, a fully functional device is developed, of low size and weight, anthropomorphic appearance, silent operation, low fabrication and maintenance cost, which is capable of satisfying to a great extent the needs of the upper limb amputees. The physical implementation of the chassis of this artificial hand has been performed using rapid prototyping technology. Each of its five digits is independently controlled via a tendon-driven underactuated mechanism. For the position control of each digit, a novel control scheme is devised and implemented based on the inherent position feedback capability of these actuators via the measurement of their electrical resistance. In addition, the necessary algorithm is developed for the formation of various hand postures and prehension patterns. In order to improve the overall hand control, the hand is equipped with tactile sensors at its fingertips, and is also capable of driving optical and tactile feedback devices. All the necessary electronics for driving the actuators and controlling the hand are developed and embedded inside the physical prototype. Using a special programming package, a graphical user interface is designed, through which the grasp capabilities of the developed hand are studied and evaluated for various objects. Finally, several user control techniques of the hand are proposed, and a control algorithm based on the use of electromyographic signals is also developed.

Systemic inflammation involves a complex array of cytokines that can result in organ dysfunction. Mortality remains high despite the vast amount of research conducted to find an effective biomarker. The cause of systemic inflammation can be broad and non-specific; therefore, this research investigates using transendothelial electrical resistance (TEER) measurements to better define systemic inflammatory response syndrome (SIRS)/sepsis within a patient. Results show a difference in TEER measurements between healthy individuals and SIRS-rated patients. This research also displays correlations between TEER measurements and biomarkers currently studied with systemic inflammation (tumor necrosis factor-α, C- reactive protein, procalcitonin). Furthermore, this research also presents the groundwork for developing a microfluidic cell-based biosensor using low temperature co-fired ceramic materials. An LTCC TEER-based microfluidic device has the potential to aid in a more effective treatment strategy for patients and potentially save lives.

Springtails (Collembola) are wingless arthropods that are impressively adapted to cutaneous respiration in temporarily rain-flooded habitats by non-wetting skin morphology. Recapitulating the robust and effectively liquid-repellent surface characteristics of springtail skin in engineered materials may offer exciting opportunities for demanding applications. Herein, we present a strategy for mimicking morphological surface features of springtail skin in polymer membranes produced by reverse imprint lithography. We report the fabrication of multi-level silicon masters that, in turn, serve as templates for the replication of flexible polymer membranes. We examined the robust wetting characteristics of polymer membranes by in situ plastron collapse tests and condensation tests. The mechanical stability of the polymer membranes was tested using a tribometer set-up and compared with needle-shaped pillar structures made from similar material. The fabricated membranes are flexible, free-standing, and adaptable to various substrate materials and shapes that allow for emerging applications.

This thesis describes the development of highly selective fiber optic sensors using molecularly imprinted polymers (MIPs) as recognition elements associated with fluorescence for detection. Additionally, we extended the study to the development of other MIP-based optical sensors and sensing methods. MIPs are synthetic biomimetic receptors possessing specific cavities designed for a target molecule. Produced by a templating process at the molecular level, MIPs are capable of recognizingand binding target molecules with selectivities and affinities comparable to those of natural receptors. Compared to biological recognition elements, MIPs are more stable, cheaper and easier to integrate into standard industrial fabrication processes. Hence, MIPs have become interesting alternatives to biomolecules as recognition elements for biosensing. In the first part of this thesis (Chapter 2), MIPs were synthesized by in-situ laser-induced photopolymerization in only a few seconds, as a micrometer-sized tip at the extremity of a telecommunication optical fiber. Photonic and physico-chemical parameters were optimized to tailor the properties of the polymer micro-objects. Gold nanoparticles were incorporated into the MIP microtip for signal enhancement. To prove the efficiency of the sensor, initial studies were performed with a MIP templated with N-carbobenzyloxy-L-phenylalanine (Z-L-Phe) and the fluorescent amino acid derivative dansyl-L-phenylalanine as analyte. The fluorescence was collected either externally at the tip level by an optical fiber connected to a spectrofluorimeter or by collection of the fluorescent signal re-emitted into the fiber through the second arm of a Y-shaped bifurcated fiber. The fluorescent analyte could be detected in the low nM concentrations. In order to monitor nonfluorescent analytes, a naphthalimide-based fluorescent monomer was incorporated into the MIP during its synthesis; fluorescence enhancement was observed when analyte binding occurs. Using this system, the sensor containing a MIP specific for the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D), could detect and quantify this analyte at concentrations as low as 2.5 nM. The signaling MIP-based sensor was also applied to analytes of interest for food safety and biomedical applications, such as the mycotoxin citrinin and the sphingolipid, D-erythro-sphingosine-1-phosphate. In the second part of the thesis (Chapter 3), a different type of fiber optic sensor: cheap, fast and made for "single-use", was developed by using 4-cm long disposable polystyrene evanescent wave optical fiber waveguides. The coating of the MIP was either performed ex-situ, by dip-coating the fiber in a suspension of MIP particles synthesized beforehand, or in-situ by evanescent-wave photopolymerization directly on the fiber. The resulting fiber optic sensor could detect 2,4-D in the low nM range and demonstrated specific and selective recognition of the herbicide over its structural analogues and other non-related carboxyl-containing analytes. Additionally, we demonstrated the versatility of the system by applying the evanescent wave fiber optic sensor to detect citrinin, a mycotoxin, by simply coating the waveguide with a MIP specific for citrinin. This type of technology could possibly be extended to detect other carboxyl-containing analytes, as long as a specific MIP for the concerned analyte is available. In parallel, the technique of evanescent-wave photopolymerization was used for the synthesis of signaling MIP microdots on continuous and nanostructured gold films. This study lays the foundations for future development of plasmonic MIP nanosensors and microchips. In the last part of the thesis (Chapter 4), an innovative sensing method, based on the use of MIPs and analysis by fluorescence polarization, was developed in order to allow the fast and directquantification of analytes in food and environmental samples.

This thesis describes the development of highly selective fiber optic sensors using molecularly imprinted polymers (MIPs) as recognition elements associated with fluorescence for detection. Additionally, we extended the study to the development of other MIP-based optical sensors and sensing methods. MIPs are synthetic biomimetic receptors possessing specific cavities designed for a target molecule. Produced by a templating process at the molecular level, MIPs are capable of recognizingand binding target molecules with selectivities and affinities comparable to those of natural receptors. Compared to biological recognition elements, MIPs are more stable, cheaper and easier to integrate into standard industrial fabrication processes. Hence, MIPs have become interesting alternatives to biomolecules as recognition elements for biosensing. In the first part of this thesis (Chapter 2), MIPs were synthesized by in-situ laser-induced photopolymerization in only a few seconds, as a micrometer-sized tip at the extremity of a telecommunication optical fiber. Photonic and physico-chemical parameters were optimized to tailor the properties of the polymer micro-objects. Gold nanoparticles were incorporated into the MIP microtip for signal enhancement. To prove the efficiency of the sensor, initial studies were performed with a MIP templated with N-carbobenzyloxy-L-phenylalanine (Z-L-Phe) and the fluorescent amino acid derivative dansyl-L-phenylalanine as analyte. The fluorescence was collected either externally at the tip level by an optical fiber connected to a spectrofluorimeter or by collection of the fluorescent signal re-emitted into the fiber through the second arm of a Y-shaped bifurcated fiber. The fluorescent analyte could be detected in the low nM concentrations. In order to monitor nonfluorescent analytes, a naphthalimide-based fluorescent monomer was incorporated into the MIP during its synthesis; fluorescence enhancement was observed when analyte binding occurs. Using this system, the sensor containing a MIP specific for the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D), could detect and quantify this analyte at concentrations as low as 2.5 nM. The signaling MIP-based sensor was also applied to analytes of interest for food safety and biomedical applications, such as the mycotoxin citrinin and the sphingolipid, D-erythro-sphingosine-1-phosphate. In the second part of the thesis (Chapter 3), a different type of fiber optic sensor: cheap, fast and made for "single-use", was developed by using 4-cm long disposable polystyrene evanescent wave optical fiber waveguides. The coating of the MIP was either performed ex-situ, by dip-coating the fiber in a suspension of MIP particles synthesized beforehand, or in-situ by evanescent-wave photopolymerization directly on the fiber. The resulting fiber optic sensor could detect 2,4-D in the low nM range and demonstrated specific and selective recognition of the herbicide over its structural analogues and other non-related carboxyl-containing analytes. Additionally, we demonstrated the versatility of the system by applying the evanescent wave fiber optic sensor to detect citrinin, a mycotoxin, by simply coating the waveguide with a MIP specific for citrinin. This type of technology could possibly be extended to detect other carboxyl-containing analytes, as long as a specific MIP for the concerned analyte is available. In parallel, the technique of evanescent-wave photopolymerization was used for the synthesis of signaling MIP microdots on continuous and nanostructured gold films. This study lays the foundations for future development of plasmonic MIP nanosensors and microchips. In the last part of the thesis (Chapter 4), an innovative sensing method, based on the use of MIPs and analysis by fluorescence polarization, was developed in order to allow the fast and directquantification of analytes in food and environmental samples.

At low Reynolds number flows, the effect of inertia becomes negligible and the fluid motion is dominated by the effect of viscous forces. Understanding of the behaviour of low Reynolds number flows underpins the prediction of the motion of microorganisms and particle sedimentation as well as the development of micro-robots that could potentially swim inside the human body to perform targeted drug/cell delivery and non-invasive microsurgery. The work in this thesis focuses on developing an understanding in the mathematical analysis of objects moving at low Reynolds numbers. A boundary element implementation of the Method of regularized Stokeslets (MRS) is applied to analyse the low Reynolds number flow field around an object of simple shape (sphere and cube). It also showed that the results obtained by a boundary element implementation for an unbounded cube, where singularities are presented in the corners of the cube, agrees with more complex solutions methods such as a GBEM and FEM.A methodology for analysing the effect of walls by locating collocation points on the surface of the walls and the object is presented. First at all, this methodology is validated with a boundary element implementation of the method of images for a sphere at different locations. Then, the method is extended when more than one wall is presented. This methodology is applied to predict the velocity filed of a cube moving in a tow tank at low Reynolds numbers for two different cases with a supporting rod similar to an experimental set-up, and without the supporting rod as in the CFD simulations based on the FVM. The results indicate a good match between CFD and the MRS, and an excellent approximation between the MRS and experimental data from PIV measurements. The drag, thrust and torque generated by helices moving at low Reynolds numbers in an unbounded medium is analysed by the resistive force theory, a slender body theory, and a boundary element method of the MRS. The results show that the resistive force theory predict accurately the drag, thrust and torque of moving helices when the resistive force coefficients are calculated from a slender body theory approximation by calculating independently the resistive force coefficients for translation and rotation, because it is observed that the resistive force coefficients depend also of the nature of motion. Moreover, the thrust generated by helices of different pitch angles is analysed calculated by a CFD numerical simulation based on the FVM and a boundary element implementation, an compared with experimental data. The results also show an excellent prediction between the boundary element implementation, the CFD results and the experimental data. Finally, a boundary element implementation of the MRS is applied to predict swimming of a biomimetic swimmer that mimics the motion of E.coli bacteria in an unbounded medium. The results are compared with the propulsive velocity and induced angular velocity measurement by recording the motion of the biomimetic swimmer in a square tank. It is observed that special care needs to be taken when the biomimetic swimmer is modelled inside the tank, as there is an apparent increment in the calculate thrust propulsion which does not represent a real situation of the biometic swimmer which propels by a power supply. However, this increment does not represent the condition of the biomimetic swimmer and a suggested methodology based on the solution from an unbounded case and when the swimmer is moving inside the tank is presented. In addition, the prediction of the free-swimming velocity for the biomimetic swimmer agrees with the results obtained by the MRS when the resistive force coefficients are calculated from a SBT implementation. The results obtained in this work have showed that a boundary element implementation of the MRS produces results comparable with more complex numerical implementations such as GBEM, FEM, FVM, and also an excellent agreement with results obtained from experimentation. Therefore, it is a suitable and easy to apply methodology to analyse the motion of swimmers at low Reynolds numbers, such as the biomimetic swimmer modelled in this work.

Notre équipe a développé un protocole d’ingénierie de l’organe dentaire basé sur la biomimétique et l’utilisation de cellules dentaires embryonnaires dissociées. La recherche de ressources cellulaires permettant d’éviter le recours aux cellules embryonnaire reste un défi majeur, et nécessite une meilleure connaissance des paramètres limitants. Nous avons testé les potentialités odontogènes de lignées cellulaires, dentaires ou non, embryonnaires ou adultes. Le développement dentaire étant contrôlé par des interactions réciproques entre ectomésenchyme dérivé des crêtes neurales et épithélium, ces cellules ont été réassociés à un épithélium dentaire compétent. Nous avons recherché la formation de dents in vitro et/ou après implantation chez la souris adulte et étudié un certain nombre de paramètres biologiques et techniques. Ainsi, nous avons étudié l’impact de l’âge, de la mise en culture, et de l’hétérogénéité cellulaire sur les potentialités odontogènes des cellules mésenchymateuses. Nos résultats montrent que le potentiel odontogène des différentes lignées mésenchymateuses testées pouvait être lié à l’âge des cellules et qu’il est perdu lorsque les cellules mésenchymateuses sont cultivées avant d’être ré-associées. Ceci pouvant s’expliquer par un changement phénotypique, nous avons testé un certain nombre de gènes essentiels au développement dentaire, et suivi l’expression de marqueurs de surface. Les changements observés peuvent être liés à une sélection cellulaire in vitro pouvant conduire à des modifications de l’hétérogénéité des cellules en monocouche. / Our laboratory has developed a protocol for tooth organ engineering, based on biomimetic and the use of dissociated embryonic dental cells. Searching for mesenchymal cell sources avoiding the use of embryonic cells still remains a major challenge. We tested the odontogenic potential of several cell lines, dental or not, embryonic or adult. These cells were re-associated with a competent intact dental epithelium. We searched for tooth formation in vitro and/or after implantation in adult mice and studied different biological and technical parameters. For this purpose we analyzed the effects of the age, of a pre-culture step, and of the cellular heterogeneity on the odontogenetic potential of mesenchymal cells. To test the heterogeneity, we compared the patterns of expression of cell surface markers in cultured and implanted re-association with those observed during tooth development..Our results show that the absence of odontogenetic potential in the different cell lines that were tested, in part depends on the age of cells and that it is lost when mesenchymal cells are cultured in monolayer before their re-association. This could be explained by phenotypical changes as shown by testing several genes involved in tooth development, and tracing cell surface markers expression. Changes were observed, wich could be related to a cell selection in vitro, leading to variations in cellular heterogeneity. Indeed, pulpal cellular heterogeneity shows specific patterns of expression, that are space-time defined during tooth development, and is controlled by epithelial-mesenchymal interactions.

Hydroxyapatite (HAP) coatings are used in orthopaedic surgery for bone regeneration. Current methods of phase quantification of HAP coatings suffer from drawbacks. A novel methodology of quantitative phase analysis of HAP coatings has been devised and validated. This method, based on whole pattern fitting with a fundamental parameters approach, incorporates amorphous calcium phosphate (ACP) and apatite phases into structural refinements. A comparison of the structural and chemical properties of plasma sprayed (PS) and novel electrodeposited (ED) HAP coatings has been conducted. ED coatings contained less ACP and more preferred orientation than the PS coatings, although the stoichiometry was similar. In vitro investigations of PS and ED coatings in simulated body fluid and foetal calf serum revealed that both are bioactive. A carbonated apatite layer produced on the ED coatings was -0.7μm thick with a stoichiometry and chemical constituents similar to that of natural bone apatite. PS coatings produced a nanocrystalline carbonated apatite layer (-4μm). For the first time it has been possible to model crystalline HAP and nanocrystalline apatite as independent phases and obtain accurate lattice parameters for each. A positive linear correlation has been made between microstrain and the solubility of HAP and carbonated apatites. Dissolution studies have shown that the behaviour of HAP and carbonated apatite is dominated by crystallite size at low undersaturation and by crystallite size and microstrain at high undersaturation for crystallites between -30OA- 1000A. Metastable equilibrium occurred for crystallites <_400A at low undersaturation. Carbonate content did not affect the solubility or dissolution behaviour. A novel technology for coating polymeric tape with HAP for potential use in anterior cruciate ligament reconstruction has been devised. Mechanical tests have demonstrated that no adverse properties are induced by the coating technology. Cell culture studies have shown that the HAP layer is capable of enhanced attachment, proliferation and differentiation of osteoblast cells compared to uncoated tape.

L’objectif de ce travail est l’obtention de mimes efficaces d’hémoprotéines telles le cytochrome P450, la myoglobine ou la cytochrome c oxydase, grâce à des variations synthétiques autour d’une porphyrine à anse phénanthroline (Porphen). Un nouveau modèle de cytochrome c oxydase a plus particulièrement été analysé ici. Il est préparé par substitution des deux positions meso d’une Zn-Porphen. Des phénomènes dynamiques ont été observés et étudiés par RMN 1H, mettant en évidence la présence d’atropoisomères et la coordination-décoordination de la pyridine proximale sur le zinc. Le remplacement du zinc par du fer a ensuite permis l’étude de la coordination d’un sixième ligand exogène dans un site distal dynamique. L’évolution de la géométrie du complexe a été suivie par spectrophotométrie UV-Visible et RPE. En présence de ligands azotés de type midazoles, il se forme dans tous les cas des complexes [1 récepteur/ 1 substrat]. La forte affinité de notre modèle pour le dioxygène a été montrée à la fois par spectrophotométrie UV-Visible, RMN 1H et par résonance Raman. Que ce soit en UV-Visible ou en RMN, la réversibilité du dioxygène a été montré par son remplacement par du CO. La souplesse de cette nouvelle architecture a été mise en évidence, par l’observation d’une relative flexibilité lors des études par spectroscopie IR de la fixation de CO dans le site distal. Cette adaptabilité est également à l’origine d’un comportement assez surprenant en électrochimie, où la réduction du fer(III) et l’oxydation du cuivre(I) en présence de O2 sont facilitées. En électrocatalyse, la réduction de O2 par ce nouveau modèle de cytochrome c oxydase n’est pas facilitée en terme de potentiel, mais efficace quant à la contribution d’un mécanisme à 4 électrons. / The purpose of this work was to prepare efficient models of cytochrome P450, hemoglobin and cytochrome c oxidase, by various synthetic modifications on a phenanthroline-strapped porphyrin (Porphen). In particular, a new model of cytochrome c oxidase was analyzed here. This compound was obtained by substitution of both meso positions of a Zn-Porphen. Dynamics phenoma were observed and analyzed by 1H NMR, showing the presence of atropoisomers and coordination-decoordination of the proximal pyridine on zinc. Zinc was then replaced by iron, which allows the coordination of a sixth exogenous ligand in the dynamic distal site. The evolution of the complexes’ geometry was monitored by UV-Visible spectrophotometry and EPR. In the presence of imidazolesligands, complexes [1 receptor/ 1 substrate] were observed in all cases. Our model’s high affinity for dioxygen was shown by UV-Visible and 1H NMR spectroscopy and Raman resonance. In UV-Visible and NMR studies, the reversibility of dioxygen binding was demonstrated by replacement with CO.The versatility of this new architecture was demonstrated during IR studies by the relative flexibility of the CO binding in the distal site. This versatility also led to surprisingly behavior in electrochemistry, where the reduction of iron(III) and the oxidation of copper(I) were easier in the presence of O2. In electrocatalysis, the reduction of O2 by this new cytochrome c oxidase model was not easier in terms of potentiel, but was efficient in a 4-electrons mechanism.