Tesis sobre el tema "Gels and Hydrogels"

Molecular self-assembly provides a simple and efficient route of constructing well-defined nanostructures which may serve as extra cellular matrix (ECM) mimics. This work focuses on two specific octapeptides: FEFEFKFK and FEFKFEFK (F: phenylalanine, E: glutamic acid, K: lysine) with alternating charge distribution. The peptides were shown to self-assemble in solution and form β-sheet rich nanofibres which, above a critical gelation concentration (CGC), entangle to form self-supporting hydrogels. The fibre morphology of the hydrogels was analysed using TEM and Cryo-SEM illustrating the dense fibrillar network of nanometer size fibres. Oscillatory rheology results showed that the hydrogels possesses viscoelastic properties. By varying peptide concentration and type hydrogel stiffness, viscosity, water content, fibre density and other mechanical properties were tailored to control cell interactions and subsequent tissue growth. Bovine chondrocytes were used to assess the biocompatibility of these novel scaffolds over 21 days under 2D and 3D cell culture conditions, particularly looking into cell morphology, proliferation and matrix deposition. 2D culture resulted in cell viability and collagen type I deposition. In 3D culture, the mechanically stable gel was shown to support viability, retention of cell morphology and collagen type II deposition. Subsequently, the scaffold may serve as a template for cartilage repair. In addition, this research also focused on developing novel injectable scaffold design with in situ gelation properties to encapsulate chondrocytes for cell culture applications.

This diploma thesis deals with hyaluronan modified by palmitoyl and its gelation. Gels were created from palmitoyl hyaluronan with molecular weight 216 kDa and degree of substitution 11 % in concentrations 15 and 20 g dm-3 in water and concentrations 10, 15, 20 g dm-3 in NaCl and TSB. Also gel from palmitoyl hyaluronan with molecular weight 35 kDa and degree of substitution 10 % in concentrations 20, 30 g dm-3 in NaCl was created. Gels were investigated concerning medical applications. Gels were rigid and had very good properties, which was confirmed by rheology. The physical properties (pH, water content) of gels and stability were investigated. On the grounds of the MTT test, three methods of cell incorporation were suggested. Gels are nontoxic, biocompatible, and biodegradable with nontoxic degradation products and that is why they are excellent aspirants for use in biomedicine.

Un hydrogel est un matériau mou, largement gonflé d’eau, rendu élastique via un réseau de chaînes de polymère. Un gel est intrinsèquement fragile. On peut remédier à cette fragilité grâce à l’ajout de liaisons sacrificielles dynamiques. L’ingénierie macromoléculaire a permis au XXIème siècle de formuler des gels à destination de la biologie afin de proposer des matériaux de synthèse tout en remédiant aux problèmes de biocompatibilité, à la compatibilité des interfaces tissu/matériau et des propriétés mécaniques dont le corps a besoin. Pourtant, la fracture de ces matériaux hautement déformables et parfois viscoélastiques reste un sujet mal compris et assez peu investigué expérimentalement. Le défi aujourd’hui est de mieux comprendre les mécanismes mis en jeu en pointe de fissure mais les techniques expérimentales qui permettent une approche locale et avec des cadences d’acquisition rapides sont limitées. Notre travail vise à développer une méthode innovante pour sonder la fracture des gels. L’eau étant leur principal composant, ces matériaux, comme les tissus biologiques, sont une excellente plateforme pour l’étude de la propagation d’ondes acoustiques, i.e. de cisaillement (S) ou de compression (P). Dans les matériaux composés principalement d’eau, les ondes de compression, typiquement les ultrasons, se propagent à environ 1500 m/s (vitesse des ondes P dans l’eau) alors que les ondes de cisaillement sont de l’ordre du m/s (entre environ 1-8 m/s) et leur vitesse augmente avec la rigidité du matériau. Il est donc possible de voir les ondes S se propager grâce à la différence de vitesse entre ces deux ondes. C’est le principe de l’élastographie par onde de cisaillement, technique d’imagerie utilisée dans cette étude pour comprendre la mécanique et la fracture des hydrogels.La fracture des gels a été étudiée localement en pointe de fissure de manière quasi-statique. Ensuite, les phénomènes physiques mis en jeu lors de la propagation d’une fissure ont été investigués grâce à l’imagerie ultrarapide.Il est important de comprendre comment la fracture se propage et s’il est possible de l’éviter ou de la stopper. Le but de tout matériel est d’éviter de casser et donc de résister à la propagation de fracture<br>A hydrogel is a soft material, largely swollen with water, made elastic via a network of polymer chains. A gel is inherently fragile. This brittleness can be overcome by adding dynamic sacrificial bonds. Macromolecular engineering of the 21st century has made possible the formulation of gels for use in biology in order to provide synthetic materials while addressing biocompatibility issues, tissue/material interface compatibility, and mechanical properties that the body requires. However, the fracture of these highly deformable and sometimes viscoelastic materials remains a poorly understood subject that has been little investigated experimentally. The challenge today is to better understand the mechanisms involved at the crack tip but the experimental techniques that allow a local approach and with fast acquisition rates are limited. Our work aims at developing an innovative method to probe the fracture of gels. Water being their main component, these materials, like biological tissues, are an excellent platform to study the propagation of acoustic waves, i.e. shear (S) or compression (P) waves. In materials composed mainly of water, compressional waves, typically ultrasound, propagate at about 1500 m/s (P-wave velocity in water) while shear waves are of the order of m/s (between about 1-8 m/s) and their velocity increases with the rigidity of the material. It is therefore possible to see the S waves propagating through the difference in speed between these two waves. This is the principle of shear wave elastography, an imaging technique used in this study to understand the mechanics and fracture of hydrogels.The gel fracture was studied locally at the crack tip in a quasi-static way. Then, the physical phenomena involved during crack propagation were investigated using ultrafast imaging.It is important to understand how the fracture propagates and if it is possible to avoid or stop it. The goal of any material is to avoid breaking and therefore to resist fracture propagation

Les hydrogels sont des polymères de plus en plus utilisés dans le domaine des biomatériaux. À cause de leur faible différence de densité avec le milieu environnant, ils sont difficiles à visualiser en imagerie par résonnance magnétique (IRM). Des agents de contraste sont couramment utilisés pour faciliter la distinction entre les tissus pendant la visualisation en IRM. L'objectif de ce projet de thèse est donc de développer un agent de contraste paramagnétique et de l'encapsuler dans des hydrogels afin d'en permettre la visualisation en IRM. Des nanoparticules de silice mésoporeuses (MSN) ont été synthétisées et fonctionnalisées avec un agent de contraste cliniquement approuvé, le DTPA-Gd (acide diethylènetriamine pentaacetique complexé avec du gadolinium). Ces nanoparticules ont été caractérisées et leurs propriétés relaxométriques ont été mesurées. Le ratio r2/r1 = 1.46 démontre leur efficacité comme agent de contraste "positif". Ces nanoparticules sont ensuite encapsulées dans des hydrogels biocompatibles par différentes stratégies : dans un hydrogel de poly (éthylène glycol) (PEG) par agitation, ou dans un hydrogel d'alginate par émulsion dans un réacteur. Ces produits ont ensuite été mis en forme, respectivement pour des applications en chirurgie interventionnelle pour des aiguilles de biopsies et en microencapsulation pour le traitement du diabète de type 1. Les performances relaxométriques de l'hydrogel de PEG ont été confirmées par NMRD (Nuclear Magnetic Relaxation Dispersion) à différents champs magnétiques. Cet hydrogel a ensuite été déposé par trempage-retrait sur des substrats de titane simulant des aiguilles de biopsie. Les substrats ont été préalablement lavés et fonctionnalisés avec du phosphate acrylate. En modulant la viscosité à l'aide d'un agent gélifiant, un hydrogel de PEG de 40 à 70 µm d’épaisseur a été obtenu en surface de tubes de titane. Les aiguilles recouvertes ont révélé un contour brillant en IRM. Des séquences d’acquisition en gradient d’écho de moins de 3 min ont permis d’obtenir un rehaussement de signal de 178% par rapport à l’eau. Pour les hydrogels d'alginate, un rehaussement de contraste jusqu'à 113 % a été quantifié par rapport aux billes sans agent de contraste, avec une séquence d'écho de spin de 4 min. Un suivi par IRM sur plusieurs mois a aussi permis de confirmer que les deux hydrogels ne relarguent pas de gadolinium au cours du temps. Ces résultats confirment la possibilité de suivre ces hydrogels à long terme sans perte de signal, ce qui est essentiel à la poursuite de procédures in vivo. Ce travail présente donc des hydrogels paramagnétiques à fort rehaussement de contraste en IRM grâce à l'insertion de nanoparticules de silice mésoporeuses fonctionnalisées avec un agent de contraste. Les résultats obtenus démontrent l'efficacité des MSN-DTPA-Gd encapsulées dans des hydrogels pour visualiser ces derniers par IRM. Ces travaux permettront une visualisation des hydrogels à long terme après implantation dans le corps humain.<br>Hydrogels are polymers increasingly used in the field of biomaterials. Due to their low density difference with the surrounding middle, they are very difficult to visualize with magnetic resonance imaging (MRI). Contrasts agents are widely used in MRI to differentiate the different biological tissues during the imaging. The main objective of this project was the development of a paramagnetic contrast agent trapped in biocompatible hydrogels enabling their visualization in MRI. Mesoporous silica nanoparticles (MSN) were synthesized and functionalized with a clinically approved contrast agent, DTPA-Gd (gadolinium-diethylenetriamine pentaacetic acid). The nanoparticles were characterized and their relaxometric properties were evaluated. The r2/r1 relaxometric ratio of 1.46 revealed an efficient “positive” MRI contrast agent. Then, different entrapment strategies were performed in biocompatible polymers forming hydrogels: in a poly (ethylene glycol) (PEG) hydrogel (by stirring) or in an alginate hydrogel (by emulsion). These products were designed for applications in interventional surgery for biopsy needles and in microemulsion for type 1 diabetes treatment, respectively. The relaxometric performances of the PEG hydrogel were assessed by NMRD (Nuclear Magnetic Relaxation Dispersion) at different magnetic field strengths. Then, the paramagnetic hydrogel was coated on titanium substrates as substitute of biopsy needles. The substrates were cleaned and functionalized with phosphate acrylate prior to dip-coating. With a thickening agent in the suspension, PEG hydrogels of 40 to 70 µm were deposited on titanium tubes. These samples showed bright outline in MRI. A signal enhancement of 178 %, in regard with water, was obtained with gradient echo sequences shorter than 3 min. For the alginate hydrogels, beads with contrast agent showed a contrast 113 % enhanced, compared to beads without contrast agents, with a spin echo sequence of 4 min. MRI monitoring over months was done to confirm the persistence of the nanoparticles entrapment in both the PEG and alginate hydrogels. These results settled the possibility to use these hydrogels in the long term with no signal decrease, which is essential for in vivo processes. This work introduced paramagnetic hydrogels with a high contrast enhancement in MRI due to the entrapment of mesoporous silica nanoparticles functionalized with a contrast agent. Results confirmed the efficiency of the MSN-DTPA-Gd trapped in the hydrogels to visualize them in MRI. This work could lead to a long term visualization of hydrogels after implantation in the body.

A variety of “clickable” self-assembling peptide-amphiphile hydrogel systems are reported. Covalently linked hydrogels were prepared using alkyne-azide “click” chemistry and thiolene photochemistry, which were used in combination with short self-assembling peptideamphiphile compounds. For alkyne-azide “click” hydrogels, samples were formed from a mixture of two peptideamphiphiles which were separately disubstituted with alkyne or azide functionalised amino acids. This allowed for production of an extensively triazole-linked hydrogel product when gelation was performed in combination with the appropriate “click” pre-catalyst and reductant. For thiol-ene photochemical hydrogels samples were formed from a mixture of two peptideamphiphiles which were separately disubstituted with cysteine or Alloc-protected lysine. UV exposure was used to catalyse the covalent linking of the asembled hydrogel. Michael addition thiol-ene reactions were also investigated as a potential covalent linking method. A number of model reactions were attempted on specially synthesised amino acids. Results were promising, but a number of difficulties were encountered which made them unsuitable for incorporation in a hydrogel system. The hydrogel samples produced were all found to be viscoelastic hydrogels through analysis by rheological methods. A variation in the stiffness of the samples was observed, with samples having Young’s modulus values in the soft to intermediate range when compared to that of various tissues. SEM analysis indicated the hydrogels exhibited a fibrous nanostructure. The biological activity of the hydrogel samples was investigated by 2D seeding of cells on hydrogel samples. A LIVE/DEAD assay was performed which indicated hydrogel samples were able to support cell attachment and growth in vitro.

Cette thèse a exploré l'intégration de moteurs moléculaires synthétiques photoactivables dans des réseaux de gels supramoléculaires. L'objectif principal était d'obtenir un mouvement macroscopique réversible en exploitant à la fois la rotation unidirectionnelle des moteurs moléculaires et la nature réversible des interactions supramoléculaires. Des moteurs moléculaires hautement fonctionnalisés ont été synthétisés et intégrés comme unités de réticulation dans des réseaux de gel supramoléculaire de peptides de diphénylalanine et de poly(γ-benzyl-L-glutamate) et d'oligonucléotides d'ADN. L'activation de la rotation unidirectionnelle des moteurs moléculaires par la lumière a permis de produire un travail nanomécanique suffisant pour perturber les interactions supramoléculaires dans les réseaux de gel à base de peptides, ce qui entraîne la contraction ou la fonte du gel à l'échelle macroscopique. Grâce aux interactions supramoléculaires réversibles, le matériau gélifié initial a pu être récupéré dans l'obscurité, soit spontanément, soit par l'application d'un stimulus thermique. Les systèmes étudiés dans cette thèse représentent une nouvelle classe de matériaux fonctionnant dans des conditions dissipatives hors équilibre, promettant des applications dans divers domaines tels que la biologie, la médecine et la science des matériaux<br>This thesis explored the integration of light-driven synthetic molecular motors in supramolecular gel networks. The main goal was to achieve reversible macroscopic motion by exploiting both the unidirectional rotation of molecular motors and the reversible nature of supramolecular interactions. Highly functionalized molecular motors have been synthesized and integrated as crosslinking units in supramolecular gel networks of diphenylalanine and poly(γ- benzyl-L-glutamate) peptides, as well as DNA oligonucleotides. Activation of the unidirectional rotation of molecular motors by light, allowed the production of nanomechanical work which is sufficient to disrupt supramolecular interactions in peptide-based gel networks leading to contraction or melting of the gel material at the macroscopic scale. Thanks to the reversible supramolecular interactions, the initial gel material was recovered in the dark, either spontaneously or by applying a thermal stimulus. The systems studied in this thesis represent a novel class of materials operating in dissipative out-of-equilibrium conditions, holding promise of applications in various fields such as biology, medicine and material science

Les assemblages colloïdaux représentent une nouvelle piste très prometteuse dans le domaine de l'ingénierie tissulaire. Idéalement, ce type d'assemblage permet l'obtention de matériaux injectables et gélifiants sur le site lésionnel, favorisant par la suite le développement de néo-tissus viables. Ce travail porte sur la formation de tels assemblages à base de chitosane et de poly(acide lactique) (PLA). Deux types d'assemblages ont été conçus et étudiés dans ce travail. Dans une première approche, le mélange de particules anioniques de poly (acide lactique) (PLA) avec du chitosane en solution faiblement acide conduit à la formation de « gels composites », résultant des interactions colloïde-polymère. Des analyses rhéologiques et de diffusion des rayons X aux petits angles ont permit de mettre en évidence le mode de formation et l'influence de plusieurs paramètres sur les propriétés finales de ces gels. Notamment, ils présentent des propriétés rhéofluidifiantes et un caractère réversible, c'est-à-dire que le gel peut se reformer après déstructuration mécanique. Le second type d'assemblage résulte du mélange de particules anioniques de PLA et de nanogels cationiques de chitosane, conduisant à la formation de « gels colloïdaux », par interactions colloïde-colloïde. L'influence de plusieurs facteurs sur la formation et les propriétés de ces gels a également été étudiée par mesures rhéologiques. Notre étude s'est notamment orientée sur la caractérisation et la stabilité des hydrogels physiques de chitosane sous forme colloïdale, ainsi que sur l'optimisation de leur cohésion<br>Colloidal assemblies may be a promising pathway to obtain injectable scaffolds favoring the development of neo-tissue in regenerative medicine. This work investigates the formation of such assemblies composed of chitosan, soluble or in suspension (nano-hydrogel), and poly(lactic acid) (PLA) nanoparticles. Two types of assemblies are studied. As a first approach, mixing negatively charged PLA particles and chitosan solution leads to the formation of “composite gels”, based on colloidpolymer interactions. Rheological and Small Angle X-Ray Scattering measurements highlighted the formation process and the influence of various parameters on final properties of these gels, which features shear-thinning and reversibility behavior, that is, the capacity to gel again after yielding. PLA nanoparticles could also be mixed with cationic chitosan nanoparticles, which are crosslinker free nano-hydrogels, leading to the formation of “colloidal gels”, based on colloid-colloid interactions. Influence of various parameters on gel synthesis and properties are investigated through rheological measurements. The study also focuses on the characterization and control of the morphological and cohesion properties of chitosan nanogel

Les matériaux polymères multi stimulables sont d’ores et déjà utilisés dans différents domaines d’applications, tels que le relargage de principes actifs sur commande, l’ingénierie tissulaire, les matériaux auto réparants ou les senseurs. Depuis une vingtaine d’années, la chimie supramoléculaire s’est révélée être un outil de choix pour créer ce type de matériaux dits « intelligents ». Elle permet en effet de moduler voir de programmer les propriétés des matériaux en contrôlant le caractère dynamique des interactions supramoléculaires via l’application de stimuli adaptés. Les travaux réalisés dans le cadre de cette thèse financée par l’ANR (projet STRAPA) avaient pour principal objectif d’exploiter des complexes à base de cyclobis paraquat paraphénylène (CBPQT4+) et d’entités riches en électrons (tétrathiafulvalène, naphtalène) pour concevoir des hydrogels supramoléculaires multi-stimulables. Deux types d’hydrogels ont été développés : des hydrogels physiques (réticulés de manière supramoléculaire) capables de présenter une transition sol-gel sous stimuli (température, ajout de molécules compétitrices) et des hydrogels chimiques (réticulés de manière permanente) dotés de motifs de reconnaissance moléculaire riches en électrons dont les propriétés de gonflement peuvent être finement contrôlées. En particulier, nous avons montré que celles-ci pouvaient être manipulées très facilement via le nombre d’unités riches en électrons présents au sein des hydrogels, en contrôlant le pourcentage de complexes formés, ainsi qu’en appliquant divers stimuli (température, red/ox, macromolécules compétitrices, tensioactifs)<br>Multistimuli-responsive polymer materials play an important role in various fields of applications, (drug delivery system, tissue engineering, and self-healing materials. In the last past decades, supramolecular chemistry has emerged as a powerful tool to build such smart materials. Indeed, thanks to the inherent and/or induced dynamic behavior of supramolecular interactions, materials properties can be potentially tuned or even programmed. The main objective of this thesis, that have been carried out in the framework of the STRAPA ANR project, was to exploit host-guest interactions formed from the cyclobis paraquat paraphenylene (CBPQT4+) host molecule and electron-rich entities (tetrathiafulvalene, naphthalene) to conceive multi-stimuli responsive hydrogels. Two kind of smart hydrogels have been developed : physical hydrogels in which the sol-gel transition can be controlled upon heating or by adding competitive molecules, and chemical hydrogels with programmable swelling properties. In the last case, we have notably shown that the actuating behavior of hydrogels could be finely triggered by applying various environmental stimuli (T, red/ox, competitive macromolecules and surfactants)

Made available in DSpace on 2014-10-09T12:43:27Z (GMT). No. of bitstreams: 0<br>Made available in DSpace on 2014-10-09T14:09:46Z (GMT). No. of bitstreams: 1 06632.pdf: 4097419 bytes, checksum: 1cde4c2787112f93fcca894974c568fd (MD5)<br>Tese (Doutoramento)<br>IPEN/T<br>Instituto de Pesquisas Energeticas e Nucleares - IPEN/CNEN-SP

Doubly cross-linked gels were fabricated based on tetra-poly(ethylene glycol) (Tetra-PEG) by shear strain. These are gels with two network structures present in the same polymeric network. The second network structure is introduced by applying a mechanical field to the first natural network structure. These doubly cross-linked gels indicated a negative energy elasticity supporting earlier findings where the energy elasticity was found significantly negative for Tetra-PEG gel. Acquired results indicate implications for past research on the elasticity of polymer gels where the energy contribution was approximated to zero. Obtained results also indicated that the modulus of rigidity for the doubly cross-linked gels is constant regardless of applied shear strain during fabrication. This would indicate that the same second network structure is formed for the interval of 25-800% applied shear strain. The residual strain for the fabricated gels can be well-described using an exponential fitting of the apparent shear modulus of the first network structure and an expression derived from the two-network theory and classic rubber theory. These theories also seem to predict the experimental residual strains for lower strain regions (&lt;100%) quite well. However for larger strain regions (&gt;100%) non-linear effects seem to affect the results causing a deviation. A slight increased modulus of rigidity was noted for the doubly cross-linked gels compared to the regular Tetra-PEG gel. However as the reproducibility of the concluded measurements could not be confirmed during this thesis the results are not conclusive and only indicate the conclusions mentioned above.

Les gels de protéine nanocomposites sont un sujet encore peu développé dans la littérature malgré de nombreuses applications allant de l’immobilisation d’enzyme aux prothèses en passant par les gels alimentaires. La protéine permet d’assurer la biocompatibilité des gels tandis que l’ajout des nanoparticules a pour but de moduler les propriétés mécaniques des gels. Nous avons donc décidé de nous intéresser aux gels d’hémoglobine réticulée chimiquement et dopés aux nanoparticules. L’hémoglobine (Hb) a été choisie pour sa grande abondance et ses propriétés de fixation du dioxygène. Les gels seront obtenus par réticulation par le glutaraldéhyde (GTA), un dialdéhyde très réactif. Les gels seront dopés par des nanoparticules de silice (NP) afin de comprendre déjà l’effet sur le gel du dopage par des nanoparticules modèles. La première partie de la thèse portera sur l’adsorption de l’hémoglobine sur les nanoparticules de silice afin de lever les dernières inconnues sur ce phénomène déjà étudié. Il sera mesuré les isothermes d’adsorption ainsi que l’activité de l’hémoglobine adsorbée. Les structures de l’hème, de la globine et de l’assemblage Hb/NP seront étudiées avec détails. Par la suite, les études se porteront sur les gels sans et avec nanoparticules afin d’élucider les effets de la gélification et du dopage respectivement. On déterminera les concentrations en Hb, GTA et NP permettant d’obtenir un gel. Puis, comme pour les assemblages Hb/NP, nous nous intéresserons à l’activité et à la structure de Hb (hème et globine). La structuration du gel sera de plus étudiée. Des études sur les propriétés élastiques des gels seront aussi menées et nous finirons sur la dynamique de la protéine gélifiée. Quand il sera possible, l’effet des concentrations des différents composants sera déterminé. Pour toutes ces études, il a été utilisé un vaste panel de techniques de caractérisation classique des protéines ou des gels. Beaucoup d’expériences ont été effectuées sur grands instruments (diffusion de rayonnement, spectroscopie d’adsorption X, dichroïsme circulaire). Des techniques plus accessibles comme la résonance paramagnétique électronique, la rhéologie ou la microscopie électronique ont aussi été employées. Les aspects les plus novateurs de cette thèse ont été l’effet de l’adsorption sur l’hème et la compréhension de la structure de la protéine gélifiée, deux aspects qui n’avaient pas été traités<br>Nanocomposite protein gels are still an underdeveloped subject in the literature despite many applications ranging from enzyme immobilization to prostheses to food gels. The protein ensures the gel biocompatibility while the addition of the nanoparticles will modulate the gel mechanical properties. We decided to focus on chemically cross-linked hemoglobin gels doped with nanoparticles. Hemoglobin (Hb) was chosen for its high abundance and its oxygen binding properties. The gels will be obtained by crosslinking with glutaraldehyde (GTA), a very reactive dialdehyde. The gels will be doped with silica nanoparticles (NP) in order to understand the effect of doping with model nanoparticles on the gel. The first part of the work will focus on the hemoglobin adsorption on silica nanoparticles in order to resolve the remaining unknowns on this phenomenon, which has already been studied. The adsorption isotherms as well as the activity of the adsorbed hemoglobin will be measured. The structures of the heme, globin and the Hb/NP assembly will be studied in details. Subsequently, works will focus on gels without and with nanoparticles in order to respectively elucidate the effects of gelation and doping. We will determine the concentrations of Hb, GTA and NP to obtain a gel. Then, as with the Hb/NP assemblies, we will look at the activity and structure of Hb (heme and globin).The structuring of the gel will also be studied. Works on the gel elastic properties will also be carried out and we will finish on the dynamics of the gelled protein. When possible, the concentration effect for the different components will be determined. For all these studies, a large panel of conventional technics to characterize proteins or gels was used. Many experiments have been performed in synchrotrons and neutron research centers (radiation scattering, X-ray absorption spectroscopy, circular dichroism). Electronic paramagnetic resonance, rheology or electron microscopy, which are more accessible technics have also been employed. The most innovative aspects of this work were the effect of adsorption on heme and the understanding of the gelled protein structure, two aspects that had not been addressed until now

L’association de la science des polymères et de la chimie supramoléculaire a conduit au développement de matériaux polymères supramoléculaires dotés de propriétés structurelles, mécaniques et fonctionnelles atypiques. Ces matériaux sont d’ores et déjà exploités dans divers domaines d’applications, tels que les matériaux auto réparants,l’ingénieur tissulaire ou encore la libération contrôlée de principes actifs. Ainsi, la chimie supramoléculaire s’est avérée être un outil puissant pour moduler les propriétés des matériaux en contrôlant le caractère dynamique des interactions supramoléculaires via l’application de stimuli adaptés. Les travaux réalisés dans le cadre de cette thèse s’inscrivent dans ce contexte et avaient pour principal objectif de développer de nouveaux assemblages (macro)moléculaires à base de complexes intra- et inter-moléculaires de CBPQT4+. Pour ce faire, un nouveau dérivé CBPQT4+-Fu intégrant une unité furane connectée de manière covalente à la partie hôte CBPQT4+ a été développé.Ce dérivé se présente en milieu aqueux sous une conformation auto-incluse dans laquelle l’unité furane au sein de la cavité témoigne d’une extrêmement faible réactivité(Diels-Alder) vis-à-vis de diènophiles. Néanmoins, celle-ci peut être libérée en ajoutant une molécule invitée (Naphtalène) présentant une forte affinité pour le macrocycle.Cette synergie, mise en évidence à l’échelle moléculaire, permettant de déclencher la réaction de Diels-Alder par la formation d’un complexe intramoléculaire a ensuite été exploitée pour concevoir divers hydrogels physiques ou réticulés physiquement et chimiquement<br>The combination of polymer science and supramolecular chemistry has led to thedevelopment of supramolecular polymer materials with unusual structural, mechanical,and functional properties. These materials have already been exploited in manyapplications, including self-repairing materials, tissue engineering, and the controlledrelease of active ingredients. Supramolecular chemistry has proved to be a powerful toolfor modulating the properties of materials by controlling the dynamic nature ofsupramolecular interactions using appropriate stimuli. The work carried out within theframework of this thesis falls within this context, and its main objective was to developnew (macro)molecular assemblies based on intra- and inter-molecular CBPQT4+complexes. To this end, a new CBPQT4+-Fu derivative was developed, integrating a furanunit covalently connected to the CBPQT4+ host moiety. This derivative presents itself inaqueous media a self-included conformation in which the furan unit within the cavityexhibits extremely low reactivity (Diels-Alder) towards dienophiles. However, this can bereleased by adding a guest molecule (naphthalene) with a strong affinity for themacrocycle. This synergy, demonstrated at the molecular scale, enabling the Diels-Alderreaction to be triggered by forming an intramolecular complex, was then exploited to design various physical and chemically cross-linked hydrogels

This diploma thesis deals with humic acid gels and their ability to bind metallic ions on their surface. In the thesis, there was studied the adsorption of copper ions on the provided gels. The gels were prepared by dissolving the humic acid in sodium hydroxide or sodium tripolyphosphate and then precipitated with hydrochloric acid or metal chlorides. Adsorption was studied at different concentrations of the copper (II) chloride and measured on UV-VIS spectrophotometer. It was proved from measuring that gels made from sodium tripolyphosphate are adsorbing less copper ions than the ones prepared by sodium hydroxide. Also, the copper ions are bound with smaller force on gels prepared with polyphosphate. Gels precipitated with magnesium chloride adsorb much more than gels precipitated with acid, thus in the case of gels sequestered metal ions extracted higher.

The presented thesis on thermosensitive polymer gel is focused especially on a thermosensitive triblock copolymer, which is composed of hydrophobic polylactide, polyglycolid and hydrophilic polyethylene glycol (PLGA-PEG-PLGA). Thermosensitive copolymers are very attractive for their phase sol-gel transitions and gel-suspension transitions. The aqueous solution of this copolymer behaves like a sol at laboratory temperature and like a gel at body temperature. These systems are used as injectable carriers for targeted drug delivery with controlled release. However, the influence of the resulting polymer concentration and temperature on the thermosensitive hydrogel nanostructure was not yet fully studied. In the experimental part, the viscoelastic behavior of hydrogels was observed by dynamic rheological analysis at different polymer concentrations and temperature conditions. The average size and distribution of micelles of triblock copolymer in aqueous solution were measured using dynamic light scattering technique. Characterization of fibrous micelles was complemented by imaging technique, cryogenic transmission electron microscopy.

Biomedical hydrogels are defined as biocompatible solid-liquid systems in which polymeric chains (fibers) are crosslinked to form a 3D-network swollen by a huge water amount. Their use as controlled drug release systems is in continuous growth. However, a critical step in their development is the characterization of their 3D nano/micro structure and the correlation with fabrication parameters. Indeed, hydrogels structure is complex and depends on fibers diameter, concentration, mesh/pore size and degree of crosslinking. Historically, hydrogel structure have been imaged using AFM, SEM, TEM and X-ray microscopy. Although these methods provide high-resolution images, they require a significant sample manipulation that can lead to shrinkage and collapse of the fibers structure. Moreover, information is strictly localized and poorly suited for bulk properties. The target of this thesis is to propose the combined use of not-destructive, economic and fast technologies like rheology and low field NMR (LF-NMR) to understand the macro-, micro- and nanoscopic characteristic of several hydrogels. We aim to drastically reduce the need for time-consuming, expensive measurement, rather to select optimal sample for more complex characterization. Reliability of rheological and LF NMR approach were tested to: • follow hydrogel gelation process in i) a sonicated nanocellulose solution ii) a thermo-sensitive chitosan gel. In particular, the effect of i) salt addition and sonicated time and ii) temperature on the gelation process were considered, respectively. • interpret the characteristics of cross-linked gels system relaying on polymer blends [PVP (poly-vinyl-pirrolidone) and alginate]. In particular gels mechanical strength, 3D nano/micro structure and mesh size distribution were determined. Some of these systems, suitable for liposomes delivery, were also characterized by TEM and this technique confirmed our findings. • correlate mesh size and release rate in a Diels-Alder poly(ethylene glycol) based hydrogel for controlled antibodies release. Our estimations well fitted with test of in vitro release of fluorescein isothiocyanate labeled Dextran and Bertuzimab. For what concerns biological tissues, the focus is on two innovative applications of LF-NMR relying on the different conditions experienced by water confined in three-dimensional structures. Indeed, from the LF-NMR point of view, we can distinguish between free water that is not affected by the solid surface, and bound water that undergoes the effect of solid surface: • The first application considers the analysis on the expectorate of patients affected by cystic fibrosis (CF). This pulmonary disease is mainly characterized by a dehydrated and hyper concentrated mucus in airways. We analysed these voluntary samples to reveal mucus dehydration and pathological components, which are strictly correlated to disease severity. As this approach is less expensive, faster, non-invasive and does not require highly qualified personnel, it has the potential to become a valuable monitoring tool. • The second application regards the evaluation of trabecular bone extracts from osteoporosis and osteoarthrosis patients who underwent hip replacement. These two pathological conditions differ for the quality of bone tissue, and both of them are typical of elders. Water mobility inside the trabecular network is connected to the pore size distribution characterising the bone tissue. Therefore we expected that osteoporosis samples present higher water mobility than osteoarthrosis ones. It could be a new method to rapidly and easily know the severity of osteoporosis.

Under specific conditions short peptides modified with an N-terminal fluorenyl-9-methoxycarbonyl (Fmoc) group can self-assemble into hydrogel scaffolds similar in properties to the natural extracellular matrix. Fmoc-diphenylalanine (Fmoc-FF) for instance, has been shown to form hydrogels at physiological pH that have the ability to support 2D and 3D cell culture. The aim of this investigation is to provide further understanding of the self-assembly mechanism of such systems in order to progress towards the establishment of design rules for the preparation of scaffolds with tuneable properties.First, Fmoc-dipeptides composed of a combination of hydrophobic aromatic residues phenylalanine (F) and glycine (G) were studied with a particular emphasis on the effect of pH variations. The systems were investigated in order to assess what influence the position of such residues in the peptide sequence had on the physical properties of the molecules, and what impact the chemical structure had on the self-assembly behaviour and the gelation properties of the materials. Subsequently, phenylalanine was replaced by leucine (L), a non-aromatic amino acid that had the same relative hydrophobicity in order to determine whether the self-assembly of such molecules is driven by aromatic interactions or hydrophobic effects.Using potentiometry, the behaviour of the systems in solution has been investigated, revealing that they were all characterised by pKa shifts of up to six units above the theoretical values. Fmoc-FF exhibited two transitions whereas the other Fmoc-dipeptides only displayed one. These transitions were found to coincide with the formation of distinct self-assembled structures with differing molecular conformations and properties that were characterised using transmission electron microscopy, infrared and fluorescence spectroscopy, X-ray scattering and shear rheometry.π-stacking of the aromatic moieties was thought to be the driving force of the self-assembly mechanism, generating dimers that corresponded to the building blocks of the supramolecular structures formed. On the other hand, the peptide components were stabilised via hydrogen bonding and could form antiparallel β-sheets depending on the amino acid sequence and the associated influence on the rigidity of the molecules. Below their (first) apparent pKa transition, Fmoc-FF, Fmoc-LL, Fmoc-FG, Fmoc-LG and Fmoc-GG formed hydrogels, with the mechanical properties and stability varying depending on the amino acid sequence. Fmoc-FF and Fmoc-LL exhibited the lowest storage modulus values (G′ ~ 0.5–5 Pa) of the studied systems while Fmoc-LG displayed the highest (G′ ~ 1000–2100 Pa). Fmoc-FG and Fmoc-LG had the peculiarity of being obtained upon heating and where found to be particularly stable, as opposed to Fmoc-GG gels which showed a tendency to crystallise. On the microscopic scale, these gels were all associated with the presence of entangled fibrillar networks of different size and morphology, which in some cases could self-assemble further through a lamellar organisation. Again, Fmoc-FG and Fmoc-LG distinguished from the other systems as they were the only Fmoc-dipeptides to show a supramolecular chirality in the form of twisted ribbons under specific pH conditions. In contrast, Fmoc-GF and Fmoc-GL did not form hydrogels below their apparent pKa due to the formation of sheet-like and spherical structures respectively.

Certain hydrogel forming de novo proteins that utilize different crosslinking methods are studied experimentally on a rheometer. The stress reaxation modulus of CRC, a telechelic, triblock protein, is shown to be that of a stretched exponential function with a value of β ≅ 0.5. The insertion of an integrin binding domain and changes in pH within the range 6.5–8.5 are shown not to significantly affect the resulting rheological behavior. A selective chemical crosslinker is used on CRC hydrogel systems and is shown to change the rheological behavior of the system to that of a combination of a chemically and physically crosslinked system. Chemically crosslinked hydrogels composed of W6, a wheat gluten-based protein, demonstrate a storage modulus weakly dependent on the angular frequency that is much greater than the loss modulus, with a modulus concentration dependence of c^9/4.

Kyoto University (京都大学)<br>0048<br>新制・課程博士<br>博士(工学)<br>甲第13850号<br>工博第2954号<br>新制||工||1436(附属図書館)<br>26066<br>UT51-2008-C766<br>京都大学大学院工学研究科合成・生物化学専攻<br>(主査)教授 濵地 格, 教授 青山 安宏, 教授 木村 俊作<br>学位規則第4条第1項該当

La "cryopolymérisation" permet d'obtenir des gels de polymère macroporeux ou "cryogels". Cette méthode a été utilisée pour la synthèse d'hydrogels thermosensibles à base de pNIPA. La température critique TC correspondant à la transition de volume a été déterminée par des mesures de taux de gonflement et par DSC. La macroporosité (distribution de la taille des pores et épaisseur des parois) et son évolution en fonction de T ont été étudiées par la microscopie biphotonique donnant des informations à l'échelle du µm à plusieurs dizaines de µm. La diffusion de rayons X (SAXS et WAXS) a été utilisée pour caractériser la structure multi-échelle (de quelques dixièmes à quelques dizaines de nm) du gel constituant les parois des macropores. Les courbes de diffusion ont été décrites analytiquement. L'évolution des dix paramètres contenus dans l'équation a été étudiée en fonction de T et discutée. Enfin, des expériences utilisant les phonons hyperfréquences générés par la technique des réseaux transitoires avec détection hétérodyne (HD-TG) ont été réalisées. Ces mesures ont permis de déterminer la vitesse de propagation de l'onde ultra-sonore (à 340 MHz), son atténuation, et la constante de diffusion thermique à différentes températures.

Nous avons imaginé et développé une méthode pour la préparation d’hydrogels par procédé sol-gel à partir de blocs hybrides (bio)organiques-inorganiques. Les blocs hybrides sont obtenus par introduction de groupements silylés, triéthoxysilanes ou hydroxydiméthylsilanes, sur des polymères synthétiques ou des molécules d’intérêt biologique telles que des peptides. Ces blocs hybrides peuvent être combinés dans des proportions choisies pour former des hydrogels multifonctionnels. Le procédé de gélification se déroule à 37°C à pH 7.4 dans un tampon physiologique. L’hydrolyse et la condensation des précurseurs silylés conduit à la formation d’un réseau tridimensionnel covalent dans lequel les entités organiques sont reliées par des liaisons siloxanes. Dans un premier temps, cette méthode a été appliquée à la synthèse d’hydrogels à base de PEG. Nous avons ensuite montré que ces hydrogels pouvaient être fonctionnalisés de façon covalente par des entités bioactives au cours de leur préparation. Des hydrogels possédant des propriétés antibactériennes ou favorisant l’adhésion cellulaire ont ainsi été préparés. Dans un deuxième temps, un peptide hybride inspiré du collagène naturel a été synthétisé et a permis l’obtention d’hydrogels qui présentent des propriétés de prolifération cellulaire similaires à celles observées sur des substrats de collagène naturel. La biocompatibilité du procédé sol-gel a été démontrée par l’encapsulation de cellules souches dans l’hydrogel au cours de sa formation. Enfin, l’impression 3D d’hydrogels hybrides a été réalisée. Ce travail de thèse met donc en lumière le potentiel de la chimie sol-gel pour la conception à façon de matériaux biomimétiques particulièrement prometteurs pour des applications en ingénierie tissulaire<br>We designed and developed a method for the preparation of hydrogels through the sol-gel process. It is based on (bio)organic-inorganic hybrid blocks obtained by functionalization of synthetic polymers or bioactive molecules, such as peptides, with silyl groups (triethoxysilanes or hydroxydimethylsilanes). These hybrid blocks can be combined in desired ratio and engaged in the sol-gel process to yield multifunctional hydrogels. Gelation proceeds at 37°C at pH 7.4 in a physiological buffer. Hydrolysis and condensation of silylated precursors result in a three-dimensional covalent network in which molecules are linked through siloxane bonds. First, this method was applied to the synthesis of PEG-based hydrogels. Then, we demonstrated that hydrogels could be covalently functionalized during their formation. Thus, hydrogels exhibiting antibacterial properties or promoting cell adhesion were obtained. Secondly, a hybrid peptide whose sequence was inspired from natural collagen was synthesized and used to prepare hydrogels that provided a cell-friendly environment comparable to natural collagen substrates. Stem cells could be encapsulated in these hydrogels with high viability. Finally, hybrid hydrogels were used as bio-inks to print 3D scaffolds. This PhD work highlights the potential of the sol-gel chemistry for the design of tailor-made biomimetic scaffolds that could be particularly promising for tissue engineering applications

Des hydrogels hybrides ont été développés comme alternative aux hydrogels d’agar utilisés en microbiologie. Notre stratégie repose sur la fonctionnalisation de polymères avec des groupements triéthoxysilanes, puis leurs mises en jeu dans le procédé sol-gel afin de fabriquer un hydrogel hybride organique-inorganique. Ce procédé est bio-orthogonal et biocompatible. Il se déroule en milieu aqueux, à pH physiologique et température ambiante. Dans une première partie, nous avons développé des hydrogels à base de PEG bisilylé. Nous avons montré que l’incorporation de PEG monosilylé permettait un relâchement du réseau de l’hydrogel. Dans une seconde partie, nous avons développé des hydrogels à base d’hydroxypropyl méthyl cellulose (HPMC). L’optimisation de la silylation de ce composé a été réalisée. L’étude de la composition de l’hydrogel (masse molaire de l’HPMC, concentration, taux de silylation) a été étudiée et a permis la préparation d’hydrogels aux propriétés similaires aux références commerciales d’agar. Nous avons ensuite étendu notre étude aux hydrogels hybrides à base d’autres polysaccharides. Le chitosan, la dextrine, la pectine et l’acide hyaluronique ont ainsi été silylés et des hydrogels hybrides ont été préparés à partir de ces précurseurs. Les hydrogels de dextrine silylée se sont révélés les plus prometteurs pour une application à la microbiologie. La composition a été optimisée et les tests microbiologiques ont validé ce composé comme une alternative à l’agar.Nous avons montré que les hydrogels synthétiques obtenus par le procédé sol-gel constituaient une alternative solide aux hydrogels d’agar. La maitrise des différents paramètres (ex : silylation, mise en forme, composition) permet d’adapter leurs propriétés<br>Hybrid hydrogels have been developed as an alternative to agar hydrogels used in microbiology. Our strategy is based on the functionalization of polymers with triethoxysilane groups, and then their use in the sol-gel process to produce an organic-inorganic hybrid hydrogels. This process is bio-orthogonal and biocompatible. It takes place in aqueous medium, at physiological pH and ambient temperature. In a first part, we developed hydrogels based on bisilylated PEG. We have shown that the incorporation of monosilylated PEG allowed a loosening of the hydrogel network. In a second part, we developed hydrogels based on hydroxypropyl methyl cellulose (HPMC). Silylation of HPMC silylation has been optimised. The study of hydrogel composition (HPMC molecular weight, concentration, silylation rate) allowed the production of hydrogels with properties similar to the agar commercial references. We then extended our study to hybrid hydrogels made from others polysaccharides. Chitosan, dextrin, pectin and hyaluronic acid were thus silylated and hybrid hydrogels were prepared from these precursors. Silylated dextrin hydrogels proved to be highly suitable for microbiology applicationWe showed that synthetics hydrogels obtained by sol-gel process constituted a solid alternative to agar hydrogels. The control of the various parameters (e.g. silylation, shaping, composition) makes it possible to prepare hydrogels with tunable properties

In this work, thermosensitive hydrogels having tunable thermo-mechanical properties were synthesized. Generally the thermal transition of thermosensitive hydrogels is based on either a lower critical solution temperature (LCST) or critical micelle concentration/ temperature (CMC/ CMT). The temperature dependent transition from sol to gel with large volume change may be seen in the former type of thermosensitive hydrogels and is negligible in CMC/ CMT dependent systems. The change in volume leads to exclusion of water molecules, resulting in shrinking and stiffening of system above the transition temperature. The volume change can be undesired when cells are to be incorporated in the system. The gelation in the latter case is mainly driven by micelle formation above the transition temperature and further colloidal packing of micelles around the gelation temperature. As the gelation mainly depends on concentration of polymer, such a system could undergo fast dissolution upon addition of solvent. Here, it was envisioned to realize a thermosensitive gel based on two components, one responsible for a change in mechanical properties by formation of reversible netpoints upon heating without volume change, and second component conferring degradability on demand. As first component, an ABA triblockcopolymer (here: Poly(ethylene glycol)-b-poly(propylene glycol)-b-poly(ethylene glycol) (PEPE) with thermosensitive properties, whose sol-gel transition on the molecular level is based on micellization and colloidal jamming of the formed micelles was chosen, while for the additional macromolecular component crosslinking the formed micelles biopolymers were employed. The synthesis of the hydrogels was performed in two ways, either by physical mixing of compounds showing electrostatic interactions, or by covalent coupling of the components. Biopolymers (here: the polysaccharides hyaluronic acid, chondroitin sulphate, or pectin, as well as the protein gelatin) were employed as additional macromolecular crosslinker to simultaneously incorporate an enzyme responsiveness into the systems. In order to have strong ionic/electrostatic interactions between PEPE and polysaccharides, PEPE was aminated to yield predominantly mono- or di-substituted PEPEs. The systems based on aminated PEPE physically mixed with HA showed an enhancement in the mechanical properties such as, elastic modulus (G′) and viscous modulus (G′′) and a decrease of the gelation temperature (Tgel) compared to the PEPE at same concentration. Furthermore, by varying the amount of aminated PEPE in the composition, the Tgel of the system could be tailored to 27-36 °C. The physical mixtures of HA with di-amino PEPE (HA·di-PEPE) showed higher elastic moduli G′ and stability towards dissolution compared to the physical mixtures of HA with mono-amino PEPE (HA·mono-PEPE). This indicates a strong influence of electrostatic interaction between –COOH groups of HA and –NH2 groups of PEPE. The physical properties of HA with di-amino PEPE (HA·di-PEPE) compare beneficially with the physical properties of the human vitreous body, the systems are highly transparent, and have a comparable refractive index and viscosity. Therefore,this material was tested for a potential biological application and was shown to be non-cytotoxic in eluate and direct contact tests. The materials will in the future be investigated in further studies as vitreous body substitutes. In addition, enzymatic degradation of these hydrogels was performed using hyaluronidase to specifically degrade the HA. During the degradation of these hydrogels, increase in the Tgel was observed along with decrease in the mechanical properties. The aminated PEPE were further utilised in the covalent coupling to Pectin and chondroitin sulphate by using EDC as a coupling agent. Here, it was possible to adjust the Tgel (28-33 °C) by varying the grafting density of PEPE to the biopolymer. The grafting of PEPE to Pectin enhanced the thermal stability of the hydrogel. The Pec-g-PEPE hydrogels were degradable by enzymes with slight increase in Tgel and decrease in G′ during the degradation time. The covalent coupling of aminated PEPE to HA was performed by DMTMM as a coupling agent. This method of coupling was observed to be more efficient compared to EDC mediated coupling. Moreover, the purification of the final product was performed by ultrafiltration technique, which efficiently removed the unreacted PEPE from the final product, which was not sufficiently achieved by dialysis. Interestingly, the final products of these reaction were in a gel state and showed enhancement in the mechanical properties at very low concentrations (2.5 wt%) near body temperature. In these hydrogels the resulting increase in mechanical properties was due to the combined effect of micelle packing (physical interactions) by PEPE and covalent netpoints between PEPE and HA. PEPE alone or the physical mixtures of the same components were not able to show thermosensitive behavior at concentrations below 16 wt%. These thermosensitive hydrogels also showed on demand solubilisation by enzymatic degradation. The concept of thermosensitivity was introduced to 3D architectured porous hydrogels, by covalently grafting the PEPE to gelatin and crosslinking with LDI as a crosslinker. Here, the grafted PEPE resulted in a decrease in the helix formation in gelatin chains and after fixing the gelatin chains by crosslinking, the system showed an enhancement in the mechanical properties upon heating (34-42 °C) which was reversible upon cooling. A possible explanation of the reversible changes in mechanical properties is the strong physical interactions between micelles formed by PEPE being covalently linked to gelatin. Above the transition temperature, the local properties were evaluated by AFM indentation of pore walls in which an increase in elastic modulus (E) at higher temperature (37 °C) was observed. The water uptake of these thermosensitive architectured porous hydrogels was also influenced by PEPE and temperature (25 °C and 37 °C), showing lower water up take at higher temperature and vice versa. In addition, due to the lower water uptake at high temperature, the rate of hydrolytic degradation of these systems was found to be decreased when compared to pure gelatin architectured porous hydrogels. Such temperature sensitive architectured porous hydrogels could be important for e.g. stem cell culturing, cell differentiation and guided cell migration, etc. Altogether, it was possible to demonstrate that the crosslinking of micelles by a macromolecular crosslinker increased the shear moduli, viscosity, and stability towards dissolution of CMC-based gels. This effect could be likewise be realized by covalent or non-covalent mechanisms such as, micelle interactions, physical interactions of gelatin chains and physical interactions between gelatin chains and micelles. Moreover, the covalent grafting of PEPE will create additional net-points which also influence the mechanical properties of thermosensitive architectured porous hydrogels. Overall, the physical and chemical interactions and reversible physical interactions in such thermosensitive architectured porous hydrogels gave a control over the mechanical properties of such complex system. The hydrogels showing change of mechanical properties without a sol-gel transition or volume change are especially interesting for further study with cell proliferation and differentiation.<br>In der vorliegenden Arbeit wurden thermosensitive Hydrogele mit einstellbaren thermo-mechanischen Eigenschaften synthetisiert. Im Allgemeinen basiert der thermische Übergang thermosensitiver Gele auf einer niedrigsten kritischen Löslichkeitstemperatur (LCST) oder kritischer Mizellkonzentration bzw. –temperatur(CMC/ CMT). Der temperaturabhängige Übergang von Sol zu Gel mit großer Volumenänderung wurde im ersten Fall bei thermosensitiven Hydrogelen beobachtet und ist vernachlässigbar für CMC/ CMT abhängige Systeme. Die Änderung des Volumens führt zum Ausschluss von Wassermolekülen, was zum Schrumpfen und Versteifen des Systems oberhalb der Übergangstemperatur führt. Die Volumenänderung kann unerwünscht sein, wenn Zellen in das Gel eingeschlossen werden sollen. Die Gelierung im zweiten Fall beruht hauptsächlich auf der Mizellbildung oberhalb der Übergangstemperatur und weiterem kolloidalem Packen von Mizellen im Bereich der Gelierungstemperatur. Weil die Gelierung hauptsächlich von der Polymerkonzentration abhängt, kann sich das Gel bei Zugabe von Lösungsmittel leicht wieder lösen. Hier sollten thermosensitive Gele entwickelt werden, die auf zwei Komponenten beruhen. Eine Komponente sollte aus einem ABA-Triblockcopolymer mit thermosensitiven Eigenschaften bestehen, dem Poly(ethylen glycol)-b-Poly(propylenglycol)-b-Poly(ethylen glycol) (PEPE), dessen Sol-Gel-Übergang auf Mizellierung und kolloidalem Jamming der gebildeten Mizellen basiert, und einer weiteren makromolekularen Komponente, einem Biopolymer, dass die Mizellen vernetzt. Auf diese Weise sollten thermosensitive Gele realisiert werden, die keine oder nur eine kleine Volumenänderung während der Änderung der mechanischen Eigenschaften zeigen, die stabiler gegenüber Verdünnung sein sollten als klassische Hydrogele mit einem CMC-basierten Übergang und die jedoch gezielt abgebaut werden können. Die Hydrogele wurden auf zwei Arten vernetzt, entweder durch physikalisches Vermischen, bei dem die Vernetzung durch elektrostatische Wechselwirkungen erfolgte, oder durch kovalente Kopplung der beiden Komponenten. Als makromolekulare Komponente zur Vernetzung der Mizellen wurden Biopolymere (hier: die Polysaccharide Hyaluronsäure (HA), Chondroitinsulfat oder Pektin oder das Protein Gelatin) verwendet, um die Hydrogele enzymatisch abbaubar zu gestalten. Um eine starke ionische/elektrostatische Wechselwirkung zwischen dem PEPE und den Polysachariden zu erzielen, wurde PEPE aminiert, um hauptsächlich monoaminiertes bzw. diaminiertes PEPE einsetzen zu können. Die Gele, die auf der physikalischen Mischung von aminierten PEPE mit HA bestehen, zeigten im Vergleich zu PEPE bei gleicher Konzentration eine Zunahme der mechanischen Eigenschaften, wie beispielsweise dem elastischem Modulus (G′) und dem Viskositätsmodulus (G′′) bei gleichzeitiger Abnahme der Gelierungstemperatur (Tgel). Durch Variation des Gehalts an aminierten PEPE-, konnte die Tgel in einem Bereich von 27-36 °C eingestellt werden. Interessanterweise zeigten die physikalischen Mischungen mit diaminierten PEPE (HA·di-PEPE) höhere mechanische Eigenschaften (elastischer Modulus G′) und eine höhere Stabilität gegenüber Verdünnungseffekten als Mischungen mit monoaminiertem PEPE (HA·mono-PEPE). Dies zeigt den starken Einfluss der elektrostatischen Wechselwirkungen zwischen der Carboxylgruppe der HA und der Amingruppe von PEPE. Die physikalischen Eigenschaften HA·di-PEPE sind vergleichbar mit den physikalischen Eigenschaften des Glaskörpers im Auge hinsichtlich Transparenz, Brechungsindex und Viskosität. Deswegen wurde das Material hinsichtlich seiner biologischen Anwendung getestet und zeigte sich sowohl im Überstand als auch im direkten Kontakt als nichtzytotoxisch. Zukünftig wird dieses Material in weiteren Untersuchungen bezüglich seiner Eignung als Glaskörperersatz geprüft werden. Zusätzlich konnte der enzymatische Abbau der Hydrogele mit Hyaluronidase gezeigt werden, die spezifisch HA abbaut. Beim Abbau der Hydrogele stieg Tgel bei gleichzeitiger Abnahme der mechanischen Eigenschaften. Aminiertes PEPE wurde zusätzlich zur kovalenten Bindung unter Verwendung von EDC als Aktivator an Pektin und Chondroitinsulfat eingesetzt. Tgel konnte auf 28 – 33 °C eingestellt werden durch Variation der Pfropfungsdichte am Biopolymer bei gleichzeitiger Zunahme der thermischen Stabilität. Die Pec-g-PEPE Hydrogele waren enzymatisch abbaubar, was zu einer leichten Erhöhung von Tgel und zu einer Abnahme von G′ führte. Die kovalente Bindung der aminierten PEPE an HA erfolgte unter Verwendung von DMTMM als Aktivator, der sich in diesem Fall als effektiver als EDC herausstellte. Die Reinigung mittels Ultrafiltration führte zu einer deutlich besseren Aufreinigung des Produkts als mittels Dialyse. Die gegrafteten Systeme waren in Nähe der Körpertemperatur bereits im Gelstadium und zeigten eine Erhöhung der mechanischen Eigenschaften bereits bei sehr geringen Konzentrationen von 2.5Gew.%. Die höheren mechanischen Eigenschaften dieser Hydrogele erklären sich durch die Kombination der Mizellbildung (physikalische Wechselwirkung) des PEPE und der Bildung kovalenter Netzpunkte zwischen PEPE und HA. PEPE bzw. entsprechende physikalische Mischungen derselben Komponenten zeigten kein thermosensitives Verhalten bei einer Konzentration unterhalb von 16 Gew%. Diese thermosensitiven Hydrogele zeigten auch eine Löslichkeit auf Abruf durch enzymatischen Abbau. Das Konzept der Thermosensitivität wurde in 3D strukturierte, poröse Hydrogele (TArcGel)eingeführt, bei dem PEPE kovalent an Gelatin gebunden wurde und mit LDI vernetzt wurde. Das gepfropfte PEPE führte zu einer Erniedrigung der Helixbildung der Gelatinketten. Nach Fixierung der Gelatinketten durch Vernetzung zeigte das System eine Erhöhung der mechanischen Eigenschaften bei Erwärmung (34-42 °C). Dieses Phänomen war reversibel beim Abkühlen. Eine mögliche Erklärung der reversiblen Änderungen bezüglich der mechanischen Eigenschaften sind die starken physikalischen Wechselwirkungen zwischen den Mizellen des PEPE, die kovalent an Gelatin gebunden wurden. Ferner wurde durch AFM Untersuchungen festgestellt, dass bei Temperaturerhöhung (37 °C) die örtlichen elastischen Moduli (E) der Zellwände zugenommen haben. Zusätzlich wurde die Wasseraufnahme der TArcGele durch PEPE und die Temperatur (25 °C und 37 °C) beeinflusst und zeigte eine niedrigere Wasseraufnahme bei höherer Temperatur und umgekehrt. Durch die niedrigere Wasseraufnahme bei hohen Temperaturen erniedrigte sich die Geschwindigkeit des hydrolytischen Abbaus im Vergleich zu dem strukturierten Hydrogel aus reiner Gelatin. Diese temperatursensitiven ArcGele könnten bedeutsam sein für Anwendungen im Bereich Stammzellkultivierung, Zelldifferenzierung und gerichteter Zellmigration. Zusammenfassend konnte bei den thermosensitiven Hydrogelen gezeigt werden, dass die Vernetzung von Mizellen mit einem makromolekularen Vernetzer die Schermoduli, Viskosität und Löslichkeitsstabilität im Vergleich zu reinen ABATriblockcopolymeren mit CMC-Übergang erhöht. Dieser Effekt konnte durch kovalente und nichtkovalente Mechanismen, wie beispielsweise Mizell- Wechselwirkungen, physikalische Interaktionen von Gelatinketten und physikalische Interaktionen von Gelatinketten und Mizellen, realisiert werden. Das Pfropfen von PEPE führte zu zusätzlichen Netzpunkten, die die mechanischen Eigenschaften der thermosensitiven architekturisierten, porösen Hydrogele beeinflussten. Insgesamt ermöglichten die physikalischen und chemischen Bindungen und die reversiblen physikalischen Wechselwirkungen in den strukturierten, porösen Hydrogelen eine Kontrolle der mechanischen Eigenschaften in diesem sehr komplexen System. Die Hydrogele, die eine Veränderung ihrer mechanischen Eigenschaften ohne Volumenänderung oder Sol-Gel-Übergang zeigen sind besonders interessant für Untersuchungen bezüglich Zellproliferation und –differenzierung.

Nous avons développé des hydrogels hybrides pour l’encapsulation de cellules souches mésenchymateuses dans la perspective de réparation du cartilage. Notre stratégie repose sur la fonctionnalisation de biopolymères et de molécules actives avec des groupements triéthoxysilanes afin de préparer des hydrogels par un procédé sol-gel. Ce procédé bio-orthogonal, se déroule dans l’eau, à pH physiologique et 37 °C. Dans un premier temps, nous avons mis au point un système de catalyseurs sol-gel biocompatible et étudié les paramètres influençant la cinétique de gélification. Dans un second temps, des peptides hybrides silylés de différentes tailles mimant la séquence du collagène ont été synthétisés. Des hydrogels ont été développés à partir de ces nouveaux blocs biomimétiques. La composition de ces hydrogels a été optimisée afin d’obtenir la meilleur viabilité cellulaire et différentiation chondrocytaire possible après encapsulation. Les propriétés mécaniques de ces hydrogels ont été étudiées révélant un fort impact de la composition. Enfin, ces hydrogels ont été imprimés en 3D par extrusion, et des compositions spécifiques combinant différant biopolymères, ont été développées pour faciliter ce procédé. Au travers des différents exemples d’hydrogels préparés, l’intérêt et la versatilité du procédé sol-gel pour la préparation de matériaux biologiquement actifs a été démontrée, et permet d’envisager de nombreuses applications dans le domaine de la santé<br>We developed hybrid hydrogels for mesenchymal stem cells embedding, which could be of interest for cartilage repair. Our strategy is based on the functionalization of bioactive molecules and biopolymer with triethoxysilane moieties to prepare hydrogels by a sol-gel process. This bio-orthogonal process take place in water, at physiological pH and 37 °C. First, we searched for a biocompatible catalysis method and we studied the reaction parameters influencing the gelation time. Then, collagen-like peptides of various sizes have been synthesised and silylated to prepare biomimetic hydrogels. The composition of these hydrogels has been improved to reach the best cellular viability and chondrocyte differentiation after embedding. The resulting mechanical properties were also studied. Finally, theses hydrogels have been 3D-printed by extrusion and new compositions have been developed to reach a better accuracy. Through the numerous hydrogel compositions we developed, the potential and versatility of sol-gel process for hydrogel preparation was demonstrated, paving the way to many applications in health sciences

This paper reports the study of hydrogen peroxide decomposition catalyzed by polymer-protected gold nanoparticles (AuNPs) immobilized within polyacrylamide hydrogel. The stabilization of AuNPs was achieved using hydrophilic polymers. Embedding of AuNPs stabilized with various polymers into polyacrylamide hydrogels was carried out using three ways: “in situ” polymerization, sorption and boronhydride methods. Size, shape and morphology of AuNPs were characterized by various physicochemical methods.

The thesis is focused on physical hyaluronan gels. The object of study is the interaction of hyaluronan (HyA) with oppositely charged surfactants in physiologic solution (0.15 M NaCl), leading to the formation of gel. In the first part of work have been determined the solids´ contents (X) in gels and their supernatants in percentage and their correlation with molecular weight concentration of original HyA solution and the ratio of binding sites on hyaluronan chain and surfactant CTAB. To conclude, decrease in HyA concentration results in higher values of X and vice versa. On the other hand, increase in the value of X with increasing molecular weight of HyA is not so significant. Analogous conclusions have been made for supernatants and the amount of solids in gel. Drying process has been recorded by drying curves. Swelling process has been used for the characterization of gels. The percentage of water that can be absorbed by dried gel, was determined. The results are in agreement with the measurements of solids´ content in gels. In the next part, the correlation between rheological properties of gels and HyA concentration, HyA molecular weight and concentration of CTAB have been studied by the oscillation and flow tests. The samples with the highest molecular weight and concentration have the most viscoelastic character. The flow test confirmed the assumed pseudoplastic behavior of gels. A very interesting trend arose while comparing HyA concentrations and viscosity in stock solutions and gels. Whereas in stock solution viscosity (at low shear rate) is lower with increasing of HyA concentration, the situation was exactly the opposite in gels. The results are in agreement with frequency tests and observed character of gels.

Ionic hydrogels belong to the class of polyelectrolyte gels or ionic gels. Their ability to swell or shrink under different environmental conditions such as change of pH, ion concentration or temperature make them promising materials, e.g. for microsensoric or microactuatoric devices. The hydrogel swelling exhibits nonlinear effects due to the occurrence of different interacting transport phenomena. Numerical simulations are an essential part in the ongoing development of microsensors and microactuators. In order to determine transport effects due to diffusion, migration and convection a multiphase mesoscale model based on the Theory of Porous Media is applied. The governing field equations are solved in the transient regime by applying the Finite Element Method. By means of the derived numerical framework a detailed investigation of the different transport phenomena is carried out. Numerical experiments are performed to characterize the dominating transfer phenomena for ionic gels under chemical stimulation.

L'objectif de ce travail a été d'étudier l'activité hydrolytique d'une enzyme, la pullulanase, vis à vis du pullulane dans des hydrogels fonctionnalisés ou non à base d'alginate de calcium. L'alginate a été modifié chimiquement par une polyéther amine de la famille Jeffamine® qui présente un caractère LCST "Low Critical Solubility Temperature". L'enzyme immobilisée à hauteur de 30% dans des billes d'alginate de calcium hydrolyse lentement le pullulane en raison de la pénétration de celui ci dans les billes et libère du maltotriose et ses premiers multiples alors que l'enzyme libre donne un mélange d'oligosaccharides très polydispersé. La proximité entre enzyme et substrat au sein de l'hydrogel explique ce comportement. L'activité de l'enzyme au sein des hydrogels est très peu affectée par le pH. L'enzyme est même active à pH 4 contrairement à l'enzyme libre ce qui montre la protection de l'enzyme dans ceshydrogels. Avec l'alginate fonctionnalisé, le taux d'immobilisation de l'enzyme est de 100% en raison d'interactions préférentielles entre la pullulanase et les greffons Jeffamine®. L'enzyme immobilisée dans ces matrices ne présente pas une activité enzymatique différente<br>The aim of this project has been to study the hydrolytic activity of the enzyme, pullulanase, toward its substrate pullulan into functionalized or non-functionalized hydrogels based on calcium alginate. Alginate has been chemically modified with a polyether amine, Jeffamine®, with LCST "Low Critical Solubility Temperature" property. The immobilized enzyme amounted to 30% within calcium alginate beads hydrolyses pullulan slowly owing to its penetration into beads and releases maltotriose and its multiples compared to free enzyme which a large distribution of pullulan fragments is observed during the treatment. The close relationship between enzyme and its substrate into these hydrolgels is only marginally affected by pH. The enzyme is even active at pH 4 contrary to the free enzyme indicating an enzyme protection within these hydrogels. In the case of functionalized alginate, the enzyme immobilisation amount is about 100% because of the preferential interactions between pullulanase and Jeffamines-grafted. The immobilized enzyme does not show a different enzyme activity

A novel gel-filled electrochemical hydrogen probe was developed and used to measure hydrogen concentrations in carbon-manganese steels. The results were compared with those from an electrochemical permeation technique and a volumetric method. The probe was used to determine the distribution of hydrogen in 5mm steel plates cathodically charged on one side to represent the wall of a pipe or pressure vessel used in hydrogen service. The concentration measurements obtained by the three techniques were in good agreement with each other and with those predicted from diffusion equations and this permitted the precise boundary conditions on the charged metal surface to be determined. Surface reaction kinetics were investigated to model the hydrogen distribution and these were solved using solutions to Fick's diffusion equations. After long charging times the hydrogen concentration on the efflux surface of the plate approached that on the influx side, indicating that an almost uniform hydrogen distribution had been established. Rather than rapid loss of hydrogen from the free surface, as had been assumed previously, it was clear that there was a large resistance to hydrogen transport across the metal/air interface. Microstructural damage was examined both optically and using the scanning electron microscope. Separate investigations were carried out to help understand the effect that reversible and irreversible trapping had on the diffusion of hydrogen through the steel.

In this thesis, glucose responsive hydrogels based on cross-linked dextran molecules were studied to determine the diffusion rate of an insulin analogue. Investigations of the interaction between concanavalin A and dextran, both in free solution and in the form of glucose responsive hydrogels were conducted. The free solution results have shown that there is an increase of association constant between concanavalin A and dextran when the molecular mass of the dextran is increased. Free solution viscometric tests have shown that increasing the molecular mass or the concentration of the dextran increases the viscosity. The hydrogels have been shown to form for dextrans of molecular mass 43kD or greater. Experiments conducted with hydrogel membranes in a diffusion cell have shown that the batch to batch reproducibility of hydrogel transport properties is low. However, clear evidence of glucose enhanced transport was obtained and these results were compared with predictions obtained from a theoretical model of gel permeability that accounts for competitive displacement of affinity cross links. Oscillatory rheological tests of gelation mixtures which showed an increase in complex viscosity at the gel point with increasing molecular mass of dextran were in agreement with empirical observations that gels formed from the highest molecular mass dextrans were more physically robust and easier to handle. Swelling rate experiments have shown that the rate of hydration of a hydrogel in the presence of glucose is decreased due to the osmotic pressure of the glucose. This work has shown that the multivalent nature of concanavalin A may not be a necessary pre-requisite for this type of hydrogel due to spatial constraints decreasing the number of potential affinity bonds per tetramer. In-house production of more tightly defined dextrans might be expected to reduce heterogeneity and improve the reproducibility of this type of hydrogel membrane.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Mechanical Engineering, 2018.<br>Cataloged from PDF version of thesis.<br>Includes bibliographical references (pages 207-216).<br>Solids with deformation-diffusion coupling are ubiquitous in engineering applications. Understanding and modeling the fracture of such solids is vitally important. This thesis addresses the theoretical formulation, numerical implementation, and application of fully-coupled deformation-diffusion-damage theories for two different classes of materials: (i) polymeric gels and (ii) hydrogen embrittlement in steels, as elaborated below. (i) Fracture of polymeric gels: We first introduce a field called "stretch of Kuhn segments and/or crosslinks", which is necessary for understanding and modeling of the fracture in polymeric materials. Together with this newly introduced field, we formulate a thermodynamically consistent phase-field type theory for fracture of gels. A central feature of our theory is the recognition that the free energy of polymeric materials is not entirely entropic in nature, there is also an energetic contribution from the deformation of the backbone bonds in a chain and/or the crosslinks. It is this energetic part of the free energy that drives the progressive damage and fracture of polymeric materials. We have implemented our theory in a finite element code, and used this simulation capability to study some interesting phenomena in failure of elastomers and gels. (ii) Fracture of steels due to hydrogen embrittlement: We have formulated a thermodynamically consistent theory for the diffusion of hydrogen coupled with the large elastic-plastic deformations, and a phase-field type theory to model ductile fracture of metals. The theory accounts for the macroscopic effects due to the generation and agglomeration of microscopic hydrogen-vacancy complexes. We have implemented our fully coupled theory in a finite element program, and calibrated the material parameters in the theory by using experimental data available in the literature. Finally we have utilized our simulation capability to study the process of fracture due to hydrogen embrittlement in some technically relevant notched-components made from steel.<br>by Yunwei Mao.<br>Ph. D.

This thesis focuses on the use of hydrophilic polymers for bioanalytical applications, including several microanalytical techniques encompassing nanotechnology, microarray technology and DNA gel electrophoresis. The dissertation is divided in two parts, which share the employment of dimethylacrylamide-based copolymers, developed at the laboratory of Analytical Microsystems of the Institute of Chemistry for Molecular Recognition (National Research Council of Italy) where the thesis has been carried out. PART A introduces a novel approach for surface modification of quantum dots and gold nanoparticles, based on physi-/chemisorption of two different functional dimethylacrylamide copolymers. Beside developing innovative functionalization strategies, the goal is to demonstrate the application of coated nanoparticles in highly sensitive immunoassays based on microarray technology. PART B of the dissertation presents the results of an activity, conducted in collaboration with the company Agilent Technology (UK), aimed at developing an innovative gel sieving matrix for high performance DNA electrophoresis. We introduce a new hydrogel obtained by cross-linking an alkyne modified polymer with an azide one, exploiting a copper catalysed click chemistry reaction. The alkyne functionalized polymer is based on poly(dimethilacrylamide) and it was obtained by a post-polymerization modification approach from the parent copolymer poly(DMA-NAS-MAPS), extensively used in the first part of this dissertation. The azide polymer is a polyethylenglycol terminated with azide groups at both ends, and is commercially available. A considerable part of this work is devoted to the optimization of the characteristics of the new hydrogel, in particular to the extension of its shelf-life, an important parameter in view of its industrial application.

Meniscal tears are the most common intra-articular injury of the knee joint. Due to the avascular zone with limited blood supply, treatment of the injury is a complex process. Today, research on the development of efficient treatments and meniscal replacements is of increasing interest. However, there are few alternatives of meniscal replacements available on the market and research has shown uncertain results in their ability to restore the natural biomechanics of the knee joint or prevent development of osteoarthritis. Furthermore there is no comparable method to evaluate tensile stresses caused by axial compressional load on a whole meniscus replacement. Therefore the possibility of knitted casing to contain, fixate and mechanically stabilize a cell seeded bioprinted gel and develop a methodology to characterize its compressional behaviour was analysed. By interlock knitting with segments of partial knit a 3D crescent-shaped biodegradable casing was produced mimicking the dimension of the medial meniscus. In the casing design, an Artelon® Flexband™ was incorporated functioning both as reinforcement at the peripheral rim and as fixation method. Moreover radial threads were added to the casing design by inclusion of weft inlays in the knitting pattern. In the non-destructive characterization of the compressional behaviour of the prototype, axial compressional forces of 10.82 N and 29.77 N were achieved. However the forces achieved were significantly lower if compared to the high force that is applied to the menisci in the knee joint. Furthermore a high influence of the coefficient of friction of the casing in the axial compressional force was concluded. Nevertheless refinements of the methodology are required to perform evaluation with comparable and reliable results.