Osteoarthritis (OA) is the most common type of joint disease that affects nearly 5 million people in Canada alone. OA involves degenerative processes affecting the integrity of articular cartilage, a thin soft tissue at the surface of subchondral bones in joints that facilitates smooth and friction less movement. This disease also affects the other tissues of the joint, including the synovium and its resident cells the fibroblast-like and macrophage-like synoviocytes. Consequences of this pathology include painful movement and stiff joints resulting in loss of range of motion. A broad number of factors may contribute to the development of OA, including obesity, injuries, infections, genetic predispositions and aging. Although there are a number of medications used for the treatment of OA, these only serve to manage its symptoms. An actual cure is yet to be developed. Inorganic polyphosphate (polyP) has previously been identified as a potentially interesting biomolecule for the treatment of OA because of its ability to stimulate tissue formation by chondrocytes - the cells found in the articular cartilage. In this thesis, we first aimed to evaluate the potential of polyP as a therapeutic for joint diseases such as OA further, by characterizing its effects on a number of cell processes (e.g., proliferation, metabolic activity, migration, matrix accumulation) in fibroblast-like synoviocytes (FLS) - specialized cells found in the synovium encapsulating the synovial joints. The synovium is an important tissue for joint physiology and OA pathogenesis; it is thus essential that any therapeutic introduced in the joint not impact this tissue negatively. These studies showed that polyP significantly inhibits FLS proliferation. This effect is interesting in the context of OA as FLS proliferation is associated with progression of the disease. PolyP also increased FLS migration rate and caused changes in metabolic activity, although the trends were inconsistent over time. We also optimized a new protocol for the synthesis of sub-micron calcium-polyP particles. Nanoparticle drug delivery for OA treatment has been gaining importance in recent years as a way to access the cells in cartilage through the small pores in the extracellular matrix (ECM) and increase drug retention time in the joint. The calcium-polyP particles were synthesized by drop-wise addition of polyP into a calcium solution at controlled pH, drop rate and mixing rate. Particles size and stability before and after sterilization were measured by dynamic light scattering. We showed that the addition of sodium citrate dihydrate as a capping agent largely prevented particle agglomeration and increased particle stability. Control over particle size, particularly in the nanometer scale, remains to be achieved; however, this work is a first step towards the development of polyP delivery systems for the treatment of OA.

SHIP2 (SH2 domain-containing inositol polyphosphate 5-phosphatase type 2) est un enzyme de la famille des inositol polyphosphate 5-phosphatases qui déphosphoryle le PtdIns(3,4,5)P3, second messager intervenant dans différentes voies de signalisation cellulaire et impliqué dans de nombreux processus biologiques. La surexpression de SHIP2 en cellule, de même que son invalidation chez la souris, ont montré un rôle de cet enzyme dans le contrôle négatif de la cascade de signalisation de l’insuline et dans la sensibilité à cette hormone. Par ailleurs, plusieurs études de polymorphismes chez l’homme ont montré une association entre ce gène et le diabète de type2. La découverte au sein de notre laboratoire de la délétion d’un motif semblable à ceux présents dans les régions déstabilisatrices de type AU-riche dans la région 3’non codante (3’UTR) du gène SHIP2 chez des patients atteints de diabète de type 2, nous a conduit à explorer le rôle de cette région dans le contrôle de l’expression de SHIP2. Dans ce but, nous avons entrepris d’identifier des protéines capables de lier ce motif AU-riche et d’entraîner l’ARN de SHIP2 vers la dégradation, et ce par deux techniques distinctes : l’une in vivo chez la levure (le triple hybride) et l’autre in vitro, par l’intermédiaire d’une sonde ARN biotinylée. Malheureusement, aucune de ces deux techniques ne nous a permis d’identifier des protéines se liant à l’ARNm de SHIP2. D’autre part, l’analyse de souris génétiquement modifiées présentant dans la région 3’UTR de SHIP2 une mutation similaire à celle observée chez les patients diabétiques n’a pas montré une augmentation significative d’expression de SHIP2 comme on aurait pu s’y attendre. Malgré les différentes techniques mises en place, nous ne sommes pas parvenus à caractériser le rôle joué par le 3’UTR de SHIP2 sur le contrôle de son expression. Dans le but de caractériser l’effet d’une surexpression de SHIP2 et de déterminer si une surexpression de ce gène pouvait mimer le phénotype de diabète de type 2 observé au sein de la population, nous avons généré des souris transgéniques d’addition par transgenèse lentivirale. Deux axes phénotypiques majeurs ont été explorés chez ces souris : le métabolisme du glucose et la prise de poids consécutive à divers régimes alimentaire. Les souris transgéniques présentent un retard dans la captation du glucose en réponse à une surcharge en glucose, s’accompagnant d’un défaut de sécrétion d’insuline. Par contre, aucune altération de la sensibilité à l’insuline n’est observée suite à une injection de cette hormone. Cette absence d’altération de la sensibilité à l’insuline est également soutenue par le fait qu’aucune altération de la captation de glucose n’est observée chez des souris surexprimant le transgène spécifiquement dans le muscle squelettique. Les analyses de prise de poids des souris transgéniques ont révélé une résistance à l’obésité des mâles transgéniques lorsqu’ils sont soumis à un régime alimentaire riche en graisse. Par contre, aucune différence n’est observée sous régime alimentaire conventionnel ou faible en graisse. La plus faible prise de poids des souris transgéniques sous régime riche en graisse s’accompagnant d’une plus faible prise de nourriture, un rôle de SHIP2 dans la régulation du comportement alimentaire et de l’appétit n’est pas à exclure. En conclusion, la surexpression de SHIP2 chez la souris provoque une intolérance au glucose induite, en tout cas en partie, par une plus faible sécrétion d’insuline, ainsi qu’une résistance à l’obésité induite par un régime riche en graisse.

Calcium polyphosphate (CPP) is a suitable substrate in a novel tissue-engineering strategy. The strategy aims to culture articular cartilage in vitro onto porous CPP and then implant the biphasic construct into the joint to replace damaged cartilage. CPP substrates should degrade faster to enhance repair. This project examined the structural and degradation effects of doping CPP with sodium phosphate, sodium hydroxide, and sodium carbonate. Doping concentration was narrowed to 0.01 Na2O/CaO for comparable phase composition to pure CPP. All doped groups sintered and crystallized at lower temperatures than pure CPP. Hydroxide-doped CPP did not form adequate sinter necks. At similar open porosity, Phosphate-doped CPP had similar diametral strength than pure CPP, while Carbonate-doped CPP had greater diametral strength. Degradation in vitro showed that Phosphate-doped CPP had greater strength loss, while Carbonate-doped CPP had similar strength loss, compared to pure CPP. Both doped groups degraded more slowly than pure CPP.

Calcium polyphosphate (CPP) is a suitable substrate in a novel tissue-engineering strategy. The strategy aims to culture articular cartilage in vitro onto porous CPP and then implant the biphasic construct into the joint to replace damaged cartilage. CPP substrates should degrade faster to enhance repair. This project examined the structural and degradation effects of doping CPP with sodium phosphate, sodium hydroxide, and sodium carbonate. Doping concentration was narrowed to 0.01 Na2O/CaO for comparable phase composition to pure CPP. All doped groups sintered and crystallized at lower temperatures than pure CPP. Hydroxide-doped CPP did not form adequate sinter necks. At similar open porosity, Phosphate-doped CPP had similar diametral strength than pure CPP, while Carbonate-doped CPP had greater diametral strength. Degradation in vitro showed that Phosphate-doped CPP had greater strength loss, while Carbonate-doped CPP had similar strength loss, compared to pure CPP. Both doped groups degraded more slowly than pure CPP.

L’objet de cette thèse est l’ignifugation de fibres textiles de type polyamide 6 (PA6). Parmi les différentes techniques utilisées pour modifier une fibre ou un textile synthétique, l’ajout en masse de retardateurs de flamme (RF) durant le procédé d’extrusion a été choisi. Les températures de mise en œuvre du PA6 étant assez élevées (généralement supérieures à 250 °C) une sélection rigoureuse de retardateurs de flamme à base de phosphore et de soufre a été faite. Cette sélection a également pris en compte le fait que le RF doit être efficace à de faibles taux de charge (<10% en masse) pour permettre de filer la matrice. Il est ressorti de cette étude que les sels d’acide sulfamique, tels que le sulfamate d’ammonium (AS) et le sulfamate de guanidinium (GAS) sont efficaces et que les formulations sont filables. Les modes d’action en phase condensée et en phase gaz ont été étudiés et décrits. On observe respectivement une charbonisation plus importante et une modification de la composition des gaz émis lors de la dégradation thermique des matériaux. Cependant, lors du passage de l’échelle laboratoire (micro extrudeuse) à l’échelle pilote, des problèmes de dégradation de la matrice polymère sont survenus. De manière à pallier cette difficulté, l’utilisation d’un co-additif (polyphosphate de mélamine), offrant un effet de synergie avec les sels de sulfamate, a permis de réduire la quantité de GAS tout en améliorant les propriétés feu du PA6 et la formulation a pu être extrudée à échelle pilote. / The aim of this Ph.D. work is the fire retardancy of polyamide 6 (PA6) fibers. Among the different techniques used to modify a synthetic fiber or fabric, the incorporation of fire retardants (FR) directly in the melt during extrusion was chosen. Processing temperatures of PA6 being rather high (generally > 250 °C), a rigorous selection of phosphorus and sulfur based FR was done. Moreover, FR must be stable at the processing temperature, while being spinnable and efficient at low loadings (<10 wt.-%). Sulfamate salts such as ammonium and guanidine sulfamate (AS and GAS) were found efficient and the PA6 formulations were spinnable. The modes of action were investigated through a study of the condensed and gas phase. However, when scaling up the process from microextruder to a large-scale extruder, processing issues were observed. In order to overcome this problem, the use of a co-additive (melamine polyphosphate), offering a synergistic effect with sulfamate salts, allowed to decrease the amount of GAS in the PA6 formulation while enhancing the FR performance. Thus, large-scale extrusion of the FR PA6 was performed successfully.

The main objective of this thesis is to further reduce the machining cycle time for producing Calcium Polyphosphate (CPP) implant constructs. To achieve this, the impregnation of the CPP lattice with various polymers is investigated, with the aim of improving the toughness of the material. By applying Taguchi’s orthogonal array method it was determined that CPP infiltrated with an ionic bonding polymer produces the best material for generating high quality machined surfaces and features. While there is some loss in surface porosity, in comparison to cutting uninfiltrated CPP, the porosity loss was deemed acceptable for the clinical purpose of the implant, and in many cases, would be trimmed off during a consecutive finish machining operation. The 2 fluted 4 mm diameter flat end mill at a cutting speed of 30 m/min and ¾ immersion up-milling, 0.1 mm chip load and 3 mm depth of cut were determined to be highly suitable for achieving both high productivity as well as excellent surface integrity. These conditions produced a material removal rate of 4,302 mm3/min, which was 14 times higher than the material removal rate achieved in machining pure CPP in earlier studies. The constructed machining model was highly successful in predicting the cutting forces, and therefore can be used in process planning and optimization in the production of tissue engineered implant constructs out of CPP. The Finite Element analyses predicted that the implant would not chip or break during the roughing operation, as validated experimentally. This allowed the roughing cycle time to be reduced from 159 min to 19 min, effectively achieving a productivity improvement of 8 times over the earlier work done in this area.

Cisplatin is an effective chemotheraputic agent for head and neck squamous cell carcinoma particularly in conjunction with radiation therapy. Unfortunately, its cytotoxic profile and associated systemic side effects limit its clinical efficacy. A localized delivery system was developed for cisplatin by processing calcium polyphosphate (CPP) in a multistep gelling protocol, with the goal of limiting its systemic toxicity and enhancing its overall clinical applicability. In addition, a novel method for processing the material was examined utilizing cold isostatic pressure (CIP) to allow for miniaturization of the system into an implantable device. The integration of cisplatin into the matrix was examined for efficient and dose dependent loading via dissolution of the final product and measurement of platinum concentrations by inductively coupled plasma optical emission spectrometry (ICP-OES). Drug release was measured in vitro by placing the CPP-cisplatin matrix into TRIS buffer solution while measuring the platinum concentration at given intervals from 0.5 hours to 14 days. The cytotoxicity of the cisplatin against L1210 cells was examined using an MTT assay following a 12-hour elution. The material demonstrated a predictable and dose dependent loading of cisplatin, although the release of the drug showed variability exemplified by a more pronounced burst release with aging of the stock CPP glass particulate. The CPP/cisplatin matrix exhibited cytotoxic effects after processing. This work suggests that further evaluation of this material as a matrix for cisplatin delivery should be undertaken in an attempt to normalize release, maximize the concentration within the system and further optimize the bead format in order to improve the potential for clinical usage.

The main objective of this thesis is to further reduce the machining cycle time for producing Calcium Polyphosphate (CPP) implant constructs. To achieve this, the impregnation of the CPP lattice with various polymers is investigated, with the aim of improving the toughness of the material. By applying Taguchi’s orthogonal array method it was determined that CPP infiltrated with an ionic bonding polymer produces the best material for generating high quality machined surfaces and features. While there is some loss in surface porosity, in comparison to cutting uninfiltrated CPP, the porosity loss was deemed acceptable for the clinical purpose of the implant, and in many cases, would be trimmed off during a consecutive finish machining operation. The 2 fluted 4 mm diameter flat end mill at a cutting speed of 30 m/min and ¾ immersion up-milling, 0.1 mm chip load and 3 mm depth of cut were determined to be highly suitable for achieving both high productivity as well as excellent surface integrity. These conditions produced a material removal rate of 4,302 mm3/min, which was 14 times higher than the material removal rate achieved in machining pure CPP in earlier studies. The constructed machining model was highly successful in predicting the cutting forces, and therefore can be used in process planning and optimization in the production of tissue engineered implant constructs out of CPP. The Finite Element analyses predicted that the implant would not chip or break during the roughing operation, as validated experimentally. This allowed the roughing cycle time to be reduced from 159 min to 19 min, effectively achieving a productivity improvement of 8 times over the earlier work done in this area.

L’objectif de ce travail de thèse est de mettre en évidence et de comprendre le mécanisme de synergie observé au niveau des propriétés retard au feu de polyuréthanes (PUs) intumescents par l’ajout de nanoparticules (NPs). En effet, l’addition de polyphospha te d’ammonium (APP) dans une matrice PU conduit à une amélioration de ses propriétés retard au feu. La substitution d’une petite quantité d’APP par des NPs (MgO, SiO2, octamethyl polyhedral oligomeric silsesquioxanes (OMPOSS) et or) conduit de plus à un phénomène de synergie. Il a été montré que la nature et la quantité de NPs jouent un rôle important sur les propriétés retard au feu et sur le mécanisme de protection. L’étude de la stabilité thermique des différents systèmes a premièrement mis en évidence une stabilisation entre l’APP et les NPs à l’exception de l’OMPOSS. Il a donc été proposé que la synergie intervienne en phase condensée où diverses réactions chimiques, caractérisées par RMN du solide, ont lieu lors de la dégradation des matériaux entrainant la formation d’une barrière protectrice intumescente. Les propriétés de cette barrière, telles que sa conductivité thermique, son expansion et sa morphologie, ont été étudiés dans un second temps à l’aide de techniques spécifiques développées dans le cadre de cette étude (en particulier la tomographie). Ces propriétés ont été reliées à la meilleure protection observée pour le système contenant l’APP et les NPs. La résistance mécanique de barrières intumescentes développées dans différentes conditions a finalement été étudiée mais n’intervient pas dans le mécanisme de synergie. / The purpose of this Ph.D work is to present and to understand the synergy observed in the fire performances of intumescent polyurethane (PU) formulations by the addition of nanoparticles (NP). Indeed, it was shown that the addition of ammonium polyphosphate (APP) in PU leads to interesting fire properties that can be enhanced substituting a small amount of APP by NPs (MgO, SiO2, octamethyl polyhedral oligomeric silsesquioxanes (OMPOSS) and gold). The nature and content of the NPs play an important role on the fire retardant properties and mechanisms. First, a thermal stabilization was observed between APP and the different NPs except OMPOSS. The synergy mechanism was thus attributed to a condensed phase action where a range of chemical species, characterized by solid state NMR, are created upon heating the material in different conditions. The char properties, such as thermal conductivity, expansion and morphology, were then characterized using novel techniques (in particular tomography). It was shown that they are linked with the thermal barrier effect of the residual material explaining the good fire properties obtained when combining APP and NPs. The mechanical strength of chars developed in different conditions was also investigated but do not play a significant role on the synergy mechanism.

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