Lim klassificeras som ett ämne som kan hålla ihop två ytor så att de motstår separation. Dagens lim kan anpassas efter vilken applikation de ska användas till och kan ha ett stort antal olika egenskaper. De fysikaliska och kemiska egenskaperna av limmet är de viktigaste faktorerna för att bilda en bra limbindning. I stort sett alla syntetiska lim består idag av polymerer, varav de flesta är petroleumbaserade. För att skapa ett mer miljövänligt alternativ undersökte denna studie tillämpningen av poly(3-hydroxibutyrat- co-3-hydroxivalerat), PHBV med 36 eller 56 vikt% HV, som huvudkomponent i ett lim. Huvudfokus ligger på hur väl PHBV är lämpligt för användning som lim och hur olika tillsatser kan förbättra dess egenskaperna. Flera olika limblandningar innehållande PHBV, mjukgörare (sebacinsyra, dimetylsebacat, etylbutanoat eller tributylcitrat) och förtjockningsmedel (Abalyn eller Foralyn) skapades och undersöktes. Ett single lap skjuvtest utfördes med kopieringspapper, filterpapper och träpinnar som vidhäftningsmaterialet, medan ett avskalningstest undersökte användningen av kopieringspappersetiketter och plastetiketter på en glasflaska samt frukt.  Båda testen visar att kopieringspapper har de mest lovande egenskaperna som en adherent för användningen av ett PHBV-baserat lim, både för PHBV innehållande 36 vikts% och 56 vikts% HV. Denna slutsats kunde dras då kopieringspapperet påvisade den starkaste bindningen med limmet. Ren PHBV uppvisade lovande häftstyrka och vidhäftning som ett smältlim. Tillsatsen av sebacinsyra tillsammans med Abalyn eller dimetylsebacat med Foralyn ökade limmets häftstyrka ytterligare. Vi fann också att lim som skapats med PHBV med 36 vikt% HV ger bättre hållfasthetsegenskaper. Framtida arbete innefattar mer exakta mätmetoder för att bestämma egenskaperna hos limblandningarna. / An adhesive is classified as a substance that holds two surfaces together and resists separation. Today’s adhesives can be modified according to various application demands, obtaining a large variety of properties. The most important factors of forming a good adhesive bond are the physical and chemical properties. Essentially all synthetic adhesives consist of polymers, most of them being petroleum-based. To obtain a more environmentally friendly option, this study investigated the use of poly(3-hydroxybutyrate-co-3-hydroxyvalerate), PHBV with 36 or 56 wt% HV, as the main component in an adhesive. The main focus was to investigate on how well PHBV is suited for use as an adhesive and how different additives can improve the adhesive properties. Several different adhesive formulations containing PHBV, plasticizers (sebacic acid, dimethyl sebacate, ethyl butyrate, or tributyl citrate) and tackifiers (Abalyn or Foralyn) were created and investigated using various tests, such as single lap shear test, peel test, tackiness determination, optical analysis, and application testing. Single lap shear tests were performed using printing paper, filter paper, and wooden sticks as adherents, while peel tests explored the use of printing paper labels and plastic labels on a glass bottle and on fruit. It was determined that two PHBV adhesives, containing 36 wt% and 56 wt% of HV, performed best using printing paper as adherent. This conclusion could be drawn based on the good interaction between the adherent and the adhesive, thereby creating a strong bond. Pure PHBV with 36 or 56 wt% showed promising strength and tackiness properties as a hot-melt adhesive. The addition of sebacic acid together with Abalyn or dimethyl sebacate with Foralyn further increased the adhesive’s strength. It was also found that adhesive formulations created using a PHBV with a lower amount of HV (around 36 wt% of HV) yields better strength properties when used as an adhesive for paper labels. Future work involves more precise measurement methods to determine the properties of the adhesive formulations.

Orientador: Paulo Pinto Joazeiro / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-07T01:42:36Z (GMT). No. of bitstreams: 1 Lucchesi_Carolina_M.pdf: 3174282 bytes, checksum: b63549c83e46027b51da37ffd45ad2d7 (MD5) Previous issue date: 2006 / Resumo: O copolímero Poli(hidroxibutirato-co-hidroxivalerato) (PHBV) tem sido intensamente estudado como substrato para a engenharia de tecidos, sendo conhecido como um poliéster hidrofóbico. A modificação da superfície por plasma é uma técnica efetiva e econômica para os materiais e tem ganhado crescente interesse da engenharia biomédica, por melhorar a biocompatibilidade da superfície. Neste estudo, avaliou-se as vantagens da modificação da superfície de membranas de PHBV tratadas por plasma de Oxigênio e Nitrogênio a fim de acelerar o processo de adesão e proliferação celular. O PHBV foi dissolvido em c1oreto de metileno à temperatura ambiente. Membranas de PHBV foram submetidas ao tratamento de plasma de Oxigênio e Nitrogênio, através de um gerador de plasma. As membranas foram esterilizadas por radiação UV por 30 min e colocadas em placas de 96 poços. Células Vero foram semeadas sobre as membranas, sendo determinada a proliferação celular sobre as matrizes, a citotoxicidade e adesão celular. Após 2, 24,48 e 120h de incubação, o crescimento e proliferação dos fibroblastos foram observados por microscopia eletrônica de varredura (MEV). As análises das membranas indicaram que o tratamento por plasma aumentou o ângulo de contato e a rugosidade, alterando a morfologia da superfície, e conseqüentemente, melhorou ocomportamento hidrofílico do polímero. A microscopia eletrônica de varredura das células Vero mostrou que as modificações da superfície proporcionaram melhor adesão, espalhamento e proliferação celular. O tratamento da superfície do polímero somado às suas propriedades químicas é um caminho para obtenção de estruturas aplicáveis a engenharia de tecido / Abstract: The copolymers poly(3-hydroxybutyric acid-co-3-hydroxyvaleric acid) (PHBV) are being intensely studied as a tissue engineering substrate. It is know that Poly(hydroxybutic) (PHB) and their copolymers are quite hydrophobic polyesters. Plasma-surface modification is an effective and economical surface treatment technique for many materiaIs and of growing interest in biomedical engineering. In this study we investigate the advantages of oxygen and nitrogen plasma treatment to modify the PHBV surface to enable the acceleration of Vero cell adhesion and proliferation. PHBV was dissolved in methylene chloride at room temperature. The PHBV membranes were modified by oxygen or nitrogen-plasma treatments using a plasma generator. The membranes were sterilized by UV irradiation for 30 min and placed in 96-well plates. Vero cells were seeded onto the membranes and their proliferation onto the matrices was also determined by cytotoxicity and cell adhesion assay. After 2, 24, 48 and 120h of incubation, growth of fibroblasts on matrices was observed by scanning electron microscopy (SEM). The analyses of the membranes indicated that the plasma treatment increased the contact angle and their roughness, it also changed the surface morphology, and consequently, enhanced the hydrophilic behavior of PHBV polymers. Scanning electron microscopy analysis of Vero cell adhered to plasma treated PHBV showed that the modified surface had allowed better cell attachment, spreading and growth than the untreated membrane. This combination of surface treatment and polymer chemistry is a valuable guide to prepare appropriated surface for tissue engineering application / Mestrado / Histologia / Mestre em Biologia Celular e Estrutural

L’accumulation de biopolymère de réserve (PolyHydroxyAlcanoates ou PHA) par la souche Cupriavidus necator, à partir de substrats de type acides gras volatils (acide butyrique, acide propionique et acide acétique) a été étudiée. Elle est induite par une limitation phosphore. Les performances atteintes lors des cultures se situent parmi les meilleures de la littérature pour ce type de substrat : jusqu’à 66 g.L-1 de biomasse totale avec un pourcentage d’accumulation massique de 88% en PHB –PolyHydroxyButyrate- ou en PHB-co-HV -PolyHydroxyButyrate-co-HydroxyValerate- comportant jusqu’à 52% de motifs d’HV.Pour chaque source carbonée, une caractérisation cinétique et stœchiométrique de la souche a été réalisée en l’absence d’effets inhibiteurs dus aux substrats acides grâce à des cultures de type Fed-Batch avec des apports non limitants et non inhibiteurs en carbone. Il a été dégagé :- un taux de croissance maximal de la souche de 0,33 h-1 pour les trois acides étudiés- une relation entre vitesse spécifique de production de PHA et taux de croissance fixée par la disponibilité et les flux de production de NADPH2 avec un découplage inverse pour les taux de croissance supérieurs à 0,05 h-1 et un couplage partiel pour les taux de croissance inférieurs- un optimum de 0,35 Cmole.Cmole-1.h-1, associé à un taux de croissance de l’ordre de 0,05 h-1.- une amélioration de la production de PHB en termes de vitesses spécifiques mais également en termes de rendements si une faible croissance résiduelle est maintenueLa réponse de la souche à un excès de substrat acide a été caractérisée via l’étude de régimes transitoires induits par des pulses sur des cultures continues préalablement stabilisées en régime permanent. Il a été montré qu’en excès de phosphore, face à un brusque excès de substrat, la souche est incapable d’adapter rapidement son taux de croissance. L’excès est donc dirigé vers la production de PHA dont les voies sont plus rapidement mobilisables. En conditions limitantes de phosphore, le substrat excédentaire est utilisé pour la production de PHA. L’inhibition par les acides se traduit par une diminution des capacités de biosynthèse de la biomasse et des PHA entrainant une réduction de l’assimilation du carbone puis une diminution des rendements de conversion. D’autre part la sensibilité d’un système continu à un excès de substrat dépend du point de fonctionnement choisi : plus il est optimal en termes de vitesse, moins le système est robuste. L’acide propionique est très inhibiteur comparé aux autres acides étudiés (dès 3-4 mM contre 30-40 mM). Il n’agit pas simplement via une accumulation excessive dans le cytoplasme mais il exerce également une inhibition spécifique des voies métaboliques.Un antagonisme entre les substrats (acide acétique et butyrique) a été constaté et expliqué grâce à une analyse des flux métaboliques. L’acide acétique est assimilé préférentiellement pour produire la biomasse, l’énergie et les cofacteurs nécessaires à la production de PHA, alors que l’acide butyrique est utilisé pour la synthèse de PHB. La proportion maximale d’acide acétique admise dans l’alimentation en fonction des conditions fixées en régime permanent est calculée et peut être limitée à 40% du carbone.Enfin il a été déterminé que si une croissance résiduelle est assurée grâce à un apport en phosphore, le pourcentage maximal d’HV dans le polymère dépend du taux d’acide propionique dans l’alimentation et ne peux dépasser 33 ± 5% sur acide propionique pur. Par contre, si aucune croissance résiduelle n’est assurée, il est possible de convertir l’acide propionique en motifs d’HV uniquement / Reserve Biopolymer (PolyHydroxyAlkanoates or PHA) accumulation by the strain Cupriavidus necator, from Volatile Fatty Acids (VFA, like butyric acid, propionic acid and acetic acid) was investigated. This production is induced by a phosphorus limitation. For this type of substrates, performances reached during cultures are among the best listed in the literature: up to 66 g.L-1 of total biomass with 88% (w/w) of PHB –PolyHydroxyButyrate- or PHB-co-HV -PolyHydroxyButyrate-co-HydroxyValerate- with a HV content up to 52 Mole%.For each carbon source, kinetic and stoechiometric characterization has been carried out thanks to Fed-Batch cultures with non-limiting and non-inhibitory carbon feed. It has been established:- a maximal growth rate of 0,33 h-1 for the three acid investigated- a relationship between specific PHA production rate and growth rate which is set by the availability and production flux of NADPH2. For growth rate above 0,05 h-1, there is an inverse coupling. For growth rate under 0,05 h-1, there is a partial coupling.- an optimum of 0,35 Cmole.Cmole-1.h-1 is associated with a growth rate of 0,05 h-1.- if a low residual growth rate is maintained, an improvement of PHB production is recorded in terms of specific production rate and yieldsThe response of the strain to an excess of acid substrate was characterized through the investigation of transient state induced by pulsed addition of substrate during continuous cultures stabilized in steady state. It was shown that in excess of phosphorus, when there is a substrate excess, the strain is unable to quickly adapt its growth rate, so the excess is directed to PHA production whose ways seem to be more easily mobilized. Under phosphorus limitation, an excess of substrate is used for PHA production. Acid inhibition results in a decrease in biomass and PHA production capacity which leads to a decrease in carbon assimilation and conversion yields. The sensitivity of a continuous system to an excess of substrate depends on the chosen operating point: the more it is optimal in terms of specific production rate, the less the system is robust. Propionic acid is highly inhibitory compared to the other acids studied (from 3-4 mM versus 30-40 mM). It does not act only via an excessive accumulation in the cytoplasm but also exerts a specific inhibition of metabolic pathways.An antagonism between substrates (acetic and butyric acid) has been established and explained thanks to the Metabolic Flux Analysis. Acetic acid is preferentially used to produce biomass, energy and cofactors for PHA synthesis, whereas butyric acid is used to product PHB. According to the conditions set during steady state, maximal content of acetic acid admitted in the feed can be calculated. It can be limited to 40% of the carbon in the feed.Finally if a growth rate is maintained thanks to a phosphorus supply, the maximal HV content in polymer is function of propionic acid in the feed and cannot exceed 33 ± 5 Mole% on pure propionic acid. Conversely, if there is no residual growth, a total conversion of propionic acid into HV is allowed

Alternative bone repair strategies are frequently sought after in orthopaedic surgery to address the growing need for improved morbidity and healing rates. This thesis sought to initiate and validate such an alternative, harnessing the flexible nature of a biomaterial substrate and the unique potential of glycosaminoglycan sugars. A novel, biodegradable biomaterial polymer, PHBV, has previously been identified to have the potential to mimic the characteristics of bone necessary for tissue repair and in this study, it was hypothesized that PHBV would be able to support bone formation. When tested in vitro, PHBV was found to support osteoblast cell attachment, proliferation and differentiation, despite its rougher, more hydrophobic surface characteristics compared to tissue culture plastic (TCP). However, unlike the progression of cells on TCP, PHBV caused a developmental delay at each stage of osteogenesis, suggesting a sub-optimal cell-substrate interaction. The expression profiles of genes involved in the maintenance of the extracellular matrix were monitored to investigate this phenomenon further. The results suggested that cells cultured on PHBV appeared to preference 7 against a collagen-based ECM and, instead, trigger an increase in the expression of other factors, such as osteopontin, presumably to modify the biomaterial microenvironment to optimise continued growth and differentiation. This finding led to the next hypothesis that functionalisation of PHBV with suitable compounds could optimise and enhance the osteogenic development at the implant site by facilitating the desired and appropriate cell-substrate interactions. Non-protein factors are often preferred for functionalisation to material scaffolds over proteins, as they are relatively robust and can survive many of the processes used in the manufacture of biomaterials. Glycosaminoglycan (GAG) sugars were appropriate candidates for this purpose, as they are not only abundantly expressed in bone, but more importantly, they are capable of binding and facilitating the activity of growth factors. Furthermore, they are resistant to several environmental influences including changes in pH, heat and desiccation. To identify a GAG that could be integrated with PHBV or any other biomaterial substrate, GAGs were extracted from phenotypically-distinct stages of MG-63 osteosarcoma cells. These GAGs were identified to display gross structural differences, as well as differences in the enzymes synthesising them, between immature and mature osteoblastic cells, with the increased production of a larger GAG species observed as the cells differentiated. Unexpectedly, however, when these GAGs were subsequently dosed back into the media of growing MG-63 cells, their bioactivity did not match the stage at which they had been harvested: all GAG species were able to influence cell survival and growth to varying degrees but were not capable of affecting cell differentiation. However, if these same GAGs were exposed to cells by first being attached to the growth substrate, they induced varying degrees of aggregation in human mesenchymal stem cells (hMSCs), with more mature GAGs producing the most profound effects. Interestingly, a similar phenomenon was not observed when MG-63 cells where cultured in a similar manner. A direct correlation between the GAGs expressed by osteoblasts and the specific cellular processes they functionally influence has yet to be identified. While the experiments presented here demonstrate an effect of GAGs in osteoblastic cell survival, a role for GAGs in the progression of bone formation was not revealed. Loss-of-function studies were therefore necessary to determine the role of GAGs in bone, but this was hampered by the limited availability of procedures that allow the alteration of GAGs and the subsequent detection of these effects. Therefore, a tool to screen the efficacy of a loss of GAG function was developed. TAT-EGFP, a purpose-designed fluorescent GAG-binding peptide, was able to confirm that treatment with sodium chlorate was an effective 8 strategy to hinder GAG expression in MG-63 cells with minimal cytotoxicity to the cells. Following more extensive studies with chlorate treatment, it was found that a recoverable disruption to both proliferation and mineralisation could be induced in MG-63 cells. This suggested a role for GAGs in osteogenesis. A series of experiments then carried out following gene expression microarray analysis indicated that GAG de-sulfation by chlorate gives rise to an S-phase block in the cell cycle and a disruption to the actin cytoskeleton, which appeared to be associated with a change in the activity of cell-surface proteoglycans, most likely syndecan 4. It was also found that cells up-regulated plasma membrane ALP activity and cholesterol synthesis, presumably in an attempt to recover from a chlorate-induced loss in GAG function. Cholesterol is known to be important in establishing connections between membrane elements and the actin cytoskeleton, and its up-regulation here may reflect dysfunctions in these units and a dysfunction in syndecan 4 activity. With further confirmation, this would suggest that syndecan 4 plays a pivotal role in maintaining osteogenesis, in at least MG-63 cells, and that sulfated GAGs function principally to facilitate this role. The effective use of GAGs in bone repair strategies will require further understanding of GAG/syndecan 4/osteogenesis relationship.

This thesis is inspired by a persistent limitation in the use of composite biomaterials for orthopaedic applications, namely the agglomeration of reinforcing particles in these composites, which has resulted in poor mechanical properties. The use of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) and hydroxyapatite (HA) nanoparticles to produce biodegradable nanocomposites is investigated. More specifically, the thesis investigates different methods of composite processing, and interfacial modifying agents and the effect that these have on the nano- and micro- scale structure of composites and their mechanical properties. PHBV and HA were chosen because PHBV is a biodegradable/biocompatible polymer and it has a relatively high stiffness when compared to other biodegradable polymers frequently used in orthopaedic applications. HA is chemically similar to ceramic phase found in bones and hard tissues and the inclusion of HA into biomedical materials has been shown to enhance the rate of osteoconduction. HA/PHBV composites were produced using different dispersing agents including poly(acrylic acid) (PAA), a model dispersing agent, polyethyleneimine (PEI) which allowed for the development of a single solvent system for composite preparation, and heparin (Hep), a macromolecule which is produced in vivo. Additionally, HA/PAA/PHBV composites were prepared from both sonicated and non-sonicated HA/PAA suspensions up to approximately 17% by weight (wt %) of HA content. Attempts to prepare composites with higher HA loadings led to inhomogeneous composite mixtures, which were caused by the dual solvent system used for the composite preparation. The HA/PEI/PHBV and HA/Hep/PHBV composites were produced up to approximately 75 wt % of HA content. It was found that the HA/PEI/PHBV and HA/Hep/PHBV composites could be prepared at higher loadings than HA/PAA/PHBV composites due to the single solvent system used for the preparation of the HA/PEI/PHBV composites and the better dispersion of HA/Hep particles in precursor suspensions. Finally, selected HA/PEI/PHBV composites were further processed using a twin screw extruder. All of the composites were characterised in terms of their dispersion levels as well as their compressive mechanical properties. In addition, HA/PEI/PHBV composite reinforced with 20 wt % of HA content was also tested for its mechanical properties using three different test types; compression, three-point bending, and tensile tests. Finally, the HA/PAA/PHBV, HA/PEI/PHBV, and HA/Hep/PHBV composites were tested their compressive mechanical properties in wet state. It was found that the sonicated HA/PAA suspensions in general had better colloidal stability than non-sonicated ones and that this yielded composites with superior compressive moduli than those prepared from non-sonicated suspensions. In addition, the better dispersion of the particles in the composites prepared from the sonicated suspensions, as confirmed by transmission electron microscopic (TEM) images, led to higher percentage crystallinities when compared to the composites prepared from non-sonicated suspensions. It is likely that the greater number of individual HA particles and smaller HA agglomerates observed in the composites prepared from sonication treatment are acting as nuclei for crystal growth more effectively than large HA agglomerates. The largest modulus and yield strength that could be achieved with this system were approximately 1.45 GPa and 80 MPa, respectively. Composites of HA/PEI/PHBV and HA/Hep/PHBV with approximately 55 wt % of HA content were found to exhibit the largest compressive moduli of approximately 2.5 and 2.8 GPa, respectively. Moreover, the yield strengths for the same materials were found to be approximately 123 and 120 MPa, respectively. This was found to correlate with the better levels of dispersion within the nanocomposites that could be achieved using these stabilisers. The extruded samples were found to have an even greater degree of particle dispersion when compared to the unextruded ones. This improved degree of particle dispersion of the extruded samples resulted in higher moduli in comparison to unextruded samples. The largest compressive modulus and yield strength of the extruded samples were found to be approximately 3.2 GPa and 125 MPa, respectively. The compressive moduli of the composites produced in this thesis are significantly greater than that of cancellous bone (0.4 GPa), but significantly lower than that of cortical bone (12.8–17.7 GPa). However, maximum yield strengths of the HA/PEI/PHBV and HA/Hep/PHBV composites match to cortical bone (120–180 MPa), which is a noteworthy finding in this thesis. The wet mechanical results of all composites as well as pure PHBV polymer showed a reduction in both moduli and yield strengths when compared to dry state. In addition, after 2 weeks in wet state both moduli and yield strengths of the composites and pure polymer converged to approximately the same values. Finally, the HA/PEI/PHBV composite samples tested by tensile testing showed the highest Young’s modulus and those tested by compression testing possessed the lowest Young’s modulus. This resulted from the difference in periods of time for heating exposure and void contents of the tested samples, which were prepared by different methods. However, toughness values obtained from the samples tested using three-point bending and tensile tests, was not significantly different.

This thesis is inspired by a persistent limitation in the use of composite biomaterials for orthopaedic applications, namely the agglomeration of reinforcing particles in these composites, which has resulted in poor mechanical properties. The use of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) and hydroxyapatite (HA) nanoparticles to produce biodegradable nanocomposites is investigated. More specifically, the thesis investigates different methods of composite processing, and interfacial modifying agents and the effect that these have on the nano- and micro- scale structure of composites and their mechanical properties. PHBV and HA were chosen because PHBV is a biodegradable/biocompatible polymer and it has a relatively high stiffness when compared to other biodegradable polymers frequently used in orthopaedic applications. HA is chemically similar to ceramic phase found in bones and hard tissues and the inclusion of HA into biomedical materials has been shown to enhance the rate of osteoconduction. HA/PHBV composites were produced using different dispersing agents including poly(acrylic acid) (PAA), a model dispersing agent, polyethyleneimine (PEI) which allowed for the development of a single solvent system for composite preparation, and heparin (Hep), a macromolecule which is produced in vivo. Additionally, HA/PAA/PHBV composites were prepared from both sonicated and non-sonicated HA/PAA suspensions up to approximately 17% by weight (wt %) of HA content. Attempts to prepare composites with higher HA loadings led to inhomogeneous composite mixtures, which were caused by the dual solvent system used for the composite preparation. The HA/PEI/PHBV and HA/Hep/PHBV composites were produced up to approximately 75 wt % of HA content. It was found that the HA/PEI/PHBV and HA/Hep/PHBV composites could be prepared at higher loadings than HA/PAA/PHBV composites due to the single solvent system used for the preparation of the HA/PEI/PHBV composites and the better dispersion of HA/Hep particles in precursor suspensions. Finally, selected HA/PEI/PHBV composites were further processed using a twin screw extruder. All of the composites were characterised in terms of their dispersion levels as well as their compressive mechanical properties. In addition, HA/PEI/PHBV composite reinforced with 20 wt % of HA content was also tested for its mechanical properties using three different test types; compression, three-point bending, and tensile tests. Finally, the HA/PAA/PHBV, HA/PEI/PHBV, and HA/Hep/PHBV composites were tested their compressive mechanical properties in wet state. It was found that the sonicated HA/PAA suspensions in general had better colloidal stability than non-sonicated ones and that this yielded composites with superior compressive moduli than those prepared from non-sonicated suspensions. In addition, the better dispersion of the particles in the composites prepared from the sonicated suspensions, as confirmed by transmission electron microscopic (TEM) images, led to higher percentage crystallinities when compared to the composites prepared from non-sonicated suspensions. It is likely that the greater number of individual HA particles and smaller HA agglomerates observed in the composites prepared from sonication treatment are acting as nuclei for crystal growth more effectively than large HA agglomerates. The largest modulus and yield strength that could be achieved with this system were approximately 1.45 GPa and 80 MPa, respectively. Composites of HA/PEI/PHBV and HA/Hep/PHBV with approximately 55 wt % of HA content were found to exhibit the largest compressive moduli of approximately 2.5 and 2.8 GPa, respectively. Moreover, the yield strengths for the same materials were found to be approximately 123 and 120 MPa, respectively. This was found to correlate with the better levels of dispersion within the nanocomposites that could be achieved using these stabilisers. The extruded samples were found to have an even greater degree of particle dispersion when compared to the unextruded ones. This improved degree of particle dispersion of the extruded samples resulted in higher moduli in comparison to unextruded samples. The largest compressive modulus and yield strength of the extruded samples were found to be approximately 3.2 GPa and 125 MPa, respectively. The compressive moduli of the composites produced in this thesis are significantly greater than that of cancellous bone (0.4 GPa), but significantly lower than that of cortical bone (12.8–17.7 GPa). However, maximum yield strengths of the HA/PEI/PHBV and HA/Hep/PHBV composites match to cortical bone (120–180 MPa), which is a noteworthy finding in this thesis. The wet mechanical results of all composites as well as pure PHBV polymer showed a reduction in both moduli and yield strengths when compared to dry state. In addition, after 2 weeks in wet state both moduli and yield strengths of the composites and pure polymer converged to approximately the same values. Finally, the HA/PEI/PHBV composite samples tested by tensile testing showed the highest Young’s modulus and those tested by compression testing possessed the lowest Young’s modulus. This resulted from the difference in periods of time for heating exposure and void contents of the tested samples, which were prepared by different methods. However, toughness values obtained from the samples tested using three-point bending and tensile tests, was not significantly different.

This thesis is inspired by a persistent limitation in the use of composite biomaterials for orthopaedic applications, namely the agglomeration of reinforcing particles in these composites, which has resulted in poor mechanical properties. The use of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) and hydroxyapatite (HA) nanoparticles to produce biodegradable nanocomposites is investigated. More specifically, the thesis investigates different methods of composite processing, and interfacial modifying agents and the effect that these have on the nano- and micro- scale structure of composites and their mechanical properties. PHBV and HA were chosen because PHBV is a biodegradable/biocompatible polymer and it has a relatively high stiffness when compared to other biodegradable polymers frequently used in orthopaedic applications. HA is chemically similar to ceramic phase found in bones and hard tissues and the inclusion of HA into biomedical materials has been shown to enhance the rate of osteoconduction. HA/PHBV composites were produced using different dispersing agents including poly(acrylic acid) (PAA), a model dispersing agent, polyethyleneimine (PEI) which allowed for the development of a single solvent system for composite preparation, and heparin (Hep), a macromolecule which is produced in vivo. Additionally, HA/PAA/PHBV composites were prepared from both sonicated and non-sonicated HA/PAA suspensions up to approximately 17% by weight (wt %) of HA content. Attempts to prepare composites with higher HA loadings led to inhomogeneous composite mixtures, which were caused by the dual solvent system used for the composite preparation. The HA/PEI/PHBV and HA/Hep/PHBV composites were produced up to approximately 75 wt % of HA content. It was found that the HA/PEI/PHBV and HA/Hep/PHBV composites could be prepared at higher loadings than HA/PAA/PHBV composites due to the single solvent system used for the preparation of the HA/PEI/PHBV composites and the better dispersion of HA/Hep particles in precursor suspensions. Finally, selected HA/PEI/PHBV composites were further processed using a twin screw extruder. All of the composites were characterised in terms of their dispersion levels as well as their compressive mechanical properties. In addition, HA/PEI/PHBV composite reinforced with 20 wt % of HA content was also tested for its mechanical properties using three different test types; compression, three-point bending, and tensile tests. Finally, the HA/PAA/PHBV, HA/PEI/PHBV, and HA/Hep/PHBV composites were tested their compressive mechanical properties in wet state. It was found that the sonicated HA/PAA suspensions in general had better colloidal stability than non-sonicated ones and that this yielded composites with superior compressive moduli than those prepared from non-sonicated suspensions. In addition, the better dispersion of the particles in the composites prepared from the sonicated suspensions, as confirmed by transmission electron microscopic (TEM) images, led to higher percentage crystallinities when compared to the composites prepared from non-sonicated suspensions. It is likely that the greater number of individual HA particles and smaller HA agglomerates observed in the composites prepared from sonication treatment are acting as nuclei for crystal growth more effectively than large HA agglomerates. The largest modulus and yield strength that could be achieved with this system were approximately 1.45 GPa and 80 MPa, respectively. Composites of HA/PEI/PHBV and HA/Hep/PHBV with approximately 55 wt % of HA content were found to exhibit the largest compressive moduli of approximately 2.5 and 2.8 GPa, respectively. Moreover, the yield strengths for the same materials were found to be approximately 123 and 120 MPa, respectively. This was found to correlate with the better levels of dispersion within the nanocomposites that could be achieved using these stabilisers. The extruded samples were found to have an even greater degree of particle dispersion when compared to the unextruded ones. This improved degree of particle dispersion of the extruded samples resulted in higher moduli in comparison to unextruded samples. The largest compressive modulus and yield strength of the extruded samples were found to be approximately 3.2 GPa and 125 MPa, respectively. The compressive moduli of the composites produced in this thesis are significantly greater than that of cancellous bone (0.4 GPa), but significantly lower than that of cortical bone (12.8–17.7 GPa). However, maximum yield strengths of the HA/PEI/PHBV and HA/Hep/PHBV composites match to cortical bone (120–180 MPa), which is a noteworthy finding in this thesis. The wet mechanical results of all composites as well as pure PHBV polymer showed a reduction in both moduli and yield strengths when compared to dry state. In addition, after 2 weeks in wet state both moduli and yield strengths of the composites and pure polymer converged to approximately the same values. Finally, the HA/PEI/PHBV composite samples tested by tensile testing showed the highest Young’s modulus and those tested by compression testing possessed the lowest Young’s modulus. This resulted from the difference in periods of time for heating exposure and void contents of the tested samples, which were prepared by different methods. However, toughness values obtained from the samples tested using three-point bending and tensile tests, was not significantly different.

L’objectif de cette thèse est le développement des matériaux polymères injectables innovants issus de ressources renouvelables et répondant aux exigences techniques automobiles. Pour cela, des mélanges à base de polyoléfines (POs) et du poly(3- hydroxybutyrate-co-hydroxyvalérate) (PHBV) ont été étudiés. Ces mélanges polymères étant non-miscibles, l’optimisation de leurs propriétés par compatibilisation a été réalisée selon différentes approches. Dans un premier temps, la synthèse de polypropylène fonctionnel original avec des taux de greffage relativement élevés et sans perte de masses molaires a permis, par réaction in-situ, la compatibilisation des mélanges PP/PHBV. Dans une deuxième partie, le poly(éthylène-co- alcool vinylique) greffé-poly(3- hydroxybutyrate-co- hydroxyvalérate) a été synthétisé par extrusion réactive, ce copolymère a été utilisé ensuite comme compatibilisant pour les mélanges PE/PHBV. L’ajout de ces compatibilisants a permis d’obtenir des matériaux ayant des propriétés optimisées et une morphologie nettement plus fine que les systèmes non compatibilisés démontrant ainsi leur efficacité / The main objective of this work is the development of innovative materials from renewable resources for the automotive industry. Polymer blends of polyolefins (POs) and poly(3-hydroxybutyrate-co-hydroxyvalerate) (PHBV) were prepared through twin screw extruder process and studied. These polymer blends are non-miscible, then in order to optimize their final properties, compatibilizing agents were synthesized by reactive extrusion. On the one hand, functionalized polypropylene with various polar monomers and without significant molecular weight loss was synthesized with the aim to compatibilize PP/PHBV blends, on the other hand, poly(ethylene-co- vinylalcohol)-graft- poly(3-hydroxybutyrate-co-hydroxyvalerate) was obtained by exchange reactions in the presence of an efficient catalyst and those in order to compatibilize PE/PHBV blends. Efficiency of these compatibilizers was investigated in the last part of this study which is dedicated to POs/PHBV polymer blends compatibilization. Materials with improved properties and finer morphologies than the non- compatibilized systems were obtained thanks to the compatibilizers

Orientador: Prof. Dr. Arnaldo Rodrigues dos Santos Junior / Dissertação (mestrado) - Universidade Federal do ABC, Programa de Pós-Graduação em Biotecnociência, 2014. / Na engenharia de tecidos, a utilização de uma fonte de celular juntamente com um biomaterial, representa uma alternativa clínica a ser aplicada a pacientes com graves lesões no tecido ósseo. Este estudo teve como objetivo avaliar células Vero, uma linhagem de célula fibroblásticas, e uma linhagem de células-tronco mesenquimais (Rat (SD) Mesenchymal Stem Cells) em testes de biocompatibilidade in vitro com polímeros de poli(hidroxibutirato-co-hidroxivalerato) (PHBV), poli(caprolactona) (PCL) e blendas 75/25 e 50/50 desenvolvidos para a bioengenharia de tecido ósseo. A diferenciação osteogênica das CTMs sobre os polímeros também foi analisada. Os biomateriais foram caracterizados morfologicamente através de Microscopia Eletrônica de Varredura (MEV), Estereoscópio e Micrômetro. Polímeros porosos se mostram mais espesso que os densos. Através das imagens obtidas nota-se a distribuição, tamanho e morfologia dos poros, notando que os polímeros se mostram com características condizentes a de outros trabalhos. As células Vero e células-tronco mesenquimais (CTMs) foram cultivadas sobre as amostras citadas. Realizamos o ensaio com o MTT, análise morfológica e citoquímica. Para as CTMs fizemos ainda ensaios para avaliar a diferenciação osteogênica (fosfatase alcalina e vermelho alizarina). Nenhum dos polímeros foi considerado tóxico para as células Vero e na maioria deles foi notada atividade celular, camadas de células bem distribuídas. As blendas 50/50 mostraram resultados um pouco inferiores, quanto ao MTT, essa blenda porosa demonstrou ser a uma amostra onde adesão ocorre de forma bem mais lenta, os demais polímeros apresentaram resultados semelhantes e até superiores ao controle positivo de adesão, principalmente o PCL denso. Apesar das amostras 50/50 densa e porosa não se mostrarem tóxicas, aparentemente não funcionam com bons substratos como os demais polímeros, também apresentaram várias dificuldades na técnica de preparação, sendo assim descartadas. A relação das CTMs com os biomateriais se mostrou semelhante aos resultados com Vero. As células foram capazes de se espalhar por quase toda a superfície dos polímeros inclusive entre os poros dos materiais porosos. As CTMs apresentaram resultados de adesão (MTT) sobre os polímeros, mais rápido do que a Vero, demonstrando também maior afinidade pelo PCL denso. Através da análise da atividade da enzima fosfatase alcalina (usada como marcador de diferenciação), notamos que as células-tronco se mostraram capazes de se diferenciar em contato com os polímeros. Esses dados foram confirmados com o vermelho de alizarina, que também mostrou que a diferenciação celular se mostra um pouco mais lenta nos materiais do que nas placas. De uma forma geral os polímeros com exceção das blendas 50/50 se mostraram como bons substratos para as células, com ausência de toxicidade, características morfológicas dentro do recomendado e, além disso, não bloqueiam respostas biológicas específicas, como a diferenciação osteogênica. / In tissue engineering, the use of a cell source coupled with biomaterial represents a clinical alternative which can be applied to patients with severe bone damage. This study aimed to evaluate the biocompatibility between Vero a fibroblastic cell line and Mesenquymal stem cells (Rat (SD) MSC) with biomaterials developed as scaffolds for bone tissue engineering, bioresorbable polymers composed of poly ( hydroxybutyrate-co-hydroxyvalerate ) [ PHVB ] and poly ( caprolactone ) [ PCL ] pure and ratios of (75 /25) and (50 /50) blends. The osteogenic differentiation of MSCs on the polymers was also analyzed. Morphological characterization of the materials was performed by Scanning Electron Microscopy (SEM), Stereoscope and micrometer. The porous polymers are thicker than dense. Through the images obtained it is possible to note distribution, morphology and pore size. The biomaterials seem to the same consistent characteristics of other studies. Vero cells and mesenchymal stem cells (MSCs) were cultured on the samples mentioned. We performed the assay with MTT, morphological and histochemical analysis. About MSCs we also evaluated the osteogenic differentiation (alkaline phosphatase and Alizarin red). None of the polymers was considered toxic to Vero cells and most of them presented cellular activity, layers of well-distributed cells was noted in most of them. However 50/50 blends showed no such significant results as other. In the MTT assay, this porous blend demonstrated to be the only sample where adhesion occurs more slowly, other polymers showed similar and even higher results than the adhesion positive control, especially the dense PCL. Although the dense and porous 50/50 samples do not show toxic, apparently they are not good substrates as the other polymers, also presented several difficulties in their preparation thus being discarded. The ratio of MSCs with biomaterials was similar to the Vero cells results. They were able to spread to almost all surface of the polymers including into the pores of porous materials. MSCs showed adhesion (MTT) faster than the Vero cell, also demonstrated a greater affinity for dense PCL sample. Through analysis of the enzyme alkaline phosphatase activity (used as a marker for differentiation), we noticed that the stem cells showed differentiation into contact with the polymers. These data were confirmed by alizarin red, which also showed that the cell differentiation was slower on the materials than on the plates. In general all the polymer blends but the 50/50 proved to be good substrates for cells, with no toxicity, morphological characteristics within recommended and in addition do not block specific biological responses, such as osteogenic differentiation.

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