No description available.

Este trabalho tem como objetivo a caracterização de amostras comerciais de óleo de pinho com o intuito de identificar outros componentes que apresentem ação antimicrobiana, além do α-terpineol. Também, visa avaliar a influência de três tensoativos distintos na ação antimicrobiana do o-fenil fenol, do o-benzil p-clorofenol e do p-cloro m-cresol. Os tensoativos selecionados foram o linear alquilbenzeno sulfonato de sódio (NaLAS), uma mistura de laurato de trietanolamina e de sódio (laurato de Na/Tea) e alfa olefina sulfonada (AOS). Amostras comerciais de óleo de pinho e 61 frações, isoladas por destilação a pressão reduzida de uma amostra de óleo de pinho bruto, foram avaliadas a partir de medidas de índice de refração, curvas TG/DTG, espectroscopia no infravermelho, CG-DIC e CG-EM, características organolépticas e atividade antimicrobiana com a determinação do halo de inibição (contra Staphylococcus aureus ATCC 6538 e Salmonella choleraesuis ATCC 10708). Observou-se que as amostras comerciais apresentaram composições distintas, conforme procedência. O perfil das curvas TG/DTG permitiu uma avaliação comparativa do comportamento térmico das frações isoladas na destilação. A composição do óleo de pinho apresenta diversidade de componentes com atividade antimicrobiana inibitória. Todas as amostras de óleo de pinho apresentaram atividade bacteriostática para ambos os microrganismos e as frações, na composição em que foram separadas, apresentaram melhor atividade bacteriostática para S. aureus. Foram preparadas 42 formulações considerando teores de biocida de 0, 0,7 e 1,0% (p/p) e de tensoativo a 1,5 e 5% (p/p). As amostras foram acondicionadas em frascos de PET de 30 mL e avaliadas quanto ao pH, cor, odor, aparência nas 2ª, 4ª, 8ª e 12ª semanas do teste de estabilidade, conduzido a 4, 25 o e 45o C. O sistema tensoativo empregando o laurato de Na/TEA apresentou melhor estabilidade. Os tensoativos estudados (AOS, NaLAS e laurato de Na/TEA), apresentaram ação antimicrobiana e influenciaram positivamente na atividade dos ativos fenólicos. Pôde- se identificar a influência do teor de tensoativo na estabilização do derivado fenólico em um o sistema exposto a alta temperatura (45o C) armazenado em embalagem de PET. Os teores de ativos fenólicos foram determinados por CLAE empregando detector UV. / This work aims at characterizing commercial samples of pine oil with a view at identifying other components that present antimicrobial action besides its o -terpineol. It also aims at evaluating how three different surfactants influence the antimicrobial action of the o-phenyl-phenol, of the o-benzyl-p-chlorophenol and p-chloro-m-cresol. The selected surfactants were the sodium linear alkylbenzene sulphonate (NaLAS), a mixture of triethanolamine and sodium laurate, in addition to sodium alpha-olefins sulfonate (AOS). Pine oil commercial samples and 61 fractions, which were isolated by reduced pressure distillation of a raw pine oil sample, were analyzed based on measures of index of refraction, TG/DTG curves, infrared spectroscopy, GC-FID and GC-MS, odour characterization and antimicrobial activity with the determination of the inhibition disc (against Staphylococcus aureus ATCC 6538 and Salmonella choleraesuis ATCC 10708). It was observed that commercial samples presented marked different compositions according to their origin. TG/DTG curves profile allowed a comparative evaluation of the thermal behavior of those fractions isolated during distillation. Pine oil composition presents a diversity of components with antimicrobial activity. All pine oil samples presented bacteriostatic activity when tested against the two microorganisms mentioned above, and fractions in the composition in which they were inserted presented better bacteriostatic activity against S. aureus. Forty two formulations were prepared taking into consideration the content of biocide of 0, 0.7 and 1.0% (w/w) levels and surfactant of 1.5 e 5% (w/w) levels. Samples were stored into PET nd th th bottles of 30 mL and evaluated as per the pH, color, odour, appearance during 2nd , 4th , 8th and 12th weeks of the stability test, which was carried out at 4, 25 e 45o C. When using Na/TEA laurate, the surfactant system presented better stability. Studied surfactants - AOS, NaLAS and Na/TEA laurate - presented antimicrobial action and positively influenced in active phenolics activity. It was possible to identify the influence of content of surfactant in o stabilizing the phenolic derivative while under a system exposed to high temperature (45 C) inside a PET packaging. Active phenolics content was determined by HPLC using UV detection.

Este trabalho tem como objetivo a caracterização de amostras comerciais de óleo de pinho com o intuito de identificar outros componentes que apresentem ação antimicrobiana, além do α-terpineol. Também, visa avaliar a influência de três tensoativos distintos na ação antimicrobiana do o-fenil fenol, do o-benzil p-clorofenol e do p-cloro m-cresol. Os tensoativos selecionados foram o linear alquilbenzeno sulfonato de sódio (NaLAS), uma mistura de laurato de trietanolamina e de sódio (laurato de Na/Tea) e alfa olefina sulfonada (AOS). Amostras comerciais de óleo de pinho e 61 frações, isoladas por destilação a pressão reduzida de uma amostra de óleo de pinho bruto, foram avaliadas a partir de medidas de índice de refração, curvas TG/DTG, espectroscopia no infravermelho, CG-DIC e CG-EM, características organolépticas e atividade antimicrobiana com a determinação do halo de inibição (contra Staphylococcus aureus ATCC 6538 e Salmonella choleraesuis ATCC 10708). Observou-se que as amostras comerciais apresentaram composições distintas, conforme procedência. O perfil das curvas TG/DTG permitiu uma avaliação comparativa do comportamento térmico das frações isoladas na destilação. A composição do óleo de pinho apresenta diversidade de componentes com atividade antimicrobiana inibitória. Todas as amostras de óleo de pinho apresentaram atividade bacteriostática para ambos os microrganismos e as frações, na composição em que foram separadas, apresentaram melhor atividade bacteriostática para S. aureus. Foram preparadas 42 formulações considerando teores de biocida de 0, 0,7 e 1,0% (p/p) e de tensoativo a 1,5 e 5% (p/p). As amostras foram acondicionadas em frascos de PET de 30 mL e avaliadas quanto ao pH, cor, odor, aparência nas 2ª, 4ª, 8ª e 12ª semanas do teste de estabilidade, conduzido a 4, 25 o e 45o C. O sistema tensoativo empregando o laurato de Na/TEA apresentou melhor estabilidade. Os tensoativos estudados (AOS, NaLAS e laurato de Na/TEA), apresentaram ação antimicrobiana e influenciaram positivamente na atividade dos ativos fenólicos. Pôde- se identificar a influência do teor de tensoativo na estabilização do derivado fenólico em um o sistema exposto a alta temperatura (45o C) armazenado em embalagem de PET. Os teores de ativos fenólicos foram determinados por CLAE empregando detector UV. / This work aims at characterizing commercial samples of pine oil with a view at identifying other components that present antimicrobial action besides its o -terpineol. It also aims at evaluating how three different surfactants influence the antimicrobial action of the o-phenyl-phenol, of the o-benzyl-p-chlorophenol and p-chloro-m-cresol. The selected surfactants were the sodium linear alkylbenzene sulphonate (NaLAS), a mixture of triethanolamine and sodium laurate, in addition to sodium alpha-olefins sulfonate (AOS). Pine oil commercial samples and 61 fractions, which were isolated by reduced pressure distillation of a raw pine oil sample, were analyzed based on measures of index of refraction, TG/DTG curves, infrared spectroscopy, GC-FID and GC-MS, odour characterization and antimicrobial activity with the determination of the inhibition disc (against Staphylococcus aureus ATCC 6538 and Salmonella choleraesuis ATCC 10708). It was observed that commercial samples presented marked different compositions according to their origin. TG/DTG curves profile allowed a comparative evaluation of the thermal behavior of those fractions isolated during distillation. Pine oil composition presents a diversity of components with antimicrobial activity. All pine oil samples presented bacteriostatic activity when tested against the two microorganisms mentioned above, and fractions in the composition in which they were inserted presented better bacteriostatic activity against S. aureus. Forty two formulations were prepared taking into consideration the content of biocide of 0, 0.7 and 1.0% (w/w) levels and surfactant of 1.5 e 5% (w/w) levels. Samples were stored into PET nd th th bottles of 30 mL and evaluated as per the pH, color, odour, appearance during 2nd , 4th , 8th and 12th weeks of the stability test, which was carried out at 4, 25 e 45o C. When using Na/TEA laurate, the surfactant system presented better stability. Studied surfactants - AOS, NaLAS and Na/TEA laurate - presented antimicrobial action and positively influenced in active phenolics activity. It was possible to identify the influence of content of surfactant in o stabilizing the phenolic derivative while under a system exposed to high temperature (45 C) inside a PET packaging. Active phenolics content was determined by HPLC using UV detection.

CoCr-Legierungen wurden und werden für orthopädische Implantate verwendet, da sie eine Kombination aus hoher Festigkeit, Korrosionsbeständigkeit und Biokompatibilität ermöglichen. Seit 1985 wurden metallische Implantate umfassend untersucht, die mit Calciumphosphaten beschichtet sind, um die Biokompatibilität und die Anindung an das angrenzende Knochengewebe zu verbessern. Trotz des Fortschritts und der Entwicklung vieler Technologien ist die Ausfallrate solcher Implantate immer noch ein Problem. In der vorliegenden Arbeit wurde ein neuer und einfacher Weg entwickelt, um eine Hydroxylapatit (HAp) -Beschichtung auf einer CoCr-Legierung vermittels einer Suspension aus umweltfreundlichem Kiefernöl (PO), das mit HAp-Partikeln gemischt ist, zu erhalten. Die HAp-Beschichtung wurde unter Verwendung eines Pinsels auf den CoCr-Substraten aufgebracht, gefolgt von einer Wärmebehandlung bei 800°C für 1 Stunde, um die Haftung zwischen dem Substrat und der Schicht zu verbessern. Verschiedene durch Polieren, Passivieren und einer Wärmebehandlung hergestellte Oberflächenmorphologien sowie ein unbehandeltes Substrat wurden getestet und dann beschichtet, um den Einfluss der Rauheit auf die Wechselwirkung zwischen Substrat und Beschichtung zu bewerten. Die Benetzbarkeit wurden wurde durch Kontaktwinkelmessungen bestimmt. Der Kontaktwinkel von Wasser auf den Metallimplantatoberflächen war viel höher (68°) als der von PO (23°), was einen homogeneren Beschichtungsprozess ermöglichte, wenn PO für die HAp-Suspension verwendet wurde. Rasterelektronenmikroskopie- (REM), Röntgenbeugungs- (XRD) und Fourier-Transform-Infrarotspektroskopie (FTIR) -Analysen ergaben, dass die Substrate vollständig mit reinem HAp bedeckt waren. Die Wechselwirkung zwischen Substrat und Beschichtung wurde mittels REM, optischem Profilometer und Bleistift-Kratztest analysiert. Es wurde eine gute Partikelverteilung und für alle getesteten Oberflächenmorphologien eine stabile Bindung zum Substrat nachgewiesen. Die in vitro in simulierter Körperflüssigkeit (SBF) durchgeführten Bioaktivitätstests zeigten eine hohe Beständigkeit gegen Abbau und es wurde eine verringerte Bildung von Rissen auf den Oberflächen nach 21 Tagen Auslagerung beobachtet. Potentiodynamische Polarizations- und Cyclovoltammetrieanalysen bestätigten, dass die beschichteten und unbeschichteten Proben eine gute Korrosionsbeständigkeit zeigten. Die HAp-Beschichtung auf dem passivierten Substrat zeigte im Vergleich zu unbeschichteten Proben eine überlegene Korrosionsbeständigkeit, und es kann angenommen werden, dass hierbei die HAp-Beschichtung den Oberflächenschutz gegen Korrosion verbessert. Die Hochtemperaturbehandlung förderte jedoch die Abnahme der Korrosionsbeständigkeit im Falle des beschichteten, polierten Substrats, was auch das Wachstum von menschlichen primären Zellen aus Knochengewebe beeinflusste, die für in vitro-Zytokompatibilitätstests der verschiedenen Oberflächenmodifikationen verwendet wurden. Um die hohen Kosten und die Komplexität der konventionellen Abscheidungsmethoden und das damit verbundene Risiko einer Phasenumwandlung von HAp zu überwinden, bietet diese Arbeit eine einfache und effiziente Möglichkeit, HAp als mögliche Alternative zur Verbesserung von Knochenimplantaten auf metallische Substrate aufzutragen.:List of Figures vi List of Tables ix List of Abbreviations x Summary xi 1. Introduction 1 1.1 Objective 3 2. Literature review 4 2.1 Metallic biomaterials 4 2.1.1 Co-based alloys 5 2.1.2 Corrosion of CoCr alloys 7 2.2 Surface of metallic implants 11 2.3 Interface between substrate and coating 12 2.4 Cell-surface interaction 13 2.5 Calcium phosphates (CaP) 15 2.5.1 Hydroxyapatite 15 2.6 HAp coating 16 2.6.1 HAp coating methods 16 3. Materials & methods 27 3.1 Synthesis and characterization of HAp powder 27 3.2 Preparation of metallic substrate 27 3.3 Thermal behavior of the Pine Oil 28 3.4 HAp/pine oil slurry preparation and coating process 28 3.5 Characterization of the coating 29 3.6 Surface topography and wettability 29 3.7 Scratch resistance of the coating 29 3.8 Corrosion tests 30 3.8.1 Cyclic voltammetry 31 3.8.2 Potentiodynamic polarization 31 3.9 In vitro bioactivity analyses 33 3.10 Cell culture 34 4. Results & Discussion 36 4.1 Characterization of HAp powder 36 4.2 Thermal characterization of the pine oil 38 4.3 Characterization of the coating surface 40 4.3.1 X-ray diffraction and FTIR analyses 43 4.4 Surface properties 45 4.4.1 Topography, roughness and wettability 45 4.5 Scratch resistance of the coating 50 4.6 In vitro bioactivity and morphology 53 4.7 Metallography of the Co-15Cr-15W-10Ni alloy 62 4.8 Corrosive behavior 63 4.8.1 Effect of heat treatment on corrosion resistance 66 4.9 Cell Proliferation and osteogenic differentiation 68 5. Conclusions 73 5.1 Future perspectives 74 6. References 75 Acknowledgements 90 / CoCr alloys have been used for orthopedic implants as they allow combining high strength, corrosion resistance and biocompatibility. Since 1985 metallic implants coated with calcium phosphates have been widely studied to improve the biocompatibility and adequate bonding to the adjacent bones, but despites of advances and development of many technologies, rate of failure of such implants still are a problem. In the present work, a novel and simple route was developed to obtain hydroxyapatite (HAp) coating on CoCr alloy from a slurry of eco-friendly pine oil (PO) mixed with HAp particles. The HAp coating was deposited onto CoCr substrates using a paint brushing followed by heat treatment at 800°C for 1 h in order to improve the adhesion between the substrate and coating. Several substrate surface morphologies made by polishing, passivation, heat treatment and remaining the substrate untreated were tested, and then coated to evaluate the influence of the roughness on the interaction between substrate and coating. Wettability tests were carried out by sessile drop method; the contact angle of water on the metal implant surfaces was much higher (68°) than that of PO (23°), allowing a more homogeneous coating process when PO was used for HAp suspension. Scanning Electron Microscopy (SEM), X-ray diffraction (XRD) and Fourier-transform infrared spectroscopy (FTIR) analyses indicated a substrate fully covered by pure HAp. The interaction between substrate and coating was analyzed by SEM, optical profilometer and pencil scratch tests. Good particle distribution and tight bonding to the substrate were found for all surfaces morphologies tested. The bioactivity tests in vitro performed in simulated body fluid (SBF) showed high resistance to degradation and a decreased formation of cracks was observed on the surfaces after 21 days of soaking. Potentiodynamic polarization and cyclic voltammetry analyses corroborated that the coated and uncoated samples showed good corrosion resistance. The HAp coating on the passivated substrate presented a superior corrosion resistance compared to uncoated ones and it can be assumed that the HAp coating improve the surface protection against corrosion. However, the high temperature treatment promoted decrease of corrosion resistance in case of the coated polished substrate, which also affected the growth of human primary bone-derived cells which were used for in vitro cytocompatibility tests of the different surface modifications. In order to overcome the high costs and complexity of the conventional deposition methods and the risk of phase transformation of HAp, related to those, this thesis presents a straightforward and efficient route to coat HAp onto metallic substrates as a potential alternative for improvement of bone implants.:List of Figures vi List of Tables ix List of Abbreviations x Summary xi 1. Introduction 1 1.1 Objective 3 2. Literature review 4 2.1 Metallic biomaterials 4 2.1.1 Co-based alloys 5 2.1.2 Corrosion of CoCr alloys 7 2.2 Surface of metallic implants 11 2.3 Interface between substrate and coating 12 2.4 Cell-surface interaction 13 2.5 Calcium phosphates (CaP) 15 2.5.1 Hydroxyapatite 15 2.6 HAp coating 16 2.6.1 HAp coating methods 16 3. Materials & methods 27 3.1 Synthesis and characterization of HAp powder 27 3.2 Preparation of metallic substrate 27 3.3 Thermal behavior of the Pine Oil 28 3.4 HAp/pine oil slurry preparation and coating process 28 3.5 Characterization of the coating 29 3.6 Surface topography and wettability 29 3.7 Scratch resistance of the coating 29 3.8 Corrosion tests 30 3.8.1 Cyclic voltammetry 31 3.8.2 Potentiodynamic polarization 31 3.9 In vitro bioactivity analyses 33 3.10 Cell culture 34 4. Results & Discussion 36 4.1 Characterization of HAp powder 36 4.2 Thermal characterization of the pine oil 38 4.3 Characterization of the coating surface 40 4.3.1 X-ray diffraction and FTIR analyses 43 4.4 Surface properties 45 4.4.1 Topography, roughness and wettability 45 4.5 Scratch resistance of the coating 50 4.6 In vitro bioactivity and morphology 53 4.7 Metallography of the Co-15Cr-15W-10Ni alloy 62 4.8 Corrosive behavior 63 4.8.1 Effect of heat treatment on corrosion resistance 66 4.9 Cell Proliferation and osteogenic differentiation 68 5. Conclusions 73 5.1 Future perspectives 74 6. References 75 Acknowledgements 90 / Ligas de CoCr têm sido empregadas em implantes ortopédicos por permitirem a combinação de alta resistência mecânica, resistência a corrosão e biocompatibilidade. Implantes metálicos revestidos com fosfatos de cálcio têm sido estudados desde 1985 devido a sua capacidade de aumentar a biocompatibilidade e promover a ligação adequada com os ossos adjacentes. No entanto, apesar de todos os avanços e o desenvolvimento de muitas tecnologias, a taxa de falha destes implantes ainda é um problema. No presente trabalho, uma nova e simples rota foi desenvolvida para revestir a liga de CoCr com hidroxiapatita (HAp) a partir de uma mistura óleo de pinho (PO) ecológica com partículas de HAp. O revestimento de HAP foi depositado sobre o substrato de CoCr usando uma pincel de pintura seguido de tratamento térmico a 800 °C por 1 h a fim de aumentar a adesão entre o substrato e o revestimento. Foram testadas diversas morfologias de superfície do substrato, sendo estas polidas, passivadas, tratadas termicamente, além de um substrato não tratado, e em seguida foram revestidas para avaliar a influencia da rugosidade na interação entre substrato e revestimento. Testes de molhabilidade foram realizados pelo método de gota séssil; o ângulo de contato da água na superfície metálica do implante foi muito maior (68°) que os com PO (23°), permitindo uma maior homogeneidade no processo de revestimento quando utilizado PO na suspensão de HAp. Análises por Microscopia Eletrônica de Varredura (MEV), difração de raio-x (DRX) e espectroscopia infravermelha por transforma de Fourier (FTIR) indicaram que o substrato foi completamente coberto com a HAp pura. A interação entre o substrato e o revestimento foi analisada por MEV, perfilômetro óptico e teste de arrancamento, os quais demonstraram uma boa distribuição de partículas e uma próxima ligação para todas as morfologias de superfície testadas. Testes in vitro de bioatividade foram realizados com fluido corpóreo simulado (SBF) e apresentaram alta resistência a degradação e uma redução da formação de trincas na superfície foi observada após 21 dias de imersão. Análises de polarização potenciodinâmica e voltametria cíclica comprovaram uma boa resistência à corrosão nas amostras revestidas e não revestidas. O revestimento de HAp no substrato passivado apresentou uma resistência à corrosão superior aos não revestido e pode ser assumido que o revestimento de HAp melhorou a proteção da superfície contra corrosão. No entanto, o tratamento térmico com alta temperatura promoveu uma diminuição da resistência à corrosão no caso do substrato polido revestido, no qual afetou o crescimento de células primárias derivadas de osso humano que foram usadas nos testes de citocompatibilidade in vitro nas diferentes superfícies modificadas. A fim de solucionar o alto custo e a complexidade no método convencional de deposição, além dos riscos da transformação de fase da HAp, esta tese apresenta uma direta e eficiente rota de revestimento de HAp em substratos metálicos como uma potencial alternativa para o melhoramento de implantes ósseos.:List of Figures vi List of Tables ix List of Abbreviations x Summary xi 1. Introduction 1 1.1 Objective 3 2. Literature review 4 2.1 Metallic biomaterials 4 2.1.1 Co-based alloys 5 2.1.2 Corrosion of CoCr alloys 7 2.2 Surface of metallic implants 11 2.3 Interface between substrate and coating 12 2.4 Cell-surface interaction 13 2.5 Calcium phosphates (CaP) 15 2.5.1 Hydroxyapatite 15 2.6 HAp coating 16 2.6.1 HAp coating methods 16 3. Materials & methods 27 3.1 Synthesis and characterization of HAp powder 27 3.2 Preparation of metallic substrate 27 3.3 Thermal behavior of the Pine Oil 28 3.4 HAp/pine oil slurry preparation and coating process 28 3.5 Characterization of the coating 29 3.6 Surface topography and wettability 29 3.7 Scratch resistance of the coating 29 3.8 Corrosion tests 30 3.8.1 Cyclic voltammetry 31 3.8.2 Potentiodynamic polarization 31 3.9 In vitro bioactivity analyses 33 3.10 Cell culture 34 4. Results & Discussion 36 4.1 Characterization of HAp powder 36 4.2 Thermal characterization of the pine oil 38 4.3 Characterization of the coating surface 40 4.3.1 X-ray diffraction and FTIR analyses 43 4.4 Surface properties 45 4.4.1 Topography, roughness and wettability 45 4.5 Scratch resistance of the coating 50 4.6 In vitro bioactivity and morphology 53 4.7 Metallography of the Co-15Cr-15W-10Ni alloy 62 4.8 Corrosive behavior 63 4.8.1 Effect of heat treatment on corrosion resistance 66 4.9 Cell Proliferation and osteogenic differentiation 68 5. Conclusions 73 5.1 Future perspectives 74 6. References 75 Acknowledgements 90