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Electrophoretic deposition of organic/inorganic composite coatings on metallic substrates for bone replacement applications: mechanisms and development of new bioactive materials based on polysaccharides Elektrophoretische Abscheidung von organischen/anorganischen Verbundschichten auf metallischen Substraten für Knochenersatz-Anwendungen: Mechanismen und Entwicklung neuer bioaktiver Materialien auf Basis von Polysacchariden Der Technischen Fakultät der Friedrich-Alexander-Universität Erlangen-Nürnberg zur Erlangung des Grades D O K T O R - I N G E N I E U R Vorgelegt von Luis Eduardo Cordero Arias aus Cartago, Costa Rica Als Dissertation genehmigt von der Technischen Fakultät der Friedrich-Alexander-Universität Erlangen-Nürnberg Tag der mündlichen Prüfung: 28.08.2015 Vorsitzende des Promotionsorgans: Prof. Dr.-Ing. habil. Marion Merklein Gutachter: Prof. Dr.-Ing. habil. Aldo R. Boccaccini Prof. Dr. Sannakaisa Virtanen ii Acknowledgements: I want to offer my sincerest gratitude to my supervisor Prof. Dr.-Ing. habil. Aldo R. Boccaccini for his time, efforts and giving me the opportunity to do my PhD at his institute. Also my gratitude to Prof. Dr. Sannakaisa Virtanen for her help, collaboration and for always having her door open to any discussion and help. The best of my gratitude to my friend Dr. Sandra Canañas Polo, for her professional and personal advices, unconditional support, to having always time to help me and specially for her friendship. I am also very thankful to my colleagues and dear friends Qiang Chen, Anahí Philippart, Valentina Miguez, Micael Alonso, Elena Boccardi, Kai Zheng, Domenico D´Atri, Dr.- Ing. Alexander Hoppe and Heinz Mahler for always being there and ready to share their experiences during my PhD period. I would also like to thank Dr. Alejandra Chávez, Wei Li, Dr. Menti Goudouri, Bapi Sarker, Jasmin Hum, Sigrid Seuß, Bärbel Wust, Dr.-Ing. Patcharakamon Nooeaid, Dr.- Ing. Rama Krishna Chinnam, Dr.-Ing. Gerhard Frank, Alina Grünewald and Dr.-Ing. Rainer Detsch for collaboration and willingness to offer advices and help during my PhD. I would like to thank my students Gao Haoxiang, Azim Yazici, Elias Palpanes, Lena Bartelt, Jördis Schröder and Lisa Ott for their work and the opportunity to learn together. Also my gratitude for all collaborators at other chairs and institutes. Chair of Surface Science and Corrosion (WW4): Dr.-Ing. Metehan Turhan, Dr.-Ing. Florian Seuß, Ferdinand Singer, Anja Friedrich, Helga Hildebrand, Ulrike Marten-Jahns. Institute of Polymer Science (WW5): Dr. Judith Roether. Instituto de Tecnología Cerámica: Prof. Enrique Sánchez and Ing. Jessica Gilabert Albiol. Lehrstuhl für Chemische Reaktionstechnik: Dr. Nicola Taccardi. I would also like to thank the financial support of the German Academic Exchange Service (DAAD) and their conjunct program ALECOSTA with the Costa Rican Institute of Technology (ITCR). iii I am very thankful to my friends Dr. rer.nat. Javier Bustamante, Juana Torres, Huy Doan Dac, Davide Ghirardi and Max Steimle to shear their life and experiences with me, and specially Sonja Harms for being all this years with me. Finally, I express my thanks to my family for their support, advices and love during my whole life. Without you nothing could be possible. Luis Eduardo Cordero Arias iv Dedicada a mi madre y hermana y a los hermosos años de infancia con mi papá v Abstract Regarding the need to improve the usually encountered osteointegration of metallic implants with the surrounding body tissue in bone replacement applications, bioactive organic/inorganic composite coatings on metallic substrates were developed in this work using electrophoretic deposition (EPD) as coating technology. In the present work three polysaccharides, namely alginate, chondroitin sulfate and chitosan were used as the organic part, acting as the matrix of the coating and enabling the coating attachment to the metallic substrates (stainless steel AISI 316L, titanium alloy TI6Al4V and magnesium alloy AD91D). Different types of ceramic fillers were investigated as the inorganic phase of the coatings. Different bioactive glasses were used to impart osteoconductive and osteoinductive properties to the coating, while nanoparticles of titania and zinc oxide were used to bring antibacterial properties, improve the mechanical stability and control the degradation behavior of the coatings. In this work the possibility to develop stable suitable suspensions to produce coatings by EPD is shown, based in the three selected biopolymers and containing one or more types of ceramic particles in different size ranges from 20 nm to 30 µm. The investigation of different solvents for EPD, namely water and ethanol, was carried out (single or in mixtures of both) to develop stable suspensions to reduce the negative effect of the water hydrolysis in the coating morphology. The suspension stability was studied via ζ–potential measurements finding that the suspension mechanism is controlled by the polymer, which, by esterification effect, suspends the ceramic particles in the liquid media. A variety of more than 20 coatings were studied and developed during this thesis. The major goal was to develop suitable EPD technology to produce coatings with: adequate (i) homogeneity, (ii) attachment to the substrate, (iii) ceramic/polymer ratio, (iv) wettability and morphology, (v) electrochemical behavior, (vi) bioactivity and (vi) degradation behavior. Other properties were also analyzed such as: antibacterial activity and drug delivery function (by incorporation of simvastatin). Alginate based coatings containing nanoparticles of TiO2 or ZnO were developed by anodic EPD. Bioactive glass 45S5® (BG) was successfully incorporated to those coatings with the aim to provide bioactivity to the coating by the formation of hydroxyapatite. However not all the coatings were able to show bioactivity, mainly by an vi interaction of the anodic alginate with the ions coming from the simulated body fluid (SBF) and the BG particles. It was confirmed that all coatings imparted corrosion protection to the substrate when evaluated via potentiostatic polarization curves by immersion in Dulbecco´s MEM, also to the highly reactive magnesium alloy AZ91D, in the initial immersion stages. For the first time, in this project chondroitin sulfate (CS) was deposited by EPD. Even when the deposition was successful the coating degraded considerably fast when immersed in water based fluids. To tackle the fast degradation and impart bioactivity to CS coatings, a multilayer approach was chosen, where chitosan was used in the production of "sandwich-type" multilayers with the presence of BG in some of the layers. By this method the coating degradation was considerable reduced and the development of a bioactive composite coating was possible. The most successful coatings, in terms of degradation behavior and bioactivity, were the chitosan based coatings. The bioactive glass/chitosan (BG/Ch) system was studied in a comparative study using three different bioactive glasses. In this study all the coatings exhibited bioactivity, independently of the bioactive glass composition. The best coating in terms of homogeneity, degradability and bioactivity was produced using Bioglass 45S5®. For the system Bioglass/Chitosan considerable improvements were done compared with previous reported works, obtaining more stable suspensions and better coating homogeneity. To tailor the coating degradability and improve adhesion to the substrate, titania was added to the BG/Ch coating. In addition, simvastatin, a drug currently proposed to promote bone formation, was added to the system confirming the drug delivery potential of the coatings. Cell test studies with MG-63 human osteosarcoma cells were done on selected coatings to evaluate cell vitality and the effect of the simvastatin on cell behavior. In this work EPD has shown to be a highly versatile, low-cost and convenient method to produce organic/inorganic coatings on metallic substrates based on the chosen materials. Different approaches were studied: from single to multilayers, from coatings on flat surfaces to complex 3D structures, as well as the drug delivery potential. Coatings with tailored composition and thickness were successfully produced exhibiting the versatility of EPD as coating production technique. vii Zusammenfassung Die mangelnde Osteointegration von metallischen Implantaten in das umliegende Körpergewebe stellt im Anwendungsbereich für Knochenersatz ein großes Hindernis dar. Um diese Hürde zu überwinden, wurden bioaktive organische/anorganische Verbundschichten auf metallischen Substraten mittels elektrophoretischer Abscheidung (EPD) entwickelt. In der vorliegenden Arbeit wurden drei Polysaccharide (Alginat, Chondroitinsulfat und Chitosan) verwendet, die den organischen Anteil ausmachen. Zum einen bilden diese die Matrix der Beschichtung, zum anderen ermöglichen sie auch deren Haftung auf den metallischen Substraten (Edelstahl AISI 316L, Titanlegierung Ti6Al4V und Magnesiumlegierung AZ91D). Keramische Füllstoffe übernehmen die Rolle des anorganischen Teils. Verschiedene bioaktive Gläser wurden verwendet, um der Beschichtung sowohl osteokonduktive wie auch osteoinduktive Eigenschaften zu verleihen. Nanopartikel von Titandioxid und Zinkoxid wurden hingegen eingesetzt, um antibakterielle Verhalten zu generieren, die mechanische Stabilität zu verbessern und das Abbauverhalten der Beschichtungen zu steuern. Diese Arbeit zeigt die Möglichkeit auf, stabile und geeignete Suspensionen zu entwickeln, um Beschichtungen
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