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POLITECNICO DI TORINO Repository ISTITUZIONALE POLITECNICO DI TORINO Repository ISTITUZIONALE Multifunctional surfaces for implants in bone contact applications Original Multifunctional surfaces for implants in bone contact applications / Cazzola, Martina. - (2018 Mar 22). Availability: This version is available at: 11583/2704549 since: 2018-03-27T12:37:15Z Publisher: Politecnico di Torino Published DOI: Terms of use: Altro tipo di accesso This article is made available under terms and conditions as specified in the corresponding bibliographic description in the repository Publisher copyright (Article begins on next page) 04 August 2020 Doctoral Dissertation UniTo-PoliTo Doctoral Program in Bioengineering and Medical-Surgical sciences (30th Cycle) Multifunctional surfaces for implants in bone contact applications By Martina Cazzola ****** Supervisors: Prof. Enrica Vernè, Supervisor Prof. Silvia Spriano, Co-Supervisor Dr. Sara Ferraris, Co-Supervisor Politecnico di Torino 2017 Declaration I hereby declare that, the contents and organization of this dissertation constitute my own original work and does not compromise in any way the rights of third parties, including those relating to the security of personal data. Martina Cazzola 2017 * This dissertation is presented in partial fulfillment of the requirements for Ph.D. degree in the Graduate School of Politecnico di Torino (ScuDo). Acknowledgment At the end of this journey I would like to thank all the people who have been by my side along the way. Firstly, I would like to thank my tutors Prof. Enrica Vernè, Prof. Silvia Spriano and Dr. Sara Ferraris for their support to my Ph.D research, for sharing with me their knowledge and for motivating me. Without their help all this work would not have been possible. My sincere gratitude also goes to all the people who have collaborated at this research work. My sincere thanks to Prof. Enrico Prenesti and Dr. Ingrid Corazzari of the chemistry department of the Università degli Studi di Torino, To Prof. Lia Rimondini, Dr. Andrea Cochis, Dr. Barbara Azzimonti and Ms. Rita Sorrentino from The Department of Health Sciences of The Università del Piemonte Orientale, To Prof Giuliana Banche and Dr. Valeria Allizont from the Department of Public Health and Pediatrics, Microbiology Division of the Univerità degli Studi di Torino, To Prof. Cinzia Margherita Bertea from the Plant Physiology Unit, Department Life Sciences and Systems Biology of the Università degli Studi di Torino, To Prof. Aleksandra Czyrska-Filemonowicz and Dr. Grzegorz Cempura from the International Centre of Electron Microscopy for Materials Science & Faculty of Metals Engineering and Industrial Computer Science from the AGH University of Science and Technology and thanks to Prof. Giuseppe Pezzotti from the Ceramic Physics Laboratory of the Kyoto Institute of Technology. All of these people have made their knowledge, skills and instrumentation available to make this work possible. Thank you to Cri, Vero, Gian, Dlp, Ale and the mansarda’s girls, Fabiana, Giulia, Dome and all my colleagues because I’ve bothered you a lot, but you did not kill me and thanks for all the beers. Thanks to Cri, Sara, Cla, Catalù, Andre, Fra, Fra B. and all my university friends for sharing all these years with me supporting and motivating me. Thanks to Ele, Sara, Paola and Marti because we have known each other for so long and we have shared so many things. I never tell you before, but I love you. Last but not least, I would like to thanks my family; my parents, my brother, my grandparents and my uncle for supporting me every days of my life. If I forgot someone it’s because I'm a mess with these things, but I’m really grateful to all of you. Martina Abstract The idea behind this thesis is the use of natural molecules to confer to the surface of biomaterials for bone contact applications multifunctional properties by means of functionalization. Natural molecules can be extracted from plants or food waste and wine industries, in order to obtain molecules with high added value, but low cost; local sources are preferred in order to valorize the territory, for examples Barbera grapes and Mentha Piperita of Pancalieri in the case of Piedmont. Polyphenols and essential oils are natural extracts from these types of sources and they have antitumoral, anti-inflammatory, antioxidant and osteoinductive properties and they are also reductive agents useful for the in situ reduction of silver nanoparticles in order to confer antibacterial properties to the surfaces. The substrates used for functionalization were a surface chemical treated bioactive titanium alloy and a silica based bioactive glass. Different functionalization protocols were studied and improved. The procedures of functionalization were tailored for each molecule and both substrates by varying pH, medium, solute concentration and time of functionalization in order to maximize the amount of molecules grafted to the surface and minimize any effect of degradation of the molecules. After functionalization, the samples were physically and chemically characterized and tested in vitro with bacterial and cellular assays. The presence and the activity of the natural molecules on the surface after functionalization was confirmed by means of Fourier Transform Infrared Spectroscopy, X-ray photoelectron spectroscopy, spectroscopic measurements with the Folin&Ciocalteu method, Gaschromatographic analysis and Fluorescence microscope analysis. The cellular tests highlighted the selective actions of polyphenols with tumoral and healthy cells showing cytotoxicity effects on tumoral cells and protective effects against RONS on healthy cells. Bioactivity tests of apatite precipitation in vitro in Simulated Body Fluid and of cellular mineralization show the ability of polyphenols to improve mineralization process. They influence the deposition kinetic of hydroxyapatite deposition kinetic and the formation of different apatite structure depending on the type of polyphenol used for the functionalization. Functionalization with Mentha essential oil shows the reduction of the bacterial adhesion on the treated surfaces tested with Staphylococcus Aureus and the same result was found for the surfaces with the silver nanoparticles. In both cases, it is evidenced that it is challenging to find a proper balance between an effective antibacterial action (bactericide or bacteriostatic) and cytocompatibility. Silver nanoparticles were in situ reduced on the surface of chemically-treated titanium alloy by means of the use of polyphenols as natural reducing agents in order to confer antibacterial activity to the surface. The presence, distribution and shape of the silver nanoparticles were investigated with Field Emission Scanning Electron Microscopy equipped with energy dispersive spectroscopy, as well as Transmission electron microscopy with selected area electron diffraction patterns. The nanoparticles are under the 10 nm and well dispersed on all the surface of the samples. According to the results, surface functionalization of biomaterials is a promising strategy in order to combine the properties of biomolecules with those of surfaces and also to obtain is situ synthesis of antibacterial nanoparticles. Contents Introduction ............................................................................................................ 1 Chapter 1- Introduction to critical aspects concerning surface characteristics of bone contact implants ........................................................................................ 3 1.1 Introduction .................................................................................................... 3 1.2 Osteointegration ............................................................................................. 4 1.2.1 Surfaces modifications to enhance osteointegrability ............................. 6 1.3 Infections ....................................................................................................... 9 1.3.1 Surfaces modifications in order to avoid bacterial infections ............ 11 1.4 Multifunctional surfaces .............................................................................. 14 References ......................................................................................................... 15 Chapter 2- Polyphenols ....................................................................................... 19 2.1 Introduction .................................................................................................. 19 2.2 Classification and chemical structure of polyphenols ................................. 20 2.2.1 Composition and structure of green tea leaves polyphenols ................ 23 2.2.1 Composition and structure of red grape polyphenols........................... 25 2.3 Properties of polyphenols ............................................................................ 26 2.3.1 Antitumoral properties ......................................................................... 28 2.3.2 Antioxidant properties .......................................................................... 33 2.3.3 Antibacterial properties ........................................................................ 35 2.3.4 Effects on bone health .......................................................................... 39 2.3.5 Bioavailability and stability .................................................................. 40 2.4 Functionalization with polyphenols ............................................................ 41 2.5 Materials and methods
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