Development of multifunctional dental implants

I II Copyright and terms of use of the work by third parties Third parties can use this academic work as long as the internationally accepted rules and good practices are respected, with regard to copyright and related rights. Thus, the present work may be used under the terms set out in the license below. If the user needs permission to be able to use the work under conditions not foreseen in the above-mentioned licensing, he/she should contact the author, through the RepositóriUM of the University of Minho. Atribuição CC BY https://creativecommons.org/licenses/by/4.0/ III Acknowledgements: This Thesis represents the ending of an important stage of my life. Throughout my path I was positively influenced by a number of friends, family and mentors to whom I would like to thank. To my advisor Lus Silvino Marques and my co-adviser Sandra Mariana Silva Marques for the guidance. To the group from Minho’s University to Sandra Carvalho, Nuno Bexiga, Edgar and José David and the group NOVA-FCT, to Tânia, Francisco, and Professor Isabel SáNogueira for all the help and patience to transmit to me the knowledge I needed during this journey. To my grandmother, for being great role model who inspire me every day. To my parents, for the help, strength, and motivation which they guide me with every day. And finally, to my friends, for all the love, patience, and motivation to always be a better version of me. To all, thank you. IV STATEMENT OF INTEGRITY I hereby declare having conducted this academic work with integrity. I confirm that I have not used plagiarism or any form of undue use of information or falsification of results along the process leading to its elaboration. I further declare that I have fully acknowledged the Code of Ethical Conduct of the University of Minho. V Desenvolvimento de implantes dentários multifuncionais. Resumo: Os implantes dentários são geralmente fabricados com materiais à base de titânio (Ti) devido à sua biocompatibilidade e resistência à corrosão. No entanto, a baixa capacidade de formar uma forte ligação química com o tecido vivo, conhecida como bioatividade, é uma das desvantagens deste tipo de materiais. Consequentemente, o uso de implantes dentários é às vezes acompanhado por falhas devido à fraca osseointegração e posteriores infeções causadas por microrganismos. Nesse sentido, são necessárias novas abordagens para o desenvolvimento de superfícies bioativas capazes de potencializar o crescimento ósseo e que, ao mesmo tempo, apresentem propriedades antibacterianas. O desenvolvimento de superfícies altamente bioativas e antimicrobianas, com melhor osseointegração e resistência à corrosão, pode ser alcançado por meio da deposição de revestimentos à base de TiN-M (M=Ag, Zn) por pulverização catódica reativa em magnetrão. A escolha deste tipo de material deve-se às propriedades dos seus constituintes: o nitreto de titânio por ser um biocompatível e aumentar a resistência à corrosão; e as nanopartículas de prata (Ag) e zinco (Zn) pelas suas propriedades antimicrobianas. Pela variação das condições de deposição poderemos obter revestimentos com diferentes composições de nitreto de titânio e de nanopartículas, explorando desta forma um vasto espectro de propriedades mecânicas e biológicas, sem que haja nenhum tipo de efeito citotóxico. Palavras-chave: Implante dentário; Nitreto de titânio; Osseontegração; Periimplantite; Pulverização catódica reativa em magnetrão VI Development of multifunctional dental implants Abstract: Dental implants are generally manufactured by titanium (Ti) based materials due to their biocompatibility and corrosion resistance. However, a low ability to form a strong chemical bond with living tissue, known as bioactivity, is one of the disadvantages of this type of material. Consequently, the use of dental implants is sometimes accompanied by failures due to poor osseointegration and subsequent infections caused by microorganisms. In this sense, new approaches are needed for the development of bioactive surfaces that can enhance bone growth and, at the same time, have antibacterial properties. The development of highly bioactive and antimicrobial surfaces, with better osseointegration and corrosion resistance, can be achieved through the deposition of coatings based on TiN-M (M = Ag, Zn) by reactive sputtering in magnetron. The choice of this type of material is due to the properties of its constituents: titanium nitride as it is biocompatible and increases corrosion resistance; and silver (Ag) and zinc (Zn) nanoparticles, for their antimicrobial properties. By varying the deposition conditions, coatings with different compositions of titanium nitride and nanoparticles can be obtained with different compositions of titanium nitride and nanoparticles, thus exploiting a wide spectrum of mechanical and biological properties, without any type of cytotoxic effect. Keywords: Dental implant; Osseointegration; Peri-implantitis; Reactive magnetron cathodic sputtering; Titanium nitride. VII Table of Contents: Acknowledgements: ...................................................................................................... III Resumo: .......................................................................................................................... V Abstract: ........................................................................................................................ VI List of Figures: ............................................................................................................... X List of tables: .............................................................................................................. XIII List of Abbreviations: ................................................................................................ XIV Chapter 1 - Introduction: ............................................................................................ 16 1. Introduction: .................................................................................................. 17 2. Work objectives, methodology and thesis organization: ............................ 18 3. References: ..................................................................................................... 21 Chapter 2 - State of the art: ........................................................................................ 22 1. Introduction: .................................................................................................. 23 2. Dental implants: ............................................................................................. 23 3. Titanium-based dental implants: ................................................................. 25 3.1. Biocompatibility of titanium for dental implants: ............................... 26 4. Failure of dental implants: ............................................................................ 26 4.1. Osseointegration Failure: ...................................................................... 27 4.2. Peri-implantis infection:......................................................................... 27 5. Surface modification: .................................................................................... 29 6. Bioactive materials: ....................................................................................... 30 6.1. Antibacterial activity Ag nanoparticles:............................................... 31 6.2. Antibacterial activity Zn nanoparticles: .............................................. 32 7. References: ..................................................................................................... 34 Chapter 3 – Coatings deposition TiN-Ag and TiN-Zn: ............................................. 40 1. Introduction: .................................................................................................. 41 XIV List of Abbreviations: PVD Physical vapor phase deposition TiN Titanium nitride Ti Titanium Ag Silver Zn Zinc Al Aluminium V Vanadium Si Silicon Ar Argon N2 Nitrogen ZnO Zinc oxide AgO Silver oxide WHO World Health Organization ROS Reactive oxygen species ATP Adenosine triphosphate DNA Deoxyribonucleic acid ALP Alkaline phosphatase activity cpTi Commercially pure titanium SEM Scanning electron microscope XRD X-ray powder diffraction AFM Atomic force microscopy EDS Energy-dispersive X-ray spectroscopy at.% Atomic percentage WCA Water contact angle DC Direct current PULS Pulse ICP-OES Inductively Coupled Plasma - Optical Emission Spectrometry NaCl Sodium chloride CaCl2 Calcium chloride KCl Potassium chloride NADPH Nicotinamide adenine dinucleotide phosphate Abs Absorbance XV ISO International Organization for Standardization TSA Trypticase Soy Agar CFU Colony-forming unit p-NPP p-Nitrophenyl Phosphate HCl Hydrochloric acid SD Standard deviation ICDD International Centre for Diffraction Data FCC Face centered cubic 16 Chapter 1 - Introduction: 17 1. Introduction: The impact of edentulism on the health and well-being of the population is a problem requiring intervention. Human beings have always faced problems associated with tooth loss. In the past, the inability to bite and chew minimally processed food was a threat to survival. However, with the advancement in food processing, survival is no longer a concern. Instead, the ability to enjoy a wide variety and textures of food has become the primary motivation for attempting to retain teeth or seeking methods for replacing lost teeth. Nowadays, facial aesthetic factors have gained increasing importance in the maintenance of dentition and with the increase in contemporary dental techniques, replacement of lost teeth has become possible and desirable [1]. According to “Markets and Markets” [2], the global dental implants market is expected to grow at a rate of 6.5% by 2023, reaching approximately USD 13.01 billion by then. The growth of this market is driven by the increasing elderly population and corresponding age-related dental diseases, the increasing prevalence of dental caries and periodontal diseases worldwide and the increasing desire for cosmetic dental surgery and the growing number of dentists capable of doing so [2]. Dental implants can be manufactured from various materials, each of which has different advantages and disadvantages. Due to exceptional corrosion resistance, biocompatibility, and mechanical stability, commercially pure titanium, and its alloy Ti-6Al-4V are the most commonly used materials in their manufacture [3]. However, there is still debate about its capacity to integrate with bone, since the Ti surface does not present the capacity to stimulate the formation of new tissue, delaying the biological integration of the dental implant [4] [5]. Subsequently, the slow osseointegration of the dental implant leads to a long postoperative period and increases the likelihood of microorganisms interacting with the surgical site. Microbial adhesion and colonization may induce infections with consequent surgical complications [3]. Statistically, 47% of implant failures are due to inefficient osseointegration and 45% occur during the first year of use [6]. Thus, the development of bioactive surfaces capable of enhancing and promoting bone growth and bone matrix mineralization, thus reducing the frequency of infectious complications, is a major challenge for manufacturers and the scientific community worldwide. In this work, a way to increase the probability of success of dental implants by adding a coating to the implant, having in mind that the perfect coating should possess antibacterial and osseointegrative properties [7]. Thus, considering this goal, titanium nitride (TiN) was chosen as the base coating, as it has excellent corrosion and wear 18 resistance properties and has been used as a hard coating in dental implants [8]. Moreover, in order to make it biologically active, zinc and silver nanoparticles were added, which can confer antibacterial properties to the implant surface [7]. 2. Work objectives, methodology and thesis organization: This work is inserted in the project ORAiDEA - POCI-01-0247-FEDER-039985 "Development of Multifunctional Dental Implants", by the European Regional Development Fund (ERDF) through the Operational Program Competitiveness and Internationalization (POCI) which proposes to develop multifunctional dental implants, starting with their design and subsequent production through machining of titanium blocks. The project aims to build an important step toward solving real problems and needs that affect more specifically the sector of dental implants, namely peri-implant infections, which are increasingly common and can lead to implant loss and the need for additional surgeries. In this sense, this work aims to deposited by a physical vapor phase deposition (PVD) technology, namely magnetron sputtering, multifunctional titanium nitride (TiN) coating doped with metallic nanoparticles, such as silver (Ag) and zinc (Zn) in dental implants allowing the improvement of osseointegration capacity and antibacterial activity through. In more detail, the objectives of this work are: • Deposit by PVD TiN-M [M=Ag; Zn] coatings with different amounts of metal nanoparticles on Ti-6Al-4V substrates; • Characterization of physical and chemical properties, like: roughness, surface energy, bioactivity studies, among others; • Obtain coatings with good mechanical properties and with antibacterial activity over time without cytotoxicity; • To evaluate the osseointegrative capacity, through the assessment of adhesion, proliferation and differentiation of SaOS-2 cells (osteoblasts); • Correlate the kinetics of ion release with bacterial inhibition. 19 The following scheme, figure 1, summarizes the working methodology used in the thesis, which will be described throughout the different chapters. Figure 1 - Scheme of the work methodology used for the development of the multifunctional implants. This paper is organized into 6 chapters to provide a logical sequence for understanding the production of TiN-M coatings by magnetron sputtering and its subsequent characterization. Chapter 1 introduces the topic of the thesis and the methodology followed is presented. Chapter 2 presents the state of the art. Throughout the chapter, a review of the literature on dental implants and their current problems is provided. Chapter 3 describes the production of TiN-Ag and TiN-Zn coatings by magnetron sputtering. A theoretical explanation of the technique is provided, and the methodology and conditions used to obtain the coatings are presented. Chapter 4 includes a brief description of characterisation techniques. An explanation of the theoretical concept of the physical and chemical characterisation 20 techniques used is provided and the protocols followed in the biological analysis are described. Chapter 5 presents and discusses the results. The influence of the deposition conditions on the chemical composition, morphology and ion release is discussed. Results of biological tests are also discussed. Lastly, the Chapter 6, final remarks and future research presents an overall conclusion of the thesis and discusses possible future research perspectives. 21 3. References: [1] M. S. Block, “Dental Implants: The Last 100 Years,” J. Oral Maxillofac. Surg., vol. 76, no. 1, 2018, doi: 10.1016/j.joms.2017.08.045. [2] “Dental Implants and Prosthesis Market Size, Share and Trends forecast to 2023 by Type, Material, Type of Facility | COVID-19 Impact Analysis | MarketsandMarkets™” https://www.marketsandmarkets.com/MarketReports/dental-implants-prosthetics-market-695.html (accessed Jan. 25, 2022). [3] T. Guo, K. Gulati, H. Arora, P. Han, B. Fournier, and S. Ivanovski, “Race to invade: Understanding soft tissue integration at the transmucosal region of titanium dental implants,” Dent. Mater., vol. 37, no. 5, 2021, doi: 10.1016/j.dental.2021.02.005. [4] A. Al-Noaman, S. C. F. Rawlinson, and R. G. Hill, “MgF2containing glasses as a coating for titanium dental implant. IGlass powder,” J. Mech. Behav. Biomed. Mater., vol. 125, 2022, doi: 10.1016/j.jmbbm.2021.104948. [5] D. P. Utami, D. J. Indrani, and Y. K. Eriwati, “The role of implant surface modification method on the success of osseointegration,” J. Kedokt. Gigi Univ. Padjadjaran, vol. 31, no. 2, 2019, doi: 10.24198/jkg.v31i2.17967. [6] M. Esposito, J. M. Hirsch, U. Lekholm, and P. Thomsen, “Biological factors contributing to failures of osseointegrated oral implants. (I). Success criteria and epidemiology,” European Journal of Oral Sciences, vol. 106, no. 1. 1998, doi: 10.1046/j.0909-8836..t01-2-.x. [7] S. E. A. Camargo et al., “Novel coatings to minimize bacterial adhesion and promote osteoblast activity for titanium implants,” J. Funct. Biomater., vol. 11, no. 2, 2020, doi: 10.3390/jfb11020042. [8] G. Zorn, V. Migonney, and D. G. Castner, “Grafting titanium nitride surfaces with sodium styrene sulfonate thin films,” Biointerphases, vol. 9, no. 3, 2014, doi: 10.1116/1.4878215. 22 Chapter 2 - State of the art: 23 1. Introduction: This chapter provides an overview of dental implants and the current problems they present. The most relevant works in the field are summarized with regard to the most important issues of the present thesis: Development of multifunctional dental implants. 2. Dental implants: Teeth are bony structures with numerous functions. They are used for chewing, participating in speech, and contributing to the owner's aesthetics. Over the years, teeth are exposed to various chemical and physical effects, which lead to the erosion of dental hard tissues, gum regression and other complications, and it is therefore often necessary to resort to their removal. As a result, the demand for dental implants has been increasing [1]. Dental implants are devices inserted preferably in direct contact with the maxillary and/or mandibular bone. They are composed of three main components: a cylindrical screw known as an implant; the abutment that connects the implant to the dental prosthesis; and the crown/prosthesis that is usually customized according to the patient [2], figure 2. Figure 2 - Components of restored implant. A, Implant crown. B, Abutment. C, Implant fixture. Adapted [2] 30 Figure 5 Scheme of surface functionalisation of dental implants with the aim of improving osseointegration and infection control. Adapted [30]. Dental implants made of pure titanium are prone to suffering scratches and abrasions during routine oral hygiene procedures. This low resistance to wear induces irrevocable damage to the implant surface, increasing its roughness, which facilitates the adhesion of bacteria and the early formation of biofilms [33]. In addition, these are also known to be intrinsically susceptible to corrosion and ion leaching in physiological environments, generating products that cause many adverse and toxic reactions in host tissues [34]. To overcome these problems, titanium nitride (TiN) coatings have been used since they present promising characteristics such as high hardness; wear resistance; corrosion and erosion resistance; low friction coefficient; no toxicity, and excellent biocompatibility [35]. In addition, TiN-modified surfaces enable various application opportunities, mainly as antimicrobial coatings in food technology and biomedicine through the addition of transition metal nanoparticles, such as zinc (Zn) and silver (Ag) [36]. 6. Bioactive materials: As has been discussed, to prevent failures it is necessary to use implants produced with materials that enhance osseointegration processes and at the same time, prevent or suppress bacterial colonization. These materials can present different mechanisms of action. In one hand, they may promote bone tissue formation by favoring the activity of osteoblastic cell or the differentiation of stem cells into osteogenic phenotypes, and in 31 other hand, their surface properties and composition may interfere with bacterial adhesion and viability, modifying the surface energy or releasing antibacterial agents, such as ions or antibiotics [37]. Ag and Zn nanoparticles, as well as their oxides, AgO and ZnO, prevent initial bacterial attachment, preventing biofilm formation [38]. However, it is important to note that although nanoparticles can kill bacteria and have no cytotoxic effect on osteoblastic and epithelial cells at low concentrations, this cannot be guaranteed at high concentrations. Therefore, it is necessary to develop a suitable coating, i.e., with concentrations that are below the citotoxicity limit [39]. 6.1. Antibacterial activity Ag nanoparticles: A nanoparticle can be defined as "an object of very small dimensions that behaves as a whole in terms of its properties". Nanoparticles have among them several important characteristics that can be used to classify them, such as composition, morphology, uniformity, and agglomeration [40]. Furthermore, nanoparticles are classified according to their diameter which can vary from 1 nanometer to 100 nanometers and can correspond to individual molecules or macromolecules that include higher order assemblies in nanoparticles [41]. The size of nanoparticles allows different biological interactions when compared to non-nanostructured materials, due to the surface-to-volume ratio. This property can be exploited to create nanostructures capable of diagnosing and treating numerous pathologies [42]. Silver is a transition metal that is distinguished by having high reflectivity and electrical and thermal conductivity, as well as low emissivity. This material is characterized by having only one crystalline form, presenting a face-centered cubic shape with a lattice constant of a=0.4086 nm [43]. Silver nanoparticles have a high surface/volume ratio and present different morphological characteristics. They can easily penetrate the bacterial cell membrane and act as a catalyst for bacteria destruction. Their antimicrobial properties have been widely studied revealing that there is an inverse relationship between nanoparticle size and antimicrobial activity. Particles in the 1-10 nanometer range have proven to be most effective in fighting bacteria, but smaller particles are also reported to be more toxic and this effect is most noticeable when they are in the oxide form (AgO) [44]. Therefore, reckless or excessive use of silver nanoparticles can result in potential risks to humans and the environment and it is crucial to keep the concentration of free silver nanoparticles below World Health Organization (WHO) permissible limits. [45]. 32 Although the exact mechanisms of the antibacterial effects of nanoparticles have not yet been fully elucidated, several pathways have been proposed. For example, silver nanoparticles can continuously release silver ions that contribute to the death of microbes, due to their affinity for sulfur proteins present in the cell wall and cytoplasmic membrane. The adhering ions increase the permeability of the cytoplasmic membrane and lead to the disruption of the bacterial envelope [46]. Additionally, silver ions trapped free in cells can deactivate respiratory enzymes by generating reactive oxygen species (ROS) and disrupting ATP production. As sulfur and phosphorus are important components of DNA, their interaction with silver ions can cause problems in DNA replication and consequently, cell reproduction. In addition, silver ions can also inhibit protein synthesis by denaturing ribosomes present in the cell cytoplasm [47], figure 6. Figure 6 - The antibacterial actions of silver nanoparticles (AgNPs) [48]. 6.2. Antibacterial activity Zn nanoparticles: To date, zinc has been used in oral hygiene products as an antimicrobial agent to control dental plaque, inhibit the formation of colonies and reduce halitosis. Lately, it has been incorporated into many dental materials due to the zinc ion's ability to inhibit the growth of bacteria however, it is considered a bacteriostatic agent and not bactericidal, as its effect is reversed when the cells are washed out [49]. Zinc ions have multiple inhibitory effects on bacterial cell activity, such as on glycolysis, glycosyltransferase production and polysaccharide synthesis, transmembrane proton translocation, and tolerance to acidic environments. They can increase bacterial cell membrane permeability to protons, reduce adenosine triphosphate (ATP) synthesis 33 in glucose-forming cells, and decrease F-type adenosine triphosphate (F-ATPase) activity due to their ability to inhibit the glycolytic enzymes glyceraldehyde-3-phosphate dehydrogenase and pyruvate kinase, as well as phosphoenolpyruvate [50]. Its oxidized form (ZnO), on the other hand, has also been studied, and is a biosafety material, non-toxic to human cells [51]. ZnO nanoparticles can disrupt bacterial growth by interacting with the bacterial surface or by entering the interior of the bacteria. This leads to the disruption of bacterial enzyme systems by displacing magnesium ions essential for the enzymatic activity of bacteria and subsequently showing a significant bactericidal effect [52]. 34 7. References: [1] M. S. Zafar, S. Najeeb, and Z. 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Hardes et al., “The influence of elementary silver versus titanium on osteoblasts behaviour in vitro using human osteosarcoma cell lines,” Sarcoma, vol. 2007, 2007, doi: 10.1155/2007/26539. [40] J. Njuguna, F. Ansari, S. Sachse, V. M. Rodriguez, S. Siqqique, and H. Zhu, “Nanomaterials, nanofillers, and nanocomposites: types and properties,” in Health and Environmental Safety of Nanomaterials, 2021. [41] L. Leon, E. J. Chung, and C. Rinaldi, “A brief history of nanotechnology and introduction to nanoparticles for biomedical applications,” in Nanoparticles for 38 Biomedical Applications: Fundamental Concepts, Biological Interactions and Clinical Applications, 2019. [42] J. L. Paris and M. Vallet-Reg, “Nanostructures for imaging, medical diagnostics and therapy,” in Fundamentals of Nanoparticles: Classifications, Synthesis Methods, Properties and Characterization, 2018. [43] The Chemistry of Copper, Silver and Gold. 1973. [44] R. P. Allaker and K. Memarzadeh, “Nanoparticles and the control of oral infections,” International Journal of Antimicrobial Agents, vol. 43, no. 2. 2014, doi: 10.1016/j.ijantimicag.2013.11.002. [45] R. K. Kunkalekar, “Role of Oxides (Fe3O4, MnO2) in the Antibacterial Action of Ag-Metal Oxide Hybrid Nanoparticles,” in Noble Metal-Metal Oxide Hybrid Nanoparticles: Fundamentals and Applications, 2018. [46] S. Khorrami, A. Zarrabi, M. Khaleghi, M. Danaei, and M. R. Mozafari, “Selective cytotoxicity of green synthesized silver nanoparticles against the MCF-7 tumor cell line and their enhanced antioxidant and antimicrobial properties,” Int. J. Nanomedicine, vol. 13, 2018, doi: 10.2147/IJN.S189295. [47] N. Durán, G. Nakazato, and A. B. Seabra, “Antimicrobial activity of biogenic silver nanoparticles, and silver chloride nanoparticles: an overview and comments,” Applied Microbiology and Biotechnology, vol. 100, no. 15. 2016, doi: 10.1007/s00253-016-7657-7. [48] I. X. Yin, J. Zhang, I. S. Zhao, M. L. Mei, Q. Li, and C. H. Chu, “The antibacterial mechanism of silver nanoparticles and its application in dentistry,” International Journal of Nanomedicine, vol. 15. 2020, doi: 10.2147/IJN.S246764. [49] M. M. Almoudi, A. S. Hussein, M. I. Abu Hassan, and N. Mohamad Zain, “A systematic review on antibacterial activity of zinc against Streptococcus mutans,” Saudi Dental Journal, vol. 30, no. 4. 2018, doi: 10.1016/j.sdentj.2018.06.003. [50] T. N. Phan, T. Buckner, J. Sheng, J. D. Baldeck, and R. E. Marquis, “Physiologic actions of zinc related to inhibition of acid and alkali production by oral streptococci in suspensions and biofilms,” Oral Microbiol. Immunol., vol. 19, no. 1, 2004, doi: 10.1046/j.0902-0055.2003.00109.x. [51] A. Sirelkhatim et al., “Review on zinc oxide nanoparticles: Antibacterial activity and toxicity mechanism,” Nano-Micro Letters, vol. 7, no. 3. 2015, doi: 10.1007/s40820-015-0040-x. 39 [52] S. Tavassoli Hojati et al., “Antibacterial, physical and mechanical properties of flowable resin composites containing zinc oxide nanoparticles,” Dent. Mater., vol. 29, no. 5, 2013, doi: 10.1016/j.dental.2013.03.011. 46 3.3. Closed-field unbalanced magnetron sputtering: Deposition of uniform coatings of complex components using a single spray target with an unbalanced magnetron source can be difficult, hence the choice of a multipletarget system. By installing two unbalanced magnetrons opposite each other, the magnet arrays can be configured with either identical or opposite polarities. In the first case, the configuration is described as "mirrored" and in the second case, "closed-field", as it can be seen in figure 20 [17]. Figure 9 - Dual unbalanced magnetron configurations [17] In the mirrored configuration, the field lines are directed towards the chamber walls. Consequently, the secondary electrons following these field lines are lost, leading to a low plasma density in the substrate region. On the other hand, in the "closed-field" configuration, the field lines are connected between the magnetrons. This configuration keeps the plasma density in the substrate region since few electrons are lost in the chamber walls. Thus, during the deposition process, it is possible to reach high levels of bombardment, with a rate of ions per atom 2-3 times higher than those obtained in the previous configurations referred [22]. 4. Coatings deposition: TiN-Ag and TiN-Zn: 47 4.1. Equipment used in the production of the coatings: The deposition chamber used in the production of the different coatings is in the Functional Coatings Laboratory II, of the Physics Department of the University of Minho, Azurém campus, figure 21. Figure 10 - Reactive magnetron sputter equipment from the University of Minho. In this deposition chamber, there are two opposite rectangular targets measuring 200 mm x 100 mm. For the realization of this work, one of them corresponds to pure titanium (99.99%) and the other to the element to incorporate in the film (Ag (99.99%), Zn (99.99%)). Next to the inner wall of the chamber and along its entire perimeter, there is a distributor tube that injects the gases used during the depositions (Ar and N2). The chamber is heated by an electric resistance and an attempt has been made to maintain a temperature of 200 ºC. For each series of depositions, were used, Ti-6Al-4V, Si and glass substrates, was fixed to the sample holder (hexagonal prism) using assemblies with conductive polyimide tape (Kapton tape) and copper wires to improve conductivity. The distance between the targets and the sample holder was constate at 70 mm, and the holder was always rotating at a clockwise speed of 7 rpm during the depositions. 4.2. Preparation of the substrates: The depositions were performed on Ti-6Al-4V, silicon, and glass substrates. The previous preparation conditions of the substrates are described below. 48 4.2.1. Ti-6Al-4V substrates: Before being mounted on the sample holder the Ti-6Al-4V substrates were ultrasonically cleaned in water and detergent, distilled water, acetone, and isopropyl alcohol, during 10 minutes for each solvent. Finally, they were grouped in groups of 15/16 substrates. Finally, 5 groups were glued on the sample holder, one group per face, with Kapton tape together with a copper wire connecting all of them to improve the conductivity of the substrates, figure 22. Figure 11 - Preparation of the substrates. 4.2.2. Silicon substrates: A silicon wafer was cut into squares with a diamond tip. Subsequently, these were ultrasonically cleaned with isopropyl alcohol in a for 10 minutes. Finally, these were also grouped, in groups of 4 and glued to one face of the sample holder with Kapton tape and a copper wire. 4.2.3. Glass substrates: The glass was cleaned with isopropyl alcohol and taped to the display case with Kapton tape. 4.3. Deposition Parameters of TiN, TiN-Ag, and TiN-Zn coatings: The deposition of TiN, TiN-Ag, and TiN-Zn films followed four steps: first, etching was performed to clean the substrates and the targets inside the deposition chamber; then, two interlayers, one of Ti and other of TiN, were produced to improve the adhesion of the films to the substrates; and finally, the films were deposited. The parameters used in each step are detailed below. 49 4.3.1. Etching: In this step, the argon flow rate was maintained at 80 sccm and a DC pulsed source was used to apply a current of 300 mA to the sample holder, in pulses with a duration of 1536 ns at a frequency of 200 kHz for 15 minutes; A DC source was connected to the titanium target with a current density of 0.08 A and, for doped coatings, a current density of 0.01 A (DC) was also applied to the Ag or Zn target. 4.3.2. Interlayers: To improve the adhesion of the coatings, interlayers were deposited, for the TiN coating only one Ti interlayer was deposited. In TiN-Ag and TiN-Zn coatings, two interlayers were deposited, one of Ti and one of TiN, figure 23. Figure 12 - Schematic representation and parameters of deposited interlayers. The Ti interlayer was deposited by applying a DC current density of 1.0 A was applied to the Ti target and a BIAS voltage of -75V to the substrate holder for 15 minutes. The argon flow rate was maintained at 55 sccm. The TiN interlayer was deposited by applying a DC current density of 1.0 A on the Ti target and a BIAS voltage of -50V on the substrate holder for 5 minutes, with the flow rates of argon and N2 maintained at 55 sccm and 6.25 sccm, respectively. 4.3.3. Deposition: The deposition parameters of TiN, TiN-Ag, and TiN-Zn films are summarized in table 4. They can also be found in annex 1, where they are presented in more detail. Due to the deposition rates of silver and zinc, it was decided to use a pulsed dc power supply to apply current to these targets to obtain better control over the content of the dopant 50 material to be incorporated. In addition, the BIAS potential was adjusted from -75 V for pure TiN deposition, to -50 V for coatings incorporated with nanoparticles, since in previous TiN-Ag deposition studies, it was found that a BIAS of -75 V leads to too high TiN-Ag contents in the TiN-Ag film. The system was programmed to maintain a constant current on the targets for all depositions. Table 3 - Summarized deposition parameters of TiN, TiN-Ag, and TiN-Zn coatings. Coating Top layer Interlayer Gas flow (sccm) Deposition time (h) Temperature (ºC) Current density (mA/cm2) Ti TiN Ar N2 Ti Ag Zn TiN TiN ✓ X 55 6.25 2 ≈ 200 10 X X TiN-Ag 0.15 TiN-Ag ✓ ✓ 55 6.25 1.5 ≈ 200 10 0.75 X TiN-Ag 0.18 TiN-Ag ✓ ✓ 55 6.25 1.5 ≈ 200 10 0.90 X TiN-Ag 0.21 TiN-Ag ✓ ✓ 55 6.25 1.5 ≈ 200 10 1.05 X TiN-Zn 0.18 TiN-Zn ✓ ✓ 55 6.25 1.5 ≈ 200 10 X 0.90 TiN-Zn 0.25 TiN-Zn ✓ ✓ 55 6.25 1.5 ≈ 200 10 X 1.25 TiN-Zn 0.32 TiN-Zn ✓ ✓ 55 6.25 1.5 ≈ 200 10 X 1.60 51 5. References: [1] M. P. Mughal et al., “Surface modification for osseointegration of Ti6Al4V ELI using powder mixed sinking EDM,” J. Mech. Behav. Biomed. Mater., vol. 113, 2021, doi: 10.1016/j.jmbbm.2020.104145. [2] S. M. Rossnagel, “Thin film deposition with physical vapor deposition and related technologies,” J. Vac. Sci. Technol. A Vacuum, Surfaces, Film., vol. 21, no. 5, 2003, doi: 10.1116/1.1600450. [3] X. Q. Tan, J. Y. Liu, J. R. Niu, J. Y. Liu, and J. Y. Tian, “Recent progress in magnetron sputtering technology used on fabrics,” Materials, vol. 11, no. 10. 2018, doi: 10.3390/ma11101953. [4] D. Lundin, J. T. Gudmundsson, and T. Minea, High power impulse magnetron sputtering : Fundamentals, technologies, challenges and applications. 2019. [5] Y. Deng, W. Chen, B. Li, C. Wang, T. Kuang, and Y. Li, “Physical vapor deposition technology for coated cutting tools: A review,” Ceramics International, vol. 46, no. 11. 2020, doi: 10.1016/j.ceramint.2020.04.168. [6] W. D. Sproul, D. J. Christie, and D. C. Carter, “Control of reactive sputtering processes,” Thin Solid Films, vol. 491, no. 1–2. 2005, doi: 10.1016/j.tsf.2005.05.022. [7] R. P. Howson, “The reactive sputtering of oxides and nitrides,” Pure Appl. Chem., vol. 66, no. 6, 1994, doi: 10.1351/pac199466061311. [8] V. Jokanović et al., “Detailed phisyco-chemical characterization of the multilayered thin films based on titanium oxynitride and copper doped titanium nitride obtained by different PVD techniques,” Vacuum, vol. 195, 2022, doi: 10.1016/j.vacuum.2021.110708. [9] I. Safi, “Recent aspects concerning DC reactive magnetron sputtering of thin films: A review,” Surf. Coatings Technol., vol. 127, no. 2–3, 2000, doi: 10.1016/s0257-8972(00)00566-1. [10] Q. M. Mehran, M. A. Fazal, A. R. Bushroa, and S. Rubaiee, “A Critical Review on Physical Vapor Deposition Coatings Applied on Different Engine Components,” Critical Reviews in Solid State and Materials Sciences, vol. 43, no. 2. 2018, doi: 10.1080/10408436.2017.1320648. 52 [11] R. D. Arnell and P. J. Kelly, “Recent advances in magnetron sputtering,” Surf. Coatings Technol., vol. 112, no. 1–3, 1999, doi: 10.1016/S0257-8972(98)00749X. [12] M. Audronis and V. Bellido-Gonzalez, “Hysteresis behaviour of reactive high power impulse magnetron sputtering,” Thin Solid Films, vol. 518, no. 8. 2010, doi: 10.1016/j.tsf.2009.12.011. [13] K. Strijckmans, R. Schelfhout, and D. Depla, “Tutorial: Hysteresis during the reactive magnetron sputtering process,” J. Appl. Phys., vol. 124, no. 24, 2018, doi: 10.1063/1.5042084. [14] J. Musil, P. Baroch, J. Vlček, K. H. Nam, and J. G. Han, “Reactive magnetron sputtering of thin films: Present status and trends,” in Thin Solid Films, 2005, vol. 475, no. 1-2 SPEC. ISS., doi: 10.1016/j.tsf.2004.07.041. [15] L. C. Fontana and J. L. R. Muzart, “Triode magnetron sputtering TiN film deposition,” Surf. Coatings Technol., vol. 114, no. 1, 1999, doi: 10.1016/S02578972(99)00032-8. [16] S. Kadlec, J. Musil, and H. Vyskocil, “Hysteresis effect in reactive sputtering: A problem of system stability,” J. Phys. D. Appl. Phys., vol. 19, no. 9, 1986, doi: 10.1088/0022-3727/19/9/004. [17] P. J. Kelly and R. D. Arnell, “Magnetron sputtering: A review of recent developments and applications,” Vacuum, vol. 56, no. 3. 2000, doi: 10.1016/S0042-207X(99)00189-X. [18] “Magnetron sputtering & magnetron sputtering deposition | FHR Thin film technology.” https://www.fhr.biz/en/thin-film-technology/sputteringtechnology/magnetron-sputtering/ (accessed Aug. 19, 2022). [19] S. Lobe et al., “Radio frequency magnetron sputtering of Li7La3Zr2O12 thin films for solid-state batteries,” J. Power Sources, vol. 307, 2016, doi: 10.1016/j.jpowsour.2015.12.054. [20] B. Window and N. Savvides, “Unbalanced dc magnetrons as sources of high ion fluxes,” J. Vac. Sci. Technol. A Vacuum, Surfaces, Film., vol. 4, no. 3, 1986, doi: 10.1116/1.573904. [21] R. P. Howson, H. A. J’Afer, and A. G. Spencer, “Substrate effects from an unbalanced magnetron,” Thin Solid Films, vol. 193–194, no. PART 1, 1990, doi: 10.1016/S0040-6090(05)80020-3. 53 [22] P. J. Kelly and R. D. Arnell, “The influence of magnetron configuration on ion current density and deposition rate in a dual unbalanced magnetron sputtering system,” Surf. Coatings Technol., vol. 108–109, 1998, doi: 10.1016/S02578972(98)00566-0. 54 Chapter 4 - Characterization techniques 55 1. Introduction: The developed surfaces were carefully investigated and analysed using various characterisation techniques. Importantly, the coatings were deposited on different substrate materials such as Ti-6Al-4V, Si and glass, thus ensuring that the properties of the coating would not be affected by the nature of the substrates themselves. The characterisation of the surfaces produced was carried out at the School of Science of the University of Minho, using various techniques such as Energy Dispersive Spectrometry (EDS), Scanning Electron Microscopy (SEM), X-ray Diffraction (XRD), water contact angle (WCA) and Atomic Force Microscopy (AFM). At the same time, the evaluation of the functional response of the deposited coatings was performed at the Biological Engineering department of the University of Minho, in Braga and at the NOVA School of Science and Technology, in Lisbon. The objectives of these tests are the determination of the possible response of the organism to the implant with the coatings produced; and the determination of the antibacterial activity, essential for the objective proposed in the development of the multifunctionality of the implants. For that, Inductively Coupled Plasma Optical Emission spectroscopy (ICP-OES), antibacterial, cell viability, adhesion, proliferation, and alkaline phosphatase (ALP) assays were performed. In this chapter, is given a brief description of the different methods and techniques used for the characterisation of the coatings. 2. Physical and chemical characterization techniques: Physical and chemical characterization techniques were used in order to evaluate the morphology, topography, chemical composition, crystal structure, contact angle and surface energy of the produced coatings. 2.1. Scanning electron microscopy and energy dispersive spectroscopy (SEM-EDS): SEM provides information about the microstructure of a surface, homogeneity and morphology of the particles presented in the coating [1]. SEM micrographs formation depends on the acquisition of signals produced from the incidence of a focused electron beam on the sample surface, which can cause numerous types of interactions, such as inelastic interactions with the atomic electrons and elastic interactions with the atomic nucleus [2], as it can be seen in figure 7. 62 The exponents p and d denote the polar and dispersive components while s, l and v represent the solid, liquid and vapour phases, respectively. Applying equation 3 to Young's equation gives the linear expression, equation 4: 1+cos(Θ) 2𝛾𝑙𝑣 √𝛾𝑙𝑣 𝑑=√𝛾𝑠𝑣 𝑝 √𝛾𝑙𝑣 𝑝 𝛾𝑙𝑣 𝑑+√𝛾𝑠𝑣 𝑑 (Equation 4): Where Θ is the contact angle of the liquid on the solid surface and 𝛾𝑙𝑣 𝑝, 𝛾𝑙𝑣 𝑑, 𝛾𝑙𝑣 are the surface tension parameters of the liquid. Consequently, the components 𝛾𝑠𝑣 𝑝, 𝛾𝑠𝑣 𝑑, can be determined from the slope and intercept of the linear fit to the data. The wettability of the different samples was determined using a contact angle meter apparatus (OCA 15 Plus, DataPhysics Instruments GmbH Filderstadt, Germany). All measurements were performed at room temperature and two microliters drops of pure water (polar liquid); glycerol (polar liquid, Sigma, Merck KGaA, Darmstadt, Germany) and 1-bromonaphthalene (apolar liquid, Sigma, Merck KGaA, Darmstadt, Germany), were used as reference liquids. Using the Owens, Wendt, Rabel, and Kälble (OWRK) method [15], the surface free energy was calculated. 2.4. Inductively Coupled Plasma Optical Emission Spectrometry (ICP-OES): The silver and zinc nanoparticles present in the coatings are responsible for the antibacterial activity of the coatings through ion release. One way to confirm this property is by studying their ion release kinetics. An inductively coupled plasma optical emission spectrometer (ICPOES-ICP PerkinElmer Optima 8000 spectrometer) was used to measure the release of silver and zinc ions when the coatings are in a liquid medium, in this case, in artificial saliva (since they are coatings for dental implants). This technique begins by introducing a liquid sample into a nebuliser, which transforms it into droplets. These droplets are then sprayed into a plasma torch, where the plasma excites the electrons in the samples. As with other techniques, when the electrons return to the fundamental state, they emit electrons with wavelengths specific to the characteristic element. The intensity of this emission is proportional to the amount of the element present in the sample. Figure 12 shows a typical scheme of the ICP-OES system. 63 Figure 17 - Schematic of a typical inductively coupled plasma-optical emission spectrometry (ICP-OES) system [16]. In this work, the uncoated substrates were used as control while, the Ti-6Al-4V coated samples were used to analyse the release of Ag+ and Zn+ ions and compared with the control samples. Different coatings of each condition were placed in a falcon with 50ml of artificial saliva solution. Subsequently, 2ml of this solution was removed to a new falcon at different time-setting points of immersion time in the artificial saliva solution: 2h, 6h, 12h, 48h, 120h and 168h. Before analysis, the 2ml of the solution were diluted in 4ml of nitric acid (HNO3). Finally, ICP-OES was performed, and the concentration of ions released over time was calculated. The artificial saliva solution used in the test was prepared in the laboratory according to table 3. Table 4 - Chemical composts used to make 400 ml of artificial saliva. Artificial saliva solution Chemical compounds Quantity measure NaCl 0,1458 g CaCl2 0,1080 g KCl 0,08 g H2O ultrapure 400 ml To quantify the release of silver and zinc ions in an aqueous medium, a calibration curve was calculated using a silver standard solution and a zinc standard solution (specpure, 1000 ug/ml). These calibration curves are shown in figure 13. 64 Figure 18 - ICP-OES Silver calibration: (a) silver calibration curve with nitric acid; (b) zinc calibration curve with nitric acid. 65 3. Biological analyses: The biological response of the coatings produced were assessed by tests of cell viability, antibacterial activity, cells adhesion, cells proliferation, and ALP activity. 3.1. Cell viability: Cell viability was assessed using the resazurin assay, which is based on the ability of metabolically active cells to reduce resazurin to resorufin, figure 14. Resazurin is a cell-permeable redox indicator that can be used to monitor the number of viable cells. This non-fluorescent blue dye is converted to pink-fluorescent resorufin in the presence of metabolically active cells. NADPH dehydrogenase is probably responsible for the electron transfer from NADPH to resazurin, which is reduced to resorufin. This conversion can be detected by visual observation of its pink colour or absorbance readings of the resorufin/resazurin ratio at 570/600. [16]. Figure 19 - Structures and reaction mechanism of resazurin (RZ), resorufin (RS) [18]. The methodology consisted in exposing human osteosarcoma cells (SaOS-2) to McCoy culture medium that were previously in contact with the different coatings. For this, the coatings were first sterilized in the autoclave at 180ºC, during 1h. Then, were immersed for 48h at 37ºC in a McCoy culture medium with a concentration of 100 mg/ml. At the same time, SaOS-2 cells were "seeded" in a Mccoy culture medium, in a 96-wellplate at a concentration of 30000 cells/cm2, figure 15. 66 Figure 20 - 96-well plate, "seeded" with SaOS-2 cells at a concentration of 30000 cells/cm2. After 24h from "seeding", the extracts (McCoy medium where samples were submerged), were added to SaOS-2 cells culture for 24h. Three replicates were made with five dilutions of the extracts (100, 10-2, 10-4, 10-6, 10-8) for each sample, three negative controls using DMSO, and three positive controls using SaOS-2 cells. Finally, the resazurin was added, and after waiting for 3h, the absorbance at 570 and 600 nm was read using a Biotek ELX 800 UV reader. The percentage of viability was calculated using equation 5: ⌈((𝐴𝑏𝑠570𝑐𝑒𝑙𝑙 𝑚𝑒𝑑𝑖𝑢𝑚 − 𝐴𝑏𝑠600 𝑐𝑒𝑙𝑙 𝑚𝑒𝑑𝑖𝑢𝑚)− ((𝐴𝑏𝑠570𝑟𝑒𝑠𝑎𝑧𝑢𝑟𝑖𝑛 𝑐𝑜𝑛𝑡𝑟𝑜𝑙 − 𝐴𝑏𝑠600 𝑟𝑒𝑠𝑎𝑧𝑢𝑟𝑖𝑛 𝑐𝑜𝑛𝑡𝑟𝑜𝑙) (𝐴𝑏𝑠570𝑐𝑜𝑛𝑡𝑟𝑜𝑙 − 𝐴𝑏𝑠600 𝑐𝑜𝑛𝑡𝑟𝑜𝑙) − ((𝐴𝑏𝑠570𝑟𝑒𝑠𝑎𝑧𝑢𝑟𝑖𝑛 𝑐𝑜𝑛𝑡𝑟𝑜𝑙 − 𝐴𝑏𝑠600 𝑟𝑒𝑠𝑎𝑧𝑢𝑟𝑖𝑛 𝑐𝑜𝑛𝑡𝑟𝑜𝑙) ⌉ ×100 (Equation 5) Where: 𝐴𝑏𝑠570𝑐𝑒𝑙𝑙 𝑚𝑒𝑑𝑖𝑢𝑚and 𝐴𝑏𝑠600𝑐𝑒𝑙𝑙 𝑚𝑒𝑑𝑖𝑢𝑚 means the measured absorbance at 570 and 600 nm, respectively, of the sample (cells growth in the presence of the coatings; 𝐴𝑏𝑠570𝑐𝑜𝑛𝑡𝑟𝑜𝑙 and 𝐴𝑏𝑠600𝑐𝑜𝑛𝑡𝑟𝑜𝑙 means the measured absorbance at 570 and 600nm, respectively, of the control (cells growth without coatings); and 𝐴𝑏𝑠570𝑟𝑒𝑠𝑎𝑧𝑢𝑟𝑖𝑛 𝑐𝑜𝑛𝑡𝑟𝑜𝑙 and 𝐴𝑏𝑠600 𝑟𝑒𝑠𝑎𝑧𝑢𝑟𝑖𝑛 𝑐𝑜𝑛𝑡𝑟𝑜𝑙 means the measured absorbance at 570 and 600nm, respectively, of the resazurin control (only resazurin). An important point of this methodology is that it is an indirect method for cytotoxicity evaluation, so it is only possible to evaluate if the leaching that may occur on the surface of the material is harmful to the cells. 3.2. Antibacterial Activity Assay: To evaluate the antibacterial activity of the coatings produced, three microorganisms typically involved in the formation of bacterial biofilms in the oral cavity were used: two gram-positive bacteria’s, Streptococcus mutans (DSM 20523) and 67 Staphylococcus aureus (ATCC 6538), involved in the first stage of biofilm formation; and a gram-negative bacteria, Escherichia coli (K12 DSM 498). The experimental procedure for the antibacterial activity of the produced coatings was based on the ISO22196 standard [18]. Firstly, the substrates were sterilized in the autoclave at 180ºC during 1h. Each organism was pre-inoculated and incubated at 37°C for about 16h. Streptococcus mutans was incubated in a controlled atmosphere with 5% CO2. Two independent trials were conducted for each condition, for each trial, three coatings from each condition were used. Each coating was placed in a well of 20-well plates and adeed 40 µL of the pre-inoculum of Streptococcus mutans with a concentration of 5 x 105 cells/ml (see figure 16). On the top of each coating, a sterilised plastic film (PVC thin film with 13x13 mm2) , was placed on top of the surface coating in order to spread the drop. In addition, 2 ml of water ultra-pure was placed in one well to keep the humidity atmosphere saturated. Finally, the plates were left to incubate for 24h at 37ºC. The plates with E. coli and S. aureus were sealed inside a bag with moist paper,in order to maintain a humid atmosphere. The plates with S. mutans were simply placed in a controlled atmosphere incubator with 5% CO2 during 24h. Figure 21 - 20-well plate, with the coatings produced. Each coating contains a drop of 40 µL of the pre-inoculum at a concentration of 5 x 105 cells/ml. After removing the well-plates from the incubator, 1ml of SP1X salts was placed in each well and the well-plates were placed in agitation for 10 min at 220 rpm to wash the samples. Finally, a 10 µL drop of serial dilutions was placed in a Petri dish with 68 trypticase soy agar (TSA),. then were incubates for 24h at 37ºC. S. mutans was incubated with 5% CO2.After, the Petri dishes were removed from the incubator and the number of Colony forming units (CFU), for each dilution was counted, figure 17. Figure 22 - Petri dishes were removed from the incubator and the number of colony forming units (CFU), for each dilution: (a) TiN-Zn 0.25 coating; (b) TiN coating. 3.3. Cell’s Adhesion test: To analyse the initial adhesion of cells to the implant, the coating samples were first sterilised in the autoclave at 180°C during 1h. Then "seeding" was performed with SaOS-2 in a 12-well plate, placing 80µL of the cell’s suspension on each sample and using a density of 40000 cells/cm2. Then, with the 12 well-plates the seeded samples were incubates in a controlled atmosphere of 37°C and with 4% CO2 for 90 min. After, McCoy culture medium was added to each well in order to assure a volume of 2 ml. After 24h of incubation, samples were changed to a new well-plates and 2 ml of McCoy culture medium with resazurin (ratio 1:1) was placed in each well and incubated for 3h at 37°C and a 5% CO2 atmosphere. After incubation time, 200uL of the McCoy medium with resazurin in contact with each coating,was removed for a new 96-well plate, obtaining two replicates per coating condition. Finally, the absorbance was measured at 570 and 600 nm. The medium that remained in the plates with the samples was refreshed with 2 ml of McCoy culture medium and incubated during 24h at 37ºC and an atmosphere with 5% CO2, in order to be used in the remaining biological assays. 69 3.4. Cell’s Proliferation assay: To evaluate the proliferation of SaOS-2 cells adhered to the different coatings, the medium of the samples was first replaced by McCoy medium with resazurin (ratio 1:1) and the well-plates were incubated at 37ºC, in an atmosphere of 5% CO2 for 3h. Then 200 µL of the medium was removed from each well and putted in each well of a 96wells-plate, in order to obtaining two replicates per each different coating. Then, the absorbance was measured at 570 and 600 nm. The medium remaining on the plates with the samples was replaced by 2 ml of McCoy's culture medium every 24h and the plates were placed in the incubator at 37ºC and in an atmosphere with 5% CO2 in order to be used in the remaining biological assays. This procedure was repeated for 4, 7, 11 and 14 days after the start of culture. 3.5. Cell’s Differentiation assay: Alkaline phosphatase (ALP), a glycoprotein belonging to a family of proteins anchored to the plasma membrane e through glycosylphosphatidylinositol binding, catalyses the hydrolysis of phosphate esters at alkaline pH. ALP is a common biochemical marker used to assess osteoblast differentiation and is thought to be involved in skeletal mineralisation. ALP is abundant in vesicles matrix that play a role in extracellular matrix processing and calcification of bone. ALP levels are increased immediately before mineralisation begins. Furthermore, the precise role of ALP in mineralisation remains unclear. It may be related to its calcium-binding action, generation of free phosphate, or degradation of mineralization inhibitors [19]. In this research work, to measure the ALP activity of SaOS-2 cells. First 100 µL of McCoy medium was added to each well of a a 96-well plate, in order to obtaining two replicates per each analysed condition coating. Then, the absorbance of the McCoy medium, present in the 96-well plates, was measured at 405 nm. After the measurement, 100 µL of a solution of nitrophenyl phosphate (p-NPP) in TRIS-HCL (1mg/ml), a widely used substrate for detecting alkaline phosphatase activity, was added to each well of the 96-well plate and the plates were incubated at 37ºC with an atmosphere of 5% CO2, during 20 and 30 min. After that time, a new measurement of the absorbances at 405 nm was made. This procedure was repeated 5, 8, 12 and 15 days after culture initiation. 70 3.6. Statistical analysis: The data collected during this work were analysed using R software and GraphPad Prism. Results are presented as mean ± standard deviation (SD). Cell viability, antibacterial activity and cell adhesion were analysed using student t test. Proliferation and alkaline phosphatase activity, on the other hand, was analysed by a two-way ANOVA using Dunnett's multiple comparisons test. All experiments were performed at least in triplicate. 71 4. References: [1] M. Faraldos and A. Bahamonde, “Multifunctional photocatalytic coatings for construction materials,” in Nanotechnology in Eco-efficient Construction: Materials, Processes and Applications, 2018. doi: 10.1016/B978-0-08-1026410.00023-2. [2] W. Zhou, R. Apkarian, Z. L. Wang, and D. Joy, “Fundamentals of scanning electron microscopy (SEM),” in Scanning Microscopy for Nanotechnology: Techniques and Applications, 2007. doi: 10.1007/978-0-387-39620-0_1. [3] S. Ebnesajjad, Surface treatment of materials for adhesive bonding. 2013. doi: 10.1016/C2013-0-12914-5. [4] H. Zhang, “Surface Characterization Techniques for Polyurethane Biomaterials,” in Advances in Polyurethane Biomaterials, 2016. doi: 10.1016/B978-0-08-1006146.00002-0. [5] M. Ohring, The Materials Science of Thin Films. 2013. doi: 10.1016/C2009-022199-4. [6] G. Cao, NANOSTRUCTURES AND NANOMATERIALS - Synthesis, Properties and Applications. 2010. doi: 10.1142/9781860945960. [7] K. Akhtar, S. A. Khan, S. B. Khan, and A. M. Asiri, “Scanning electron microscopy: Principle and applications in nanomaterials characterization,” in Handbook of Materials Characterization, 2018. doi: 10.1007/978-3-319-929552_4. [8] H. Wang and P. K. Chu, “Surface Characterization of Biomaterials,” in Characterization of Biomaterials, 2013. doi: 10.1016/B978-0-12-415800-9.000048. [9] F. J. Giessibl, “Advances in atomic force microscopy,” Reviews of Modern Physics, vol. 75, no. 3. 2003. doi: 10.1103/RevModPhys.75.949. [10] B. Voigtländer, “Scanning Probe Microscopy: Atomic Force Microscopy and Scanning Tunneling Microscopy,” NanoScience and Technology, 2015. [11] D. Alderton, “X-Ray Diffraction (XRD),” in Encyclopedia of Geology, 2021. doi: 10.1016/b978-0-08-102908-4.00178-8. [12] J. Epp, “X-Ray Diffraction (XRD) Techniques for Materials Characterization,” in Materials Characterization Using Nondestructive Evaluation (NDE) Methods, 2016. doi: 10.1016/B978-0-08-100040-3.00004-3. 78 The analysis SEM micrographs shows that the incorporation of silver and zinc in TiN coatings causes a drastic change in the morphology of the film. The TiN sample is homogeneous with a very smooth surface, while the silver doped coatings are composed of nanoparticles embedded in a TiN matrix. On the other hand, the zinc doped coatings saw their morphology changed, however no nanoparticles are visible. The TiN columns in the film matrix change from a "scale-like" surface morphology in the case of TiN, to a pyramidal structure when silver is added. These structures, as the silver content is increased, become progressively more rounded. It can also be observed that the homogeneity of the coating is affected. While the TiN sample is homogeneous, presenting a very smooth surface, increasing the silver content proves changes in this characteristic. Increasing the zinc content in the film induces also causes significant changes in the surface structure of the coating. The TiN columns in the film matrix evolved pyramidal structures. These structures assume a more rounded shape in the case of TiN-Zn 0.25, becoming fully amorphous and heterogeneous in the case of TiN-Zn 0.32. Several authors [1] [6], reported that TiN-Ag coatings are composed of Ag clusters inserted into the column boundaries of the TiN matrix. In this sense, TiN-Ag 0.18 and TiN-Ag 0.21 samples are composed of a heterogeneous surface being possible to observe Ag clusters. The appearance of Ag cluster on the surface can be explained by its significant diffusion throughout the film thickness and by coalescence processes, properties already known of silver-based nanocomposites [7]. The existence of these clusters on the surface is a promising aspect for the objective of the work because in this way the silver is more quickly available for the desired antibacterial activity. To elucidate these conclusions the morphology of the coatings was analysed using micrographs of cross sections, showing a columnar structure common in this type of films with a relatively low deposition temperature, according to the Thornton diagram [8], and an accumulation of silver nanoparticles (white/shiny dots) in the TiN matrix. It is noticeable that as the silver content increases, the silver is no longer as visible in the TiN matrix since, it migrates to the surface, resulting in a non-uniform distribution of Ag over the thickness of the coating. Using ImageJ software, it was possible to calculate the area of the clusters that appeared on the surface of the silver doped samples, table 6. As expected, a higher silver content led to the emergence of clusters with larger areas. 79 Table 6 - Area of the nanoparticles present in each sample. Particle size Coating Area (m2) TiN-Ag 0.18 0.054 ± 0.02 TiN-Ag 0.21 0.080 ± 0.026 In both cases, the film roughness increases along with the Ag and Zn content, figure 26. Figure 25 - AFM images of the TiN, TiN-Ag and TiNZn coatings deposited on silicon substrates. From a mechanical point of view, the primary stability of the implant will be improved by a rough surface as it provides friction between the implant surface and the bone tissue, resulting in a greater initial retention, and consequently a greater stability, achieved soon after surgery [5]. Similarly, cell adhesion also depends on the anchorage sites on the implant surface. Thus, the roughness should be carefully controlled, since if it is much smaller than the size of the cells, there will not be adequate anchorage sites. On the other hand, roughness of large dimensions compared to the size of the cells will also not provide ideal sites for biofixation. The surface hydrophobicity of the different samples was also evaluated by contact angle measurements, shown in Figure 27. The surface energy of the various coatings was calculated according to the Owens, Wendt, Rabel (OWRK) approach [9], which is a 80 standard procedure to calculate the surface energy of hydrophobic solid surfaces from the contact angle using at least two liquids, table 7. Table 7 - Water, glycerol and 1-bromonaphthalene contact angles and surface energy components of the different coatings. Coating Contact angle ± SDa (deg) Surface energy components (mN/m) Water Glycerol 1-Bromonaphthalene γ Polar γ Dispersive γ Total Ti-6Al-4V 73 ± 5 80 ± 7 55 ± 2 9.48 22.33 31.81 TiN 76 ± 2 79 ± 3 63 ± 3 9.75 19.91 29.66 TiN-Ag 0.15 85 ± 7 81 ± 6 41 ± 11 3.15 30.37 33.52 TiN-Ag 0.18 93 ± 14 77 ± 7 40 ± 4 1.27 33.81 35.09 TiN-Ag 0.21 106 ± 4 70 ± 4 31 ± 14 0.29 40.65 40.94 TiN-Zn 0.18 94 ± 6 54 ± 8 49 ± 6 1.92 37.00 39.92 TiN-Zn 0.25 114 ± 2 117 ± 2 43 ± 5 0.53 26.62 27.15 TiN-Zn 0.32 114 ± 1 107 ± 3 55 ± 3 0.13 24.54 24.66 The surfaces can be classified as superhydrophilic when in the case of liquid water Ø=0º, hydrophilic when Ø<90º and hydrophobic when Ø>90 [8]. Thus, the water contact angles obtained for the different coatings (figure 27 and table 7) indicate that TiN and TiN-Ag 0.15 are coatings with a hydrophilic character. On the other hand, the remaining coatings produced present a contact angle greater than 90° indicating that they are hydrophobic [10] [11]. It is notable that the doping of TiN with silver or zinc nanoparticles causes the increase of the hydrophobicity of the coating, i.e., as the silver or zinc content is increased, the water contact angle also increases. On the other hand, looking at the surface energies of the different coatings (table 7), and dividing it into its polar and dispersive components, the dispersive component presents higher values compared to the polar component. These results are in line with the hydrophobic character observed in the different coatings. Fangyuan Yan at al [41] analysed silver coated films by static contact angle and concluded that they are hydrophobic due to the chemical properties of silver, and the emergence of agglomerates increases hydrophobicity. 81 Figure 26 - The water contact angle of the several samples. Data was represented as mean± standard deviation. Water molecules establish the first contact with the implant surface during implant placement. Therefore, from an osseointegration point of view, a hydrophilic surface is considered desirable to promote the initial stages of bone healing. Thus, surface wettability plays an important role in early bone healing [12]. Protein adsorption occurs on the implant surface; initially by high wettability proteins present in higher concentration in plasma, which are then replaced by other proteins with greater affinity for the implant surface (Vroman effect) [13]. This process is delayed on a hydrophobic surface, thus highlighting the importance of a hydrophilic surface in preserving the tertiary structures and activities of the proteins adsorbed on the surface. On the other hand, from the point of view of antibacterial activity, it has been shown in previous studies that there is a direct correlation between the wettability of a surface and the rate of bacterial attachment: higher surface wettability results in higher bacterial attachment and a higher rate of subsequent spreading [14]. 2.2 Structural analysis: The XRD results for the crystalline structures present in the samples are presented in the form of diffractogram in figure 28. Comparing the spectra of the samples with the Power Diffraction Files from ICDD it is possible to verify the presence of the crystal 82 structures of stoichiometric FCC-TiN (ICDD 00-038-1420), FCC-Ag (ICDD 00-0040783) and hexagonal Zn (ICDD 00-004-083). Figure 27 - XRD diffractogram of several coatings: (a) TiN-Ag; (b) TiN-Zn The differences in chemical composition correlate well with the observed differences in the developed structure. The XRD pattern of the TiN coating indicates that 83 the most intense peaks are located at 36.8° and 62.1º, which are very close to the TiN (111) and TiN (220) peaks. Previous studies with TiN coatings deposited by magnetron sputtering have reported this behaviour [15] [16]. Regarding TiN-Ag coatings, one can clearly identify the presence of two crystalline phases, namely one of the stoichiometric TiN and one phase of Ag. The intensity of the TiN (111) peak decreases with increasing Ag content. On the other hand, the TiN (220) peak increases significantly as the silver content increases. Moreover, the FWHM of the TiN (220) peak becomes narrower and narrower indicating that the grain size increases [17]. The diffractogram analysis of TiN-Zn coatings, on the other hand, confirms the presence of stoichiometric titanium nitride in all the series. As the Zn content increases, the preferential phase of TiN changes from TiN (220) to (111). Also, only in TiN-Zn 0.32A series was it possible to verify the existence of Zn crystalline phase. Previous studies justify this observation with the possibility of Zn being in an amorphous form, or being present in a low concentration, or the nanoparticles are of a small size, which are not identified by XRD [18]. Using the Scherrer equation, equation 6, a grain size was calculated for the most intense peaks of TiN, Ag and Zn. The results are presented in the tables 8-9. 𝐷ℎ𝑘𝑙=𝑘𝜆 𝛽cosΘ` (Equation 6) Table 8 - Grain size was calculated on TiN-Ag coatings for the most intense TiN and Ag peaks. Peak 𝑫𝒉𝒌𝒍 (nm) TiN TiN-Ag 0.15 TiN-Ag 0.18 TiN-Ag 0.21 TiN (111) 10.5 20.9 17.4 34.7 TiN (220) 6.8 19.3 23.1 29.0 Ag (111) - 14.3 16.8 21.0 Ag (220) - - 16.8 19.4 These results are in agreement with those obtained by S. Calderon Velasco at al [22] where he reported that the main crystalline phase for silver in TiN-Ag coatings is FCC and the grain size of Ag 6-78 nm and of TiN 12-80 nm. 84 In the case of TiN-Zn coatings, it can be observed that the growth of a new phase (zinc), compromises the growth of the TiN phase, table 9. Thus, the TiN grain size decreases as the zinc content increases. Table 9 - Grain size was calculated on TiN-Zn coatings for the most intense TiN and Zn peaks. Peak 𝑫𝒉𝒌𝒍 (nm) TiN TiN-Zn 0.18 TiN-Zn 0.25 TiN-Zn 0.32 TiN (111) 10.5 34.9 14.9 9.5 TiN (220) 6.8 28.9 10.5 10.5 Zn (101) - - - 18.3 Zn (100) - - - 15.1 Finally, knowing previously through the ICDD sheets that TiN and Ag have an FCC structure and Zn a hexagonal structure, the XRD network parameters were calculated using the following equations below. The results are presented in tables 10-11. ➢ FCC Structure (TiN,Ag): 𝑎=𝑏=𝑐 1 𝑑2ℎ𝑘𝑙 =ℎ2+𝑘2+𝑙2 𝑎2 (Equation 7) ➢ Hexagonal structure (Zn): 𝑎=𝑏≠𝑐 1 𝑑2ℎ𝑘𝑙 =4 3(ℎ2+𝑘+𝑘2 𝑎2)+𝑙2 𝑐2 (Equation 8) 85 Table 10 - XRD network parameters was calculated on TiN-Ag coatings for the most intense TiN and Ag peaks. Peak a (Å) TiN TiN-Ag 0.15 TiN-Ag 0.18 TiN-Ag 0.21 TiN (111) 4.27 4.26 4.25 4.24 TiN (220) 4.27 4.26 4.25 4.24 Ag (111) - 4.09 4.09 4.08 Ag (220) - - 4.09 4.09 Table 11 - XRD network parameters was calculated on TiN-Zn coatings for the most intense TiN and Zn peaks. Peak a (Å) TiN TiN-Zn 0.18 TiN-Zn 0.25 TiN-Zn 0.32 TiN (111) 4.27 4.26 4.26 4.26 TiN (220) 4.27 4.26 4.26 4.26 a b Zn (101) - - - 2.67 4.96 Zn (100) - - - 2.67 4.96 According to the ICDD sheets, TiN and Ag have an FCC structure, with a network parameter a=4.24 Å and a=4 Å, respectively. On the other hand, according to the ICDD sheets in zinc doped TiN coatings, Zn has a hexagonal structure with a network parameter a=2.66 Å and b=4.94 Å. In this work, larger values were obtained due to the compressive stresses [42]. 86 2.3 Silver and zinc ion release: The ICP-OES results for silver and zinc ion release from samples immersed in an artificial saliva solution at room temperature for 7 days are shown in figure 29-30. Figure 28 - Ag+ release along time (2h, 6h, 12h, 24h, 48h, 120h and 168h) determined by ICP-OES analysis. Data was represented as mean± standard deviation. The silver ionization by the sample with lower silver content (TiN-Ag 0.15) over time was always lower compared to the samples with higher content, reaching a maximum of about 48 ppb/cm2 after 7 days. The samples with higher silver content (TiN-Ag 0.18 and TiN-Ag 0.21) show a different behaviour, since the sample with an intermediate silver content shows a higher silver ionisation than TiN-Ag 0.21 in the first 6h (35 and 23 ppb/cm2, respectively). 87 Figure 29 - Zn+ release along time (2h, 6h, 12h, 24h, 48h, 120h and 168h) determined by ICP-OES analysis. Data was represented as mean± standard deviation. Regarding the release of zinc ions, it can be conclude that the ionisation of zinc by the sample with lower zinc content (TiN-Zn 0.18) over time was always lower compared to the samples with a higher content. However, it can be said that the difference in zinc ionisation only becomes significant on the second day of immersion compared to TiN-Zn 0.25. On the other hand, the TiN-Zn 0.32 sample stands out from the others since it reaches 2000 ppb/cm2 in only 12h and goes up to 4300 ppb/cm2 after 7 days. This result corroborates the morphology of the samples, as mentioned previously (Section 2.1. Morphology), the TiN-Ag 0.18 and TiN-Ag 0.21 coatings present silver agglomerates on their surface (Figure 25), unlike the TiN-Ag 0.15 sample, where the silver is incorporated into the bulk coating, delaying Ag ionisation during the first days of immersion. They also meet with the EDS results (Section 2. Physical and chemical characterization:), where the silver content present in the coatings was proved to increase. Finally, this analysis also allowed us to conclude that the samples have not yet stabilized and would continue to release ions, since no stationary phase was reached in the graphs of the different samples. 94 Figure 33 - Cell proliferation of SaOS-2 cells "seeded" on the coatings produced. The control is SaOS-2 cells grown on the plates. SaOS-2 cell differentiation was assessed by histological staining for alkaline phosphatase. A two-way ANOVA analysis, using Dunnett's multiple comparisons test, was performed to statistically evaluate the results allowed some critical conclusions to be drawn. In this analysis, the results obtained in TiN coatings were compared with the doped coatings, in order to be possible to evaluate the effect of TiN doping, table 13. Table 13 - ALP activity two-way ANOVA statistical test results comparing TiN coating with doped coatings p-value summary Day 5 Day 8 Day 12 Day 15 TiN vs TiN-Ag 0.15A **** **** **** **** TiN vs TiN-Ag 0.18A **** **** **** **** TiN vs TiN-Ag 0.21A **** **** **** **** TiN vs TiN-Zn 0.18A ns ** ns ns TiN vs TiN-Zn 0.25A ns ns **** ns TiN vs TiN-Zn 0.32A * ns **** ** The results show that the silver doped coatings practically do not present ALP activity. On the other hand, TiN-Zn 0.18 coating show ALP activity similar to TiN coatings, which decreases with increasing zinc content in the coating, figure 35. 95 Figure 34 - ALP activity of SaOS-2 cells "seeded" on the coatings produced. Thus, it is concluded that silver-doped coatings decrease the differentiation of osteoblasts, despite several papers reporting that silver nanoparticles can increase mineralisation and alkaline phosphatase expression [37]. These results are also influenced by the poor adhesion demonstrated by the samples. On the other hand, zinc doped coatings show more encouraging data since TiN-Zn 0.18A and TiN-Zn 0.25A samples show similar values to that obtained in TiN. These results are in line with existing works in the literature, where it is described that in general, materials containing Zn promote osteogenic differentiation [38] [39]. Another factor that also for the increase of ALP activity is the increase of roughness [40]. 96 4. References: [1] S. M. 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Abadias, “Nanometer-scale multilayer coatings combining a soft metallic phase and a hard nitride phase: Study of the interface structure and morphology,” Surf Coat Technol, vol. 180– 181, 2004, doi: 10.1016/j.surfcoat.2003.10.139. 101 Chapter 6 – Final remarks and future research: 102 1. Final remarks: The main objective of this work was the development of multifunctional coatings to be applied to dental implants, in order to improve the osseointegration, the mechanical and antibacterial properties of these devices, based on two systems, TiN-Ag and TiN-Zn. To meet this objective, the following specific tasks had to be developed: • Magnetron sputtering deposition on Ti-6Al-4V substrates of two different systems, TiN-Ag with different Ti/Ag atomic ratios and TiN-Zn coatings with different Ti/Zn atomic ratios, where the current density applied to the Ag and Zn targets was varied. • To characterize physically and chemically the deposited coatings • To analyse the biocompatibility • To evaluate the antibacterial activity • To study cell adhesion, proliferation, and differentiation. The results show that the different processing conditions allowed to obtain different systems, where it was possible to change the physical and chemical properties of both TiN-Ag and TiN-Zn series. By varying the current density applied to the dopant element target (Ag and Zn), it was possible to obtain coatings with different rat and zinc contents. This content increases with the increase of the applied current density. According to the morphology analysis, for TiN-Ag series, only TiN-Ag 0.15 coating is homogeneous. In TiN-Ag 0.18 and TiN-Ag 0.21, agglomerates appear on the surface, which increase with increasing silver content. On the other hand, the samples of TiN-Zn series present a heterogeneous structure. The AFM analysis suggested that both series of coatings increase their roughness with increasing dopant element content. This effect is extremely noticeable in the TiN-Zn series. The water contact angles obtained for the different coatings indicate that TiN and TiN-Ag 0.15 are coatings with a hydrophilic character. On the other hand, the remaining coatings produced present a contact angle greater than 90° indicating that they are hydrophobic The XRD analysis reveals that for TiN-Ag series, the crystalline phase FCCTiN and a crystalline phase FCC-Ag are present; for TiN-Zn series there is also the crystalline phase FCC-TiN and hexagonal phase of Zn on TiN-Zn 0.32. 103 All the coatings produced are biocompatible, do not demonstrate cytotoxicity, since cell viability levels are above 90%. The antibacterial activity of the samples of both series was evaluated. TiN-Ag coatings totally inhibit bacterial activity, whereas TiN-Zn coatings show antibacterial activity, dose-dependent, only on gram-positive bacteria. Cell’s adhesion, proliferation and differentiation were also evaluated with SaOS2 cells. The results indicate that in general, fewer cells attach to silver coatings compared to TiN. in the case of TiN-Zn series, there is a dose dependent effect, where the increase in Zn concentration decreases cell adhesion. The same behaviour was verified in the proliferation and differentiation tests. Globally, considering the objective of the work, TiN-Ag 0.15 and TiN-Zn 0.32 are the coatings that present the best properties to be applied in dental implants. The TiNAg 0.15 because no dose dependent property was found in this series and therefore, TiNAg 0.15 is the most economical coating. The TiN-Zn 0.32, because it was proved that the coatings doped with zinc improve its antibacterial properties with the increase of the zinc content. 2. Future work: Future work involves the deposition of TiN, TiN-Ag 0.15 and TiN-Zn 0.32 coatings on dental implants in order to create a prototype. For this it is necessary to optimise the deposition conditions, since making depositions on flat substrates is not the same as depositing on a screw. It would also be interesting to repeat some tests, namely: the ICP-OES, performing it for a longer period of time to be able to see what the maximum concentration of Zn ions is reached; and the biological ones, conditioning the coatings with saline solution, since the adhesion of proteins on the surface could improve the adhesion and proliferation of osteoblasts. Then it would also be interesting to perform antimicrobial tests, for example with fungi and viruses, since the oral microbiota is composed of a wide variety of microorganisms, and instead of being done separately, i.e. one microorganism at a time, it would be interesting to perform the tests in consortium. Finally, it would be interesting to make a new series of TiN-Ag coatings with a lower silver content, since in antibacterial tests all the contents obtained excellent antibacterial activity, and it is not possible to find a middle ground, so that similar results could be obtained with a lower silver content, making the implant more economically reliable.