Design, development, and validation of chitosan-based coatings via catechol chemistry for modulating healthcare materials

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© Clayton Souza Campelo, 2020

Design, development, and validation of chitosan-based

coatings via catechol chemistry for modulating

healthcare materials

Thèse

Clayton Souza Campelo

Doctorat en génie des matériaux et de la métallurgie

Philosophiæ doctor (Ph. D.)

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Design, development, and validation

of chitosan-based coatings via

catechol chemistry for modulating

healthcare materials

THÈSE

Doctorat en Génie des Matériaux et de la Métallurgie

Clayton Souza Campelo

Sous la direction de :

Diego Mantovani, directeur de recherche

Rodrigo Silveira Vieira, directeur de recherche

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Résumé

Depuis la préhistoire, plusieurs matériaux ont été utilisés pour fabriquer des instruments et des appareils de santé. Au cours des dernières décennies, avec l’apparition du terme « biomatériau », les matériaux ont été conçus pour contrôler des réactions biologiques spécifiques, pour augmenter la durée de vie des biodispositifs et la qualité de vie des patients dans le monde. Cependant, indépendamment de la nature du matériau, ou au sens strict du biomatériau, et de la fonction remplie, ils sont susceptibles aux phénomènes de surface causés par son environnement. Certains phénomènes intéressants incluent l’action des protéines, des électrolytes et des cellules sur les surfaces métalliques. Ces interactions peuvent entraîner le développement de complications telles que la formation de thrombus, la corrosion et la calcification, qui affecteront le fonctionnement des dispositifs, et la contamination bactérienne qui peut transformer la surface en vecteur de propagation de maladies. Des recherches ont exploité des stratégies de modification de surface pour minimiser ou éviter ces complications. Ces approches demandent du temps et des efforts pour développer une surface efficace pour chaque cas. Sur cette base, l’objectif principal de ce travail était de concevoir et de développer des revêtements à base de chitosane à utiliser dans le revêtement de surfaces métalliques et de dispositifs utilisés dans le système de santé et de modifier ces surfaces pour moduler la réponse biologique. Pour atteindre cet objectif, le projet de recherche a été divisé en trois parties. La première était le greffage du chitosane utilisant de la dopamine comme ancre. La deuxième était le développement d’un greffage original en une étape remplaçant la dopamine par l’acide caféique. La dernière était la modification du revêtement de chitosane pour moduler la réponse biologique de la surface. À chaque étape, les surfaces revêtues ont été caractérisées par analyses biologiques et physico-chimiques. Les résultats ont démontré que la méthodologie développée produisait des revêtements de chitosane qui possédait des réponses biologiques et des performances physico-chimiques favorables et qui pouvait être modifiés pour améliorer ou conférer la propriété souhaitée. De plus, cette méthodologie permet de produire une plateforme capable d’être appliquée sur une large gamme de complications en raison de sa modulabilité. Cela représente une diminution de la consommation de temps pour créer une nouvelle surface à partir du zéro pour chaque situation.

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Abstract

Since prehistoric times, several materials have been used to make health instruments and devices. In recent decades, with the appearance of the term "biomaterial", materials have been designed to control specific biological reactions, to increase the lifespan of bio-devices and the quality of life of patients around the world. However, regardless of the nature of the material, or in the strict sense of the biomaterial, and the function fulfilled, they are susceptible to the surface phenomena caused by its environment. These phenomena include the action of proteins, electrolytes, and cells on metal surfaces. These interactions can lead to the development of complications such as thrombus formation, corrosion, and calcification, which will affect the functioning of the devices, and bacterial contamination, which can transform the surface into a vector for the spread of disease. Researches were made on the use of surface modification strategies to minimize or avoid these complications. These approaches require time and effort to develop an effective surface for each case. On this basis, the main objective of this work was to design and develop chitosan-based coatings to coat metallic surfaces and devices used in the healthcare system and to modify these surfaces to modulate the biological response. To accomplish this objective, the research project was divided into three parts. The first was the grafting of chitosan using dopamine as an anchor. The second was the development of an original one-step graft replacing dopamine with caffeic acid. The last was the modification of the chitosan coating to modulate the biological response of the surface. At each stage, the coated surfaces were characterized by biological and physicochemical analyzes. The results obtained showed that the developed methodology produced chitosan coatings that had favorable biological responses and physicochemical performances, and that it could be modified to improve or confer the desired property. Besides, this methodology makes it possible to produce a platform able to be applied to many complications due to its modularity. It represents a reduction in the consumption of time to create a new surface from scratch for each situation.

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List of tables

Table 1 – Class of healthcare materials with their properties and main applications ___________________ 4 Table 2 - Stainless steel 316L composition ____________________________________________________ 24 Table 3 - Surface coating techniques ________________________________________________________ 24 Table 4 – Modifications of chitosan and the targeted function ____________________________________ 29 Table 5 – Bacteria and fungi used to demonstrate the inhibitory and antibacterial activity of chitosan ____ 34 Table 6 - Surface characterization techniques used in this work ___________________________________ 36 Table 7 - Methodologies used to evaluate the biological performance of the coatings _________________ 38 Table 8 - Methodologies used to evaluate the physicochemical performance of the coatings ____________ 39 Table 9 – Strategies used in literature to avoid short and long-term complication for biomaterials in contact with blood ______________________________________________________________________________ 44 Table 10 – Average XPS chemical composition for electropolished stainless steel and dopamine, PEGb, natural chitosan and sulfonated chitosan modified surfaces ______________________________________ 52 Table 11 – Average roughness (Ra), root mean roughness (Rq), skewness (Rsk) from AFM images and contact angle results for electropolished stainless steel and dopamine, PEGb, natural chitosan and sulfonated chitosan modified surfaces ________________________________________________________________ 54 Table 12 – Calcium and phosphorus atomic percentages from EDS analysis for electropolished stainless steel and dopamine, PEGb, natural chitosan and sulfonated chitosan modified surfaces ___________________ 58 Table 13 - Applications of modified chitosan __________________________________________________ 68 Table 14 – Material functionalization by polyphenol derivatives and targeted functions _______________ 69 Table 15 – Average XPS atomic composition. _________________________________________________ 75 Table 16 – Chemical composition after 4 weeks of aging in PBS buffer _____________________________ 81 Table 17 - Corrosion potential, corrosion current, and corrosion rate ______________________________ 83 Table 18 – Experimental matrix for the electropolishing optimization. _____________________________ 87 Table 19 – Atomic percentage of samples during the optimization of the electropolishing process _______ 88 Table 20 - Sample index and the pH of the caffeic acid or chitosan-caffeic acid solution used for the grafting onto stainless steel substrates ______________________________________________________________ 90 Table 21 - Atomic percentage of samples during caffeic acid grafting optimization onto stainless steel substrates ______________________________________________________________________________ 90 Table 22 - Atomic percentage of samples during chitosan-caffeic acid grafting optimization onto stainless steel substrates __________________________________________________________________________ 92 Table 23 – XPS characterization of chitosan coatings before and after quaternization _________________ 95 Table 24 – XPS characterization of chitosan coatings before and after phosphorylation _______________ 98

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List of figures

Figure 1 – Examples of ancient biodevices in fiction and real life. ... 1

Figure 2 – Burnt City artificial eye. ... 2

Figure 3 – Classification of healthcare materials ... 3

Figure 4 – Structures of proteins. Artwork by Thomas Shafee. ... 8

Figure 5 – Effects of electrolytes in the surface of a stent.. ... 9

Figure 6 – Steps of the bacteria adhesion and biofilm formation. ... 9

Figure 7 – Possible interactions of the contact between the material and the tissues. ... 10

Figure 8 – Coagulation cascade. ... 11

Figure 9 – Strategies applied to the surface to control the complications of the material-tissue interactions. 14 Figure 10 – Antibacterial mechanisms related to the use of polymer surfaces. ... 16

Figure 11 - General schematic approach of the project.. ... 18

Figure 12 – Schematic representation of the coating strategy via dopamine. ... 19

Figure 13 – Scheme of chitosan grafting via dopamine. ... 20

Figure 14 – Steps of the reaction of caffeic acid-chitosan grafting. ... 21

Figure 15 – Schematic representation of the coating strategy via caffeic acid. ... 22

Figure 16 – Schematic representation of the strategy of surface modulation. ... 23

Figure 17 – Adhesiveness mechanisms of catechol group ... 26

Figure 18 – Comparison between dopamine and caffeic acid structures. ... 27

Figure 19 – Comparison between the structures of cellulose and chitosan. ... 29

Figure 20 – Sulfonated chitosan structure. ... 32

Figure 21 – Representation of grafting process ... 48

Figure 22 – Surface morphologies for electropolished stainless steel and dopamine, PEGb, natural chitosan and sulfonated chitosan modified surfaces. ... 54

Figure 23 – SEM micrographs for electropolished stainless steel and dopamine, natural chitosan and sulfonated chitosan modified surfaces after calcification. ... 57

Figure 24 – Mapping of calcium and phosphorus for natural and sulfonated chitosan. ... 59

Figure 25 – Platelet adhesion AFM micrographs for electropolished stainless steel and dopamine, natural chitosan and sulfonated chitosan modified surfaces. ... 61

Figure 26 – Average clotting times for electropolished stainless steel and dopamine, natural chitosan and sulfonated chitosan modified surfaces. ... 62

Figure 27 – XPS high resolution spectra of C1s (A- D) and O1s (E- H) ... 77

Figure 28 – Surface morphologies and hydrophilicity. ... 79

Figure 29 – Variation of composition before and after 4 weeks aging in PBS buffer. ... 81

Figure 30 – Potentiodynamic polarization. ... 83

Figure 31 – Cytotoxicity tests performed with fibroblasts. ... 84

Figure 32 – O/C and Cr/C ratios for samples in the optimization of the electropolishing process. ... 89

Figure 33 – O/C (left axis) and Cr/C (right axis) ratios for samples in the optimization of the caffeic acid grafting process. ... 91

Figure 34 – O/C, N/C, and Cr/C ratios for samples in the optimization of the chitosan-caffeic acid grafting process. ... 93

Figure 35 – Structure of glycidyl trimethyl ammonium chloride. ... 94

Figure 36 – Structure of quaternary ammonium chitosan (QAC). ... 96

Figure 37 – Structure of phosphorylcholine. ... 97

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List of abbreviations

AFM – Atomic force microscopy CA – Caffeic acid

CHI – Chitosan

ChiCa – complex chitosan-caffeic acid CR – Corrosion rate

DA – Dopamine

DMEM – Dulbecco's modified Eagle's medium DMSO – Dimethyl sulfoxide

EDAC – N-(3-dimethyl aminopropyl)-N’-ethyl carbodiimide EW – Equivalent weight

FBS – Fetum bovine serum

MES – 2-(N-morpholino) ethanesulfonic acid OCP – Open circuit potential

PBS – Phosphate buffered solution PCC – Phosphorylcholine chitosan PCL – Polycaprolactone

PDP – Potentiodynamic polarization PEG – Polyethylene glycol

PEGb – Polyethylene glycol bis carboxymethyl ether PEO-SO3 – Sulfonated polyethylene oxide

PGA – Polyglycolic acid

PGLA – Poly(glycolide-co-lactide) PLA – Polylactic acid

PLLA – Poly-L-lactic acid) PTFE – Polytetrafluoroethylene PU – Polyurethane

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xi QAC – Quaternary ammonium chitosan Ra – Arithmetical mean roughness Rq – Root mean squared roughness Rsk – Roughness skewness

SChi – Sulfonated chitosan SS – Stainless steel

Tris – tris (hydroxymethyl) amino methane XPS – X-ray photoelectron spectroscopy

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Acknowledgments

First, I would like to thank the members of the jury, Professor Mircea Mateescu, Professor Jonathan Gagnon, and Professor Jesse Greener, for their kindness in accepting to read and to judge this work and for the very much appreciated corrections and suggestions. Thanks to Professor Gaétan Laroche, for presiding the defense.

I want to thank my two thesis directors, Professor Rodrigo Vieira and Professor Diego Mantovani, for accepting me as a Ph.D. candidate and for the confidence deposited in me. Thanks for the patience and all the knowledge shared during the Ph.D. and even before.

I warmly thank Mrs. Pascale Chevallier, whose experience and kindness were indispensables to the conclusion of this work. Pascale, you will always be my third thesis director. Thanks to Mrs. Lucie Levesque and Mr. Stéphane Turgeon for the contributions and advice.

I also thank Mrs. Andrée Lord, Mrs. Martine Demers, and Mrs. Karine Fortin for their great administrative work and welcoming smile, helping me every time I had a bureaucratic issue.

Many thanks to those who contributed to this project as co-authors: Juliana Vaz and Caroline Loy. Your scientific help sure enriched work and improved the level of my communications.

Thanks to the LBB group: Sergio, Francesco, Linda, Gabriel, Samira, Malgorzata, Sergio, Majid, Essowe, Farid, Erica, Michel, Carlo, Sébastien, Ludivine, Vanessa, Agung, Mahrokh, and whoever I forgot. I also thanks Iván, Gad, Marie, Stéphanie, and Laurence. Thank you for the help, the kind words, and the good times.

My heartfelt thanks to my Powerpuff girls: Letícia (Docinho), Carolina (Florzinha), and Dimitria (Lindinha). Obrigado por me darem forças e me suportarem nos momentos em que nem eu me suportava. Vocês são demais (com ph)!

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Obrigado a Luno, João e Vinicius pelos risos nos momentos depressivos. Peçam benção à Madrinha.

Obrigado aos amigos que torceram por mim.

Many thanks to my dear friend Didier, whose silent advices helped to go through the hard times.

Merci à Véronique pour avoir briser mon orgueil en parlant que les images que j’avais fait pour mes articles étaient laides et ensuite avoir offrir de l’aide pour les faire.

Un gros merci à ma petite famille québecoise : Jean-Marc, Nathalie, Samantha e Raphaël, vous êtes la lumière qui manquait dans l’hiver où je me trouvait.

Obrigado ao meu irmão, Cristiano, e às minhas irmãs, Gisele e Silene, que sempre me apoiaram e acreditaram em mime que hoje me usam como exemplo os meus sobrinhos, Sofia, Heitor, Jamille e Leandro. Obrigado também à minha cunhada, Roberta, e aos meus cunhados, Vander e Beto. Tenho certeza que vocês torceram por mim.

Quero agradecer do fundo do meu coração ao meu pai, Gilberto, que nunca mediu esforços para que eu pudesse estudar, e à minha mãe, Fátima, minha primeira professora, responsável por me ensinar a ler e escrever e que sempre zelou para que eu aproveitasse a oportunidade que eles não tiveram. Amo vocês. Meu muito obrigado!

Pour finir, je remercie à Tommy, mon amour, pour tout le support donne au moment que j’était prêt à tout abandonner. Ta présence était un signe que tout allait bien aller. Merci pour être mon phare dans la têmpete dans laquelle je me trouvait. Mon amour pour toi = lim

𝑥→0 1

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Foreword

The necessity of new environment-material interfaces that can actively control the surface response towards a specific function have drawn the interest of the search for stable proactive coatings. These coatings can act on the interaction of the devices and its surroundings minimizing or avoiding drawbacks while promoting the enhancement of desirable features. This work presents approaches using natural molecules, chitosan and catechols, to develop covalently linked coatings that will bring wear resistance. Moreover, the use of a polymer easily modifiable such as chitosan will allow the modulation of the coating to improve its efficiency and biological response in a specific application.

The research project was executed in the Laboratory for Biomaterials and Bioengineering (LBB) at Laval University (Quebec, Canada) that has developed in the last decades the expertise in surface characterization techniques, such as X-ray photoelectron spectroscopy, atomic force microscopy, scanning electron microscopy, contact angle, and others, and cellular performance tests. Moreover, the LBB has the experience in physicochemical and mechanical tests. All this know-how, allied to skilled professionals, allowed the accomplishment of the coatings development as well as the validation of the methodology proposed. This multidisciplinary approach allowed the realization of this project and obtaining the data presented in this thesis.

This thesis was organized in seven sections. The Introduction presents a brief regard over the evolution of biomaterials, from the primitive ones, made of wood, bones, and other simple materials, to the engineered ones, which properties are tailored to the function that they will perform. In this chapter, it is presented in the context that motivated this work, the surface phenomena, the problems they carry out, and some strategies to limit, minimize, or avoid them.

The Chapter 1 brings the state of art related with the aim of this work. It presents and explains the choices of the substrate, strategies, and molecules used to produce the coating. The hypotheses and objectives that guided the development of this research are described in this chapter, along with the techniques and methodologies used in its execution.

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The chapters that follow are scientific communications written with the data obtained in the analysis. The Chapter 2 shows a 3-step methodology to produce sulfonated chitosan coatings using dopamine and poly(ethylene glycol) as anchors to bind the chitosan to the stainless steel surface. The Chapter 3 goes beyond and presents a one-step methodology to grafting chitosan using caffeic acid, avoiding the need for a linking arm to graft the chitosan to the catechol. Details of the publications are presented below:

Chapter 2: Sulfonated chitosan and dopamine based coatings for metallic implants in

contact with blood

Authors: Clayton Souza Campelo, Pascale Chevallier, Juliana Miguel Vaz, Rodrigo

Silveira Vieira, Diego Mantovani

Journal: Materials Science and Engineering: C 72 (2017) 682–691, submitted on 7

September 2016, published on 1 March 2017.

This article presents results of coating with the natural molecules dopamine and chitosan for the enhancement of the interaction of metallic devices with blood at short and long term. The manuscript focuses on the coating of metallic substrates to modulate the biological performances of the surface over the hemocompatibility and calcification resistance using polydopamine as an organic interface for posterior coupling of sulfonated chitosan. It also underlines the influence of the interface on the biological response. The blood tests were made under the CHU de Québec ethic approval for human blood collection: SCH11-09-091. The experiments and data acquisition were carried out by me, excepting the acquisition of XPS data, made by Pascale Chevallier. I also made all the data treatment and the first version of the document. Pascale Chevallier and Juliana Miguel Vaz enriched the text with their expertise and knowledge. All the authors contributed to the corrections in the final version.

Chapter 3: Development, validation, and performance of chitosan‐based coatings using

catechol coupling

Authors: Clayton Souza Campelo, Pascale Chevallier, Caroline Loy, Rodrigo Silveira

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Journal: Macromolecular Bioscience 20 (2020) 1900253, submitted on 14 July 2019,

published on 15 January 2020.

The second article explored a new approach to produce the chitosan coatings. It presents an original one-step method to grafting the chitosan into a metallic surface by using caffeic acid instead of dopamine. This method was compared with the well-known dopamine approach. A second approach using caffeic acid was also studied. The article focused on the stability and cytotoxicity of the new surfaces. The experiments, data acquisition and data treatment were made by me, while Pascale Chevallier was responsible for XPS data acquisition. Caroline Loy kindly performed the cytotoxicity tests and acquired the data. I wrote the preliminary versions with the guidance of Pascale Chevallier and all the authors contributed to the correction of the final version.

Chapter 4 presents the general discussion, showing preliminary results as well as

unpublished ones. Conclusion is the conclusion of this study and bring perspectives for future works. References is the list of bibliographies consulted for the development and discussion of the research project.

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Introduction

The use of materials for wound treatment or replacement of organs and lost limbs goes back to a distant past in human history. No one knows exactly when the humans began to develop these devices [1]. It is common to see in music, animations, games and movies, pirates carrying pegleg and hooked hand, or mages with a glass eye, without realizing that these are old examples of biodevices (Figure 1). The Rigveda, the first collection of Hindu sacred hymns, dated 1500-1200 BC, tells about how Queen Vishpala, a warrior queen who lost his leg in battle, receives from Ashvins gods an iron leg to replace the lost limb [2]. The history of artificial eyes, from antiquity to the present day, has been described [3]. In 2007, Italian and Iranians archaeologists found in the necropolis of the city of Shahr-i-Sokhta, in Sistan desert, a semi-spherical artificial eye made with a derivative of bitumen paste, with a central circle to represent the iris and golden lines "as rays of the sun" (Figure 2). The estimated age of this eye was approximately 5000 years [4].

These biodevices were made of diverse types of materials, such as glass, porcelain, wood, iron, gold, and leather. At first, the interactions that materials developed with the

Figure 1 – Examples of ancient biodevices in fiction and real life. (A) - A model of a

pirate with pegleg and hooked hand used in the game Pirates, Vikings and Knights II; (B) - Alastor "Mad Eye" Moody and his magical artificial eye from Harry Potter series; (C) - US Civil War veteran Samuel Decker, who built his own prosthetics.

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body were not taken into account; they were used only for its functionality and usability [5]. These materials were obtained through trial and error, with little chance of success. From World War II, scientists and therapists began to be more concerned about the selection of materials and the diversification of its use [1,5]. It is convenient to separate the history of materials used for healthcare in four generations [5,6].

[7]

The first generation is marked by empiricism. It goes from the pre-history to the 19th century. The devices had no aesthetic concern and were mostly produced with naturally occurring materials such as gold and ivory for dentures, glass or metal for eyeballs and wood for making lost limbs [2,5].

The second generation is the ‘Replacement Era’. In this era, ranging from the 19th century to the mid-20th century, devices start to be made from new materials, more durable and ‘inert’, which had been developed for wartime. The appearing of the anesthesia, the X-rays for diagnostics, and the aseptic practices led to the development of devices that could be implanted or fixated in long surgical procedures, with a high chance of success. Among

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the materials used in this era are silicone, polyurethanes, Teflon, nylon, polyester, methacrylates, stainless steel and titanium [5].

The third generation is marked by the appearance of bioengineered materials, i.e., materials have been developed specifically for use in contact with the human body. It is the ‘Materials by-design Era’ [5]. Many materials of the last generation were studied and modified for exclusive use as healthcare materials such as porous Teflon, polyethylene, polyethylene terephthalate, bioglass, and hydroxyapatite.

The late 1980s saw the start of the fourth generation, the ‘Proactive Biomaterials1

Era’, where materials are designed to control specific biological responses [5]. Several ideas of Materials Science began to be applied, such as surface modification, aiming this objective. These bioactive materials can be used, for instance, to improve blood interaction with medical devices, to promote osseointegration of orthopedic prostheses, or to lead antibacterial behaviors.

1.1 Healthcare materials

A vast range of materials are used as healthcare materials. These materials can be classified into three categories: metals, ceramics, and polymers. Materials of one category can be mixed with one, or more, from another to form a new type of material called

1_{In this work, the term “biomaterial” is used in a strict way as in Biomaterial Science [5], i.e., when talking}

specifically of biodevices. Otherwise, the term “material” is applied when presenting concepts in a large sense.

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composite material, which presents properties derived from both precursor materials. A general classification of materials is shown in Figure 3. The main classes of materials are briefly described below, and the most interesting components are presented in Table 1.

Ceramics are polycrystalline inorganic materials (non-metallic) normally used in dentistry and orthopedic implants [8]. They are biologically inert and possess high compressive strength. However, they have a brittle behavior, which limits its use in applications where a deformation (elongation, flexion) is expected [9].

Metals are mainly used for load-bearing implants or internal fixation devices [10] but they are also found in heart and cardiovascular implants, dentistry, cochlear implants and others [9,10]. Moreover, metals are the primary constituent of surgical instruments, surgery tables, supports, etc. The main drawback of implanted metals is the corrosion behavior and the release of corrosion products.

Polymers are long chain macromolecules constituted by monomers, smaller molecules, in repetition. Polymers can be classified in synthetic or natural depending on their source. They are a versatile material class, having been already used in surgical instruments, textiles, prostheses, device coatings, drug capsules, tissue engineering, sutures, dental implants, etc. The main advantage of polymers is easiness of processability. The possibility of polymer modification is other advantage, which make the applications of polymers virtually unlimited. However, their mechanical properties are inferior to those of metals and ceramics, which make their use difficult in applications where a high mechanical strength is required.

Table 1 – Class of healthcare materials with their properties and main applications

Material Properties Applications Ref

Ceramics

Alumina Inertness, biocompatibility, non-sensitization of

tissues, excellent wear, and friction properties, higher compressive strength to tensile strength Joint replacement; dental implants. [8]

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5 Zirconia Highly biocompatible,

high strength and fracture toughness, outstanding slow crack growth resistance, low thermal conductivity, high ionic conductivity, attractive biocompatibility, and chemical inertness

Femoral heads; dental implants, abutments, and dental crowns.

[11][8]

Hydroxyapatite Bioactivity, biocompatibility, mechanical strength, porous structure Similarity to the inorganic component of bone matrix

Replacement for bony and periodontal defects, alveolar ridge, middle ear implants, tissue engineering systems, drug delivery agent, dental materials and bioactive coating on metallic osseous implants.

[8,12]

Metals

Stainless steel Corrosion resistance, high strength, and elongation

Bone curettes, chisels, and gouges, dentistry and scalpels, Solid handles for instruments, guide pins, needles, steam sterilizers, storage cabinets, hip implants, stents, and knee implants.

[8]

Cobalt-chromium alloys

Corrosion resistance, high elastic modulus, excellent radial strength, ability to make ultra-thin struts, radiopacity

Partial denture, dental implants, and

maxillofacial implants, fracture fixation plates and screws, hip and knee prosthesis, stents.

[8,13]

Titanium alloys Superior tensile strength and

fatigue strength, chemical stability (corrosion resistance), and biocompatibility

under in vivo conditions

Pacemaker cases, housings for ventricular-assist devices, implantable infusion drug pumps, dental implants, maxillofacial and craniofacial implants, screws, and staples for

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spinal surgery, Total joint replacement arthroplasty primarily for hips and knees, Femoral hip stems, fracture

fixation plates, spinal components, fasteners, nails, rods, screws and wire, stents.

Polymers

Polyurethane Nontoxic, biocompatible,

in vivo calcification, wide

range of mechanical properties

Heart pacemaker, facial prostheses, blood bags, Blood vessels

prostheses, artificial hearts, bone

regeneration.

[6,8,14]

PTFE Chemical stability, high heat resistance, strong hydrophobicity, and high fracture toughness

Blood vessels

prostheses, prosthetic heart valves, ligaments, shunt tips, and artificial ossicles for the ear.

[6,8]

Polyester Outstanding strength, high strength-to-weight ratio, chemical resistance, ductility, stiffness, and hardness Cardiovascular implants, sutures. [8,10,15] Biodegradable polymers - PLA - PGA - PCL Biocompatibility, controllable biodegradability, relatively good processability

Stents, wound closure, sutures, drug delivery, ligament/tendon prostheses. [9] Biopolymers - Polysaccharides (chitosan, dextran, etc.) - Collagen

Nontoxic, wound healing properties, porosity, tunable properties

Tissue engineering (bone, cartilage, skin, blood vessel, nerve), delivery systems, surgical sutures, dental implants, encapsulating material, antibacterial coatings, wound healing.

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1.2 Surface phenomena

All materials are subject to certain surface phenomena when exposed to the surrounding environment. These phenomena can be classified as physical, chemical, physicochemical or biological ones. Some phenomena of interest will be first discussed, then the complications that they induce, and finally approaches to avoid these phenomena will be explored.

When a biomaterial is implanted, the first phenomena occurring are water and protein adsorption which then trigger a cascade of biological events resulting finally in the body response towards this foreign material [10]. In fact, depending on their conformation, protein adsorption will initiate other bioadhesions and influence subsequent events as cells, platelets and bacteria adhesion. Proteins are macromolecular chains of amino acids covalently linked together through a linkage known as peptide bonds [18]. Figure 4 shows the structures of proteins. The sequence of amino acids determine the primary structure of a protein, i.e., the linear sequence of amino acids is specific to a particular type of protein. Due to this sequence, the protein chain can bend or coil resulting in a specific three-dimensional conformation, which is called the secondary structure of the protein [10]. Hydrogen bonding is the responsible for this structure. The tertiary structure is formed by the folding of the secondary structure. Due to this folding, amino acids of distant sections of the protein will interact resulting in a structure that maximizes the exposure of polar groups to the aqueous environment while minimizing the exposure of hydrophobic groups. Four types of interactions play the main role to obtain the tertiary structure: covalent disulfide bonds, ionic interactions, hydrogen bonds, and hydrophobic interactions. The properties of the protein which determine the interaction with the surfaces are related to the primary structure, i.e., the amino acid sequence is crucial for the interaction behavior between protein and surface. Protein size, general charge, and stability of secondary and tertiary structures are factors that influence protein adsorption as well [10]. Furthermore, adsorbed proteins may affect the surface properties of the material, its mechanical properties making it stiff or fragile, for instance, up to induce its degradation.

Electrolytes, minerals carrying positive or negative charges such as sodium, potassium, calcium, chloride and phosphate (Na+, K+, Ca2+, Cl-, and PO43-), present in the

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body fluid can influence some surface phenomena. Electrolytes can influence the interaction of biomolecules with the surface, either enhancing or hindering it [10]. They can also chemically alter the composition in the surface of the biomaterial, leading to fractures or corrosion (Figure 5). For instance, metallic materials in contact with body fluids can suffer from corrosion, which will reduce the biocompatibility of the device, risking its lifespan while releasing corrosion products. The corrosion products can affect the function of cells in the vicinity of the biomaterial depending on the type and concentration of the products released. Moreover, cells far from the biomaterial can be affected due to the transport of corrosion products inside the body [10].

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Figure 6 – Steps of the bacteria adhesion and biofilm formation.

Figure 5 – Effects of electrolytes in the surface of a stent. (A) Wire fracture; (B)

pitting corrosion; (C) pitting corrosion and fracture. [20]

Bacteria can adhere to the surface and proliferate, forming a source of contamination. The adhesion of bacteria and colonization involve some steps (Figure 6): (1) the bacterium approaches the surface through Brownian motion or using flagella. As it approaches the surface, forces of attraction and repulsion, such as Van der Waals, hydrogen bonding, electrostatic and hydrophobic interactions come into play; (2) Once the bacterium overcomes these forces, adhesins present in the cell wall begin to regulate adhesion on the surface; (3) There is an increase in bacterial density, either by proliferation or recruitment of other cells; (4) The colony starts to produce a matrix of polysaccharides permeated by water channels through which nutrient exchanges and removal of metabolites take place [17]. From this point, the biofilm is formed [17].

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1.3 Interactions versus complications

As seen previously, the first events that take place after implantation are the interaction of ions (electrolytes) and proteins from the biological environment with the material surface leading to the formation of a protein layer. This last one will influence, at the short term, cell attachment, and at the long-term the host response towards this foreign material, including complications. Figure 7 presents some interactions that could arise from the contact between the material and the organism. Some complications may arise when specific surface phenomena take place.

The thrombogenicity, i.e., the properties of a surface to induce the thrombus formation, is a complication that impairs the well-functioning of blood-contact devices. When a foreign body is in contact with the blood, proteins will adsorb and platelets can adhere onto the surface, triggering off the coagulation cascade (Figure 8) [10]. This phenomenon is mediated by a series of enzymes and proteins, ions Ca2+_{, and platelets itself.}

In the first moment, coagulation factors adsorb onto the surface and are activated. Then, in the presence of Ca2+, they bind to the phospholipids present in the cell membrane of platelets. From this point, thrombin is activated to cleave fibrinogen in fibrin and the reaction follows in a cascade, forming a thrombus in the end [10].

Figure 7 – Possible interactions of the contact between the material and the tissues.

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As the physiological environment can be modeled as an aqueous solution at 37°C and pH 7.3, with dissolved gases, electrolytes, proteins, and cells, implanted metals are susceptible to either pitting or crevice corrosion [10]. The ionic concentration, the presence of chlorides, known to be very aggressive to metals, and the concentration of proteins in the body fluid are factors that highly influence the corrosion of metals in the human body.

Calcification, or biomineralization, is a process where deposits of calcium phosphate, or other calcium salts, are formed. It is considered normal when it occurs at

Figure 8 – Coagulation cascade. FV, FVII, FIX, FX, FXI, and FXII are coagulation

factors, enzymes and proteins that regulate the coagulation cascade. The index ‘a’ indicates that the factor is activated.

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expected sites, such as bones, teeth, and osteoinductive materials, and abnormal, or pathological, when it occurs in soft tissues and implants where calcium salts are not expected to be deposited [21]. Pathological calcification can be classified as (a) dystrophic, when it occurs in biomaterials or injured tissues, in individuals with normal calcium metabolism; or (b) metastatic: when it occurs in initially normal tissues due to abnormal metabolism of calcium. The conditions favoring both types can act synergistically, that is, under conditions of abnormal calcium metabolism, the calcification in a biomaterial can have its velocity accelerated [21]. The mineral phase formed is a low crystalline calcium phosphate called apatite, whose chemical formula is Ca5(PO4)3(OH, F, Cl), a very similar

compound with calcium hydroxyapatite, a mineral responsible for promoting structural stiffness of bones and which has chemical formula Ca10(PO4)6(OH)2 [21].

In biomaterials, calcification is responsible for failures, compromising the performance of the device and reducing its lifetime [22]. Among the devices impaired by calcification are biological and synthetic heart valves, artificial hearts, ventricular assist devices, urinary prostheses, intrauterine devices, gelatin contact lenses, and artificial bladders [21,23].

The adhesion of bacteria is a concern that has drawn the interest of researches in the recent years. Adhesion of bacteria can lead to biofilm formation [24]. Biofilm, a viscous layer of bacteria and an exopolysaccharides matrix, is commonly related to chronic pathological conditions [25]. Once formed, the removal of biofilm is very difficult. The bacterial contamination can lead to the arising of diseases that patients can develop during the time they spend in a healthcare facility receiving treatment to other diseases [25,26]. This kind of disease is called nosocomial infection, hospital-acquired infection or healthcare-associated infection (HCAI). HCAI is defined as a non-preexistential infection that a patient develops after hospitalization or procedure in a healthcare facility, i.e., the infection was not present or incubating at the time of admission [26]. HCAI increases medical costs, length of stay, complication rates, and overall morbidity and mortality [26]. It has become a very important issue in recent years. HCAI caused by methicillin-resistant

Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus (VRE), and Clostridium difficile are becoming progressively contagious and hard to treat [26].

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According to WHO, the worldwide real burden remains unknown due to the inexistence of surveillance systems in low- and middle-incoming countries, although data collected by studies estimate that hundreds of millions of patients are affected each year [27]. The estimated incidence of HCAI is 4.1 and 1.7 million affected patients in Europe and the USA, respectively, each year. In Canada, it is estimated that one in every nine patients contracts HCAI, resulting in more than 220,000 cases, with 8,500 to 12,000 deaths, being the fourth leading cause of death in Canada [28,29]. These infections result in an increased cost of around $1 billion yearly [29].

1.4 Approaches

Over the last decades, several different strategies are applied to address the complications that arise from the contact of the material with the body. These strategies could be divided in four main categories as shown in Figure 9. The first one is related to the change of the surface wettability, i.e., the ability of the surface to spread or repel a liquid that came in contact with. This strategy aims to change the wettability of the surface to obtain a surface more hydrophobic or more hydrophilic according to the target application. The second one is the surface charge. Herein, the objective is to regulate the global charge net of the surface by introduction of charges that will either attract or repel ions and molecules. The third strategy is the chemical modification of the surface. In that approach, the surface is modified by the introduction of specific chemical groups or molecules to overcome some limitations, to increase surface interactions with surrounding tissues or to give added properties. The last strategy is related to the surface topography, known to strongly impact protein/cell adhesion onto the surface. Therefore, in this strategy, the surface is tailored to enhance a desired response or to avoid an undesired effect. The control of the biomedical device surface roughness or morphology could be done by physical modification, polishing or blasting, by physical/chemical techniques such as plasma treatment, anodization, and chemical modifications such as acid etching.

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As presented before, when a device comes in contact with the blood, the coagulation cascade can be triggered. One way to prevent, or at least reduce, the thrombogenicity of the surface is to coat the device with a more hemocompatible substance playing the role of a bio-interface [30]. Concerning the interactions with blood, these coated devices should limit the adsorption of proteins, which induce platelet adhesion (fibrinogen and thrombin), or prevent the adhesion and/or activation of platelets. Moreover, the surface could adsorb antithrombin III, a serum protein that inhibits thrombin expression [31,32]. The interaction with proteins can be controlled through a modification of the surface charge through the introduction of negatively charged species. This approach aims to repel blood proteins, such as fibrinogen, reducing the positive charges of the surface, mimicking the same effect promoted by heparin [33]. Another way to improve the antithrombogenic property of the surface is changing its topography. Micropatterned surfaces could lead to a surface where endothelial cell could have more affinity, which could mean a better endothelialization and, thus, a less thrombogenic surface [34]. The use of hydrophobic surfaces has been reported. Studies that use this approach reports that

Figure 9 – Strategies applied to the surface to control the complications of the material-tissue interactions.

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hydrophobic surfaces inhibit the platelet adhesion and activation which may impair the clot formation [35].

To avoid the corrosion of metals, they can be pre-passivated by the use of an electrochemical anodizing process [36], or by an electropolishing method [37]. Another strategy applied is to coat the metal with another substance that will limit the corrosion. The use of inorganic coatings (plating, painting, enamel) is much employed worldwide [38]. It is a highly effective method to protect the metal due to the formation of a barrier, which will provide physical and chemical protection against the corrosion environment [39]. Organic coatings can inhibit selectively several factors influencing the corrosion, such as water permeability and ions/oxygen diffusion [38]. They will act through several mechanisms, such as barrier effect, inhibitive effect, anodic protection, and cathodic protection [40].

The need to obtain a surface free of calcification has motivated researches seeking the development of a treatment that inhibits calcification [41]. Researches have been made to inhibit the calcification in glutaraldehyde-treated bovine pericardium through the modification of the surface topography. In these researches, polymers were used to fill the spaces that could be sites of calcium deposition [42]. The samples treated with the polymers did not show any sign of calcification even after being immersed in a calcium-rich solution [43]. The insertion of groups that present anti-calcification properties is a strategy well explored. Several studies have dealt with the anti-calcification effects of sulfates and cations [22,41,44–48]. In this approach, cations are used to prevent the calcification through a mechanism of repulsion of ions Ca2+ [22]. While sulfate groups are expected to promote the calcification, attracting more calcium due to the electrical attraction, this phenomenon is not observed. Indeed, heparin, chondroitin sulfate, and PEO-SO3 are described in the literature as compounds able to retard the calcification [47]. The

mechanism of action proposed is that sulfate group, having strongly acid nature, could lower locally the pH, resulting in an acidic environment in a micro-region, which could dissolve the metastable calcium deposits, retarding the formation of the calcification nucleus in the early stages of the process [45,47].

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Figure 10 – Antibacterial mechanisms related to the use of polymer surfaces.

The approaches previously presented can be also used to combat the HCAI. Nowadays, in order to avoid, or at least minimize, the cases of HCAI, hand-washing is the strategy that has been applied by healthcare employers worldwide [27]. Although it is crucial to control and prevent infections, it does not avoid the problem of crossed contamination. As surfaces are the main source in the HCAI spread and contamination, an employee that has cleaned his hands can contaminate a patient when he touches a surface that is already contaminated [17]. From the surface point of view, the bacteria contamination could be minimized by a modification in the charge net or in the wettability [49] of the surface or by the introduction of chemical species that can impair the bacteria adhesion or even kill it [50,51]. The antibacterial surface could also be made through a modification of the surface topography, resulting in an antifouling surface [52]. Surfaces coated with a long-lasting polymer have been used in many researches exploring these approaches to address to this problem. In this case, the polymer needs to present non-toxicity, stability, and antibacterial property. The antibacterial mechanisms associated with the use of polymers are presented in Figure 10.

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Chapter 1 The

doctoral

research

project:

Approach and objectives

The main objective of this research project is to design of chitosan-based coatings to be used in the coverage of metallic surfaces and devices found in the healthcare field. The development of such coating has been focused on its coating adhesion and its easy processability for metallic substrates. Therefore, the choice of the substrate, its preparation, the coating chemical nature, as well as the approach to link it on the surface were key parameters. To validate and optimize the developed methodology, the coated surfaces were characterized according to the chemical composition, the roughness, and the wettability and then submitted to physicochemical and biological assays to evaluate their cytotoxicity, their antithrombogenicity, their chemical stability in simulated physiological medium, as well as their calcification and corrosion resistances. The Figure 11 presents a general scheme of the project and the next sections will present the previous mentioned elements involved in the accomplishment of this project.

1.1 Objectives and thesis structure

The general objective of this project was to develop bioactive coatings with biomolecules in order to obtain an adjustable surface with targeted functionalities whether it is a hemocompatible or antibacterial surface. To answer this main goal, natural molecules, such as catechol derivatives and chitosan, have been chosen. Indeed, thanks to catechol adsorption onto metallic surfaces, to lead to reactive terminal functional groups used as anchor points for further grafting, and thanks to catechol and chitosan molecules inherent biological properties, the biopolymer layer, catechol/chitosan, display an attractive platform that can be used in many types of application.

For that purpose, metallic surfaces were functionalized by using catechol derivatives, herein dopamine and caffeic acid, in order to form an organic interface suitable for the grafting of chitosan. Thereafter, the as-coated surface has been modified according to the desired application.

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a) Graft chitosan, using dopamine and PEG diacid as anchors, on metallic substrates and evaluate the biological properties, such as calcification, platelet adhesion and clotting time. For this, both native and sulfonated chitosan will be grafted. Thanks to sulfonate groups, it is expected an improvement of antithrombogenic and anti-calcification properties when compared to native chitosan grafted surface.

b) Graft chitosan, using caffeic acid as anchor, on metallic substrates through two different approaches and compare both surfaces with the previous one. An extensive surface characterization will permit evaluating the strengths and weakness of the surfaces produced with caffeic acid. It is expected the obtainment of a thinner and homogeneous surface, with no need for a linking arm as with dopamine approach.

c) Modify the chitosan surface previously obtained to improve the biological response, such as endothelialization and antibacterial activity. The surface was modified

Figure 11 - General schematic approach of the project. The three steps present how the

surfaces were produced/modulated, the characterization techniques and the mechanical /biological tests performed.

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with ammonium quaternary, to strengthen the antibacterial activity of the chitosan. It is expected that the insertion of ammonium quaternary will enhance the antibacterial activity of the chitosan layer.

The first step was the grafting of chitosan using dopamine as an anchor. This step is made to obtain a stable and homogeneous surface using the well-known polydopamine chemistry. This surface will be the positive control for the following step. In general lines, this step is represented in Figure 12.

After each modification step (Figure 13), the samples were analyzed by XPS, to assess its atomic surface composition, AFM, to determine the topography and roughness of the coating, and contact angle, to evidence changes in the wettability of the surface. As mentioned before, surface composition, morphology and wettability play also a crucial role in the biological answer. Therefore, it is important to assess all these surface properties.

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For this first approach, electropolishing was performed on stainless steel substrate in order to obtain a smooth metal surface, removing imperfections and non-metal compounds, and to generate a rich layer of oxides and hydroxides [53]. Also, this procedure allows to obtain reproducible samples, regarding the surface composition and topography. Then, amination of the metallic surface was performed by using polydopamine as an anchor point for further grafting. Due to the nucleophilic nature of chitosan, a linking arm is needed to promote the link between the amine groups from chitosan and dopamine. Poly(ethylene glycol) bis (carboxymethyl) ether (PEGb), a bifunctionalized PEG, was chosen to play this role. Natural and sulfonated chitosan were used to produce the chitosan coatings. The natural chitosan presented a well-known thrombogenicity while the sulfonated one was expected at least to decrease or at best to avoid this behavior. Thus, in this step, natural chitosan was chosen as the positive control. Biological tests were performed in order to evaluate the capacity of the as-coated surface to reduce both calcification and thrombogenicity.

In the second step, dopamine was substituted by caffeic acid to graft the chitosan onto the surface. As caffeic acid has already a carboxylic acid function, after its activation by EDAC leading to a stable isourea active intermediate (Figure 14), the chitosan grafting can be directly done. Thus, by this original approach, to the best of our knowledge, there is no need to use a linking arm to promote the reaction with the amine group of the chitosan. Furthermore, two different grafting ways were investigated (Figure 15). The first one is the grafting of the caffeic acid on the chitosan chain followed by the grafting onto the surface (grafting to). The second one is the grafting of the caffeic acid first onto the surface

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following with the grafting of chitosan onto the activated surface (grafting from). All obtained surfaces were fully characterized, biological properties were evaluated, and the performance of the two caffeic acid procedures were compared. Besides, by using caffeic acid approach instead of dopamine one, it is expect to achieve a more uniform, homogeneous and thinner coating which can be used as a new platform for tunable chitosan-based coating.

As stated previously, caffeic acid used as grafting agent onto surface is, to our best knowledge, an original approach, as caffeic acid is more used as antioxidant or crosslinker agent. Therefore, a grafting optimization was needed, and has been done based on the XPS high resolution O 1s spectrum, where the changes of the oxygen content on the organic ring, responsible for the adhesiveness of the catechol gave a more accurate indicative of the caffeic acid grafting efficiency.

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The last step was the modification of the chitosan layer to modulate the response of the surface. Due to the presence of amine groups in carbon-2 and hydroxyl groups in carbon-3 and carbon-6, chitosan can be promptly modified to produce derivatives with the desired properties. Using this easiness, the surface properties can be modulated, i.e., the surface obtained is modified in order to confer or enhance a biological response expected for the function it will perform. Thus, following the development of the grafting process, the surface was modified to enhance its properties, i.e., the antibacterial properties of the chitosan. This step is shown in Figure 16.

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1.2 Materials

This section describes the three main components used to obtain the coatings, i.e., the metallic substrates, the catechol derivatives responsible to covalently link the organic interface to the inorganic surface, and the biopolymer, herein chitosan, that will play the role of organic interface with the surrounding tissues when contact with human body.

1.2.1 Stainless steel 316L

Stainless steels are Fe-based alloys that present a high resistance to oxidation due to the presence of Cr in its composition, which forms an oxide layer mainly composed by Cr2O3. Several types of stainless steel are used for the fabrication of implant devices,

surgical instruments, and surfaces in general. Stainless steel 316L (SS 316L), which differs

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from the 316 type by the lower concentration of carbon, is the most used metal for medical applications, such as cardiovascular, orthopedics, dentistry, and others, due to its biocompatibility, malleability, resistance to corrosion and fatigue, work hardenability, ductility [54,55]. Moreover, stainless steel has a low cost and good availability, compared to titanium and Co-Cr alloys [54]. The SS 316L composition [56] is presented in Table 2.

Table 2 - Stainless steel 316L composition

Element C Cr Ni Mo Mn Si P S Fe

% Mass < 0.03 16-18.5 10-14 2-3 < 2 < 1 < 0.045 < 0.03 Bal.

Although it presents high corrosion resistance, the contact with body fluids can cause corrosion, which could lead to a loss of mechanical properties and the release of metallic compounds, such as nickel and molybdenum. Techniques based on surface coating can be applied to increase the corrosion resistance of the stainless steel. These techniques normally employ polymer solutions. Table 3 resumes some of these techniques.

Table 3 - Surface coating techniques

Surface coating techniques

Description Ref

Simple adsorption The simplest technique to coating a surface. The solution is cast over the surface and left to evaporate the solvent.

[17,57]

Dip coating It is a simple and cost-effective technique. It consists in a three-phase process. First, the material is immersed into the solution and left in contact with it to interactions between the surface and the solution. Then, the substrate is removed from the solution at constant speed and the solution in excess is drained. Last, the solvent evaporates from the surface to form a thin film.

[17,58]

Layer-by-layer It is a dip coating where the substrate is dipped alternately in polyelectrolyte solutions with opposite charges. The surface is washed between each dip phase.

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Spray coating This technique employs a gas flow to atomize the solution into small droplets that will be deposited over the surface forming a homogeneous film.

[17,40]

Spin coating In this technique, the solution is casted over the surface that is submitted to a rotation at high speed. The solution is uniformly spread over the surface and the excess is ejected. After evaporation of the solvent, it forms a thin and uniform film.

[17,60]

Plasma coating This technique uses highly ionized gases to deposit ultrathin adherent films over the substrate.

[17,61]

Covalent coating This technique uses a molecule that covalently bind to the surface forming a layer that can be further modified or functionalized. One group of molecules that can play this role is the catechols.

[17,62]

1.2.2 Catechol derivatives

A way to ensure the stability of the bioactive coating is to covalently link it to the substrate. The covalent approach can lead to an attachment 10 fold stronger when compared to the adsorption method [63]. To ensure this covalent attachment of the coating, it is necessary to find a molecule capable of anchoring to the metallic substrate as well as reacting with the chitosan. For that purpose, catechol molecules seem very suitable, due to their multi-functionality [64]. In addition, catecholic compounds are natural as they were found in fruits and vegetables such as onions, apples, and raw beet sugar, but also in trees, such as pine, oak, and willow. It was first isolated in 1839 by H. Reinsch by distilling catechin [65,66]. Also, catechols can act as antioxidants and chelating agents [67].

Catecholic compounds, which are ortho isomer of benzenediol derivatives, can adhere to a vast range of substrates, including mica, titanium, and even poly(tetrafluoroethylene), known for its antiadhesive property [68,69]. The exact mechanism of adhesion is not fully explained. Several types of research have proposed different forms of interaction between the catechol group and the surface (Figure 17). The

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Figure 17 – Adhesiveness mechanisms of catechol group. (a) Bidentate chelating

bonding; (b) bidentate bridging bonding inner sphere; (c) bidentate bridging bonding outer sphere. Adapted from .

most common mechanisms reported are bidentate chelating bonding (Figure 17a), where both hydroxyls bind to a single atom, and bidentate bridging bonding, where each hydroxyl do a bond with two different atoms. The last one can be divided into the inner sphere (Figure 17b) and outer sphere (Figure 17c), depending on the nature of the substrate. In the inner sphere, there is a covalent linkage between the catechol and substrate. In the outer sphere, the species remain separated, maintaining its structure intact during the electron transfer. Other mechanisms include monodentate bonding, a mix of monodentate/bidentate bondings and adsorption.

In 1981, a survey conducted by Waite and Tanzer identified catechol as the main responsible for the adhesion of mussels in the intertidal and breakwaters areas [67,68]. A detailed study of byssus proteins, the structure used by the mussels to adhere to rock, showed that in six of them it is possible to find 3,4-dihydroxyphenyl-L-alanine (DOPA), with molar percentages ranging from 3 to 30% [67]. The decarboxylation of DOPA leads to the obtainment of dopamine, an amine function of catechol group.

[70]

Several studies of surface modification use dopamine hydrochloride [53,71–74]. In alkaline conditions, the catechol is oxidized to quinone form. This oxidized form enables catechol to react with quinone itself (autopolymerization), thiol, and amine groups, by Michael addition or Schiff base reaction to produce covalently functional layers [75]. Dopamine can polymerize on various substrates, including noble metals, inorganic materials (SiO2 and Al2O3), organic polymers (polyethylene, polystyrene, polyethylene

Design, development, and validation of chitosan-based coatings via catechol chemistry for modulating healthcare materials

© Clayton Souza Campelo, 2020

Design, development, and validation of chitosan-based

coatings via catechol chemistry for modulating

healthcare materials

Thèse

Clayton Souza Campelo

Doctorat en génie des matériaux et de la métallurgie

Philosophiæ doctor (Ph. D.)

Design, development, and validation

of chitosan-based coatings via

catechol chemistry for modulating

healthcare materials

THÈSE

Doctorat en Génie des Matériaux et de la Métallurgie

Clayton Souza Campelo

Sous la direction de :

Diego Mantovani, directeur de recherche

Rodrigo Silveira Vieira, directeur de recherche

Résumé

Abstract

Contents

List of tables

List of figures

List of abbreviations

Acknowledgments

Foreword

Introduction

Chapter 1

The

doctoral

research

project:

Approach and objectives

1.1 Objectives and thesis structure

1.2 Materials

1.2.1 Stainless steel 316L

1.2.2 Catechol derivatives