Renée El

Melhem Bat

Blaise Pascal

Insa : R Gourdon

M Stéphane

M Gérard Scorletti
email: gerard.scorletti@ec-lyon.fr

J Y Toussaint

M Christian Montes

Ana-Maria Stanciuc

Quentin Flamant
email: quentin.flamant@upc.edu

Katia Biotteau-Deheuvels

Martin J Stoddart

Marc Anglada
email: marc.j.anglada@upc.edu

Alessandro A Porporati

Meinhard Kuntz

Laurent Gremillard

Mauro Alini

Dr Marianna Peroglio
email: marianna.peroglio@aofoundation.org

Christoph Martin Sprecher

Jérôme Adrien

Lucian Ioan Roiban

In vitro effect of micro-and micro-/nano-roughness of zirconiatoughened alumina Human primary osteoblast behaviour on micro-rough zirconia-toughened alumina and selectively etched micro-rough zirconia-toughened alumina

I would to take the chance to acknowledge the people without whom this thesis would not have happened.

First I would like to say a big thank you to Marianna Peroglio for her disponibility, her advices, her guidance and her confidence in my quest till the end of this journey. Working with her it was indeed a life worth experience, which I will always benefit upon my further career. Her scientific polivalence was a model that guided me along the thesis.

I am grateful to have been part of Mauro Alini's group and for his willingness to share his broad knowledge in the musculoskeletal regeneration field. His constant care for the life of each member of the group, along with the great memories from the group activities will always be proof of a model leader.

Nevertheless I would like to show my gratitude to Laurent Gremillard for his patience in broadening my horizont in the materials science field. Also along Laurent I would like to thank Jerome Chevalier for hosting me in MATEIS laboratory.

I would like to acknowledge Meinhard Kunz and Alessandro Porporati for the introduction to the different world of industry at Ceramtec and Katia Biotteau for the warm welcome and fruitful internship.

Secondly I would like to acknowledge Eduardo Saiz and the European Union for the management and financement respectively of an excellent consortium within which the main focus was on the continuous training and collaboration.

To continue I would like to mention my recognition and gratitude to the rapporteurs and members of the PhD committee for taking the time to read my thesis and to offer constructive comments as well as their presence during the defense.

Along the four years I had the chance to grow together with my AO family Marina, Dalila and GJ. I would have never make it without you. Thank you for the great memories from Davos to Spain, Italy, Netherlands and Thailand.

One of the things that I became to appreciate a lot was the Lunch Group which gave me the chance to link with people from all over the world. It was great meeting you: André, Fatemeh, Ryan, Ugo, Yishan, Fabrizio, Marta, Shan, Marto.

Here I would also like to thank Janek and Weronika Barcik that have become very close friends in my last year of PhD. I am very happy to have had the nice trip to Netherlands and enjoy good times in Davos. I hope we will be able to meet in the future as well.

At the end I would like to thank my family and Fabian for all the support and understanding especially in the last months.

Table of contents

Préface en français

L'os est un tissu hautement spécialisé avec un potentiel de régénération élevé lié au processus de remodelage osseux. Néanmoins, des implants et des prothèses osseux sont nécessaires en cas de traumatisme grave ou de troubles dégénératifs tels que l'ostéoporose.

L'osseointégration de l'implant est requise pour assurer la stabilité à long terme et la fonction réparatrice sur le site d'implantation. Aujourd'hui, le marché des implants dentaires et orthopédiques est partagé entre le titane commercialement pur et les alliages de titane. Les céramiques ont été récemment introduites, avec la zircone et les composites alumine-zircone comme principales alternatives aux métaux. À l'heure actuelle, les prothèses biocéramiques contiennent des pièces métalliques pour garantir une osséointégration adéquate. Néanmoins, une solution entièrement céramique ouvrira de nouvelles possibilités dans la conception des implants. Par exemple, dans le cas de la prothèse de la hanche, une solution complètement céramique libérera de l'espace pour une tête fémorale plus grande, réduisant ainsi le risque de dislocation et augmentant l'amplitude de mouvement de la prothèse, avec un grand bénéfice pour le patient.

Afin d'améliorer les propriétés osseointegratives, les caractéristiques de surface des implants sont considérées avec un grand intérêt car la topographie, la rugosité et la chimie de la surface auront une influence sur l'énergie de surface, la mouillabilité et plus encore les protéines adsorbées, le nombre et le type de cellules attachées, le type de matrice déposé par les cellules et finalement la réussite de l'implant. Les implants dentaires (du type à vis) varient en termes de rugosité de surface dans un spectre de rugosité de surface (S a ) de ~ 1-2 µm. En ce qui concerne le marché des implants dentaires en céramique, moins de 2% des implants dentaires globaux sont basés sur la zircone, mais il est prévu que les implants à base de zircone atteindront 6% du marché d'ici à 2020. Pourtant, les implants orthopédiques en céramique (têtes et cupules cotyloîdiennes de prothèses de la hanche) ont une part beaucoup plus importante sur le marché (et notamment le composite alumine-zircone -Biolox® depuis 1974).

En raison de leur "bio-inertie" intrinsèque, les céramiques telles que la zircone ou les composites alumine-zircone ("zirconia-toughened alumina" ou ZTA) ont besoin de modifications de surface qui permettront des changements de rugosité de surface, de chimie et d'énergie capables de guider le comportement des cellules et plus loin l'osséointégration.

Peut-être un des facteurs limitants pour l'avancement de nouvelles solutions dans le domaine des implants orthopédiques et dentaires est une faible caractérisation de la surface. Pour une meilleure corrélation entre les paramètres de surface et le comportement des cellules, la caractérisation 3D de la surface est préférrable au R a (rugosité moyenne arithmétique du profil) couramment utilisé, car les surfaces ayant les mêmes valeurs de R a peuvent grandement varier en morphologie spécifique de surface (hauteur et espacement des pics et Par conséquence, nous avons considéré un (vi) un dépistage rapide des interactions cellulesmatériau. Deux approches différentes ont été sélectionnées pour la modification de la surface de la zircone et les cellules souches mésenchymateuses (MSC) ont été utilisées pour évaluer les changements morphologiques précoces associés à différentes lignées de différenciation cellulaire. Le micro-usinage par laser femtoseconde a été utilisé pour introduire de multiples motifs sur un seul échantillon de zircone, tandis que des échantillons de zircone avec un gradient de rugosité ont été obtenus par un contrôle continu du temps d'attaque chimique.

En ce qui concerne l'effet de rugosité de surface sur le comportement des hOb, nous avons constaté que la micro-rugosité seule avait des effets mineurs et cela pourrait être lié à la méthode de fabrication (moulage par injection avec des moules rugueux) qui affecte exclusivement les paramètres de rugosité. En revanche, les surfaces de ZTA avec une combinaison de micro-et nano-rugosités ont considérablement amélioré la maturation des ostéoblastes primaires humains par rapport aux surfaces micro-rugueuses. En effet, une augmentation de l'expression de marqueurs de différenciation ostéogénique tels que la phosphatase alcaline a été observée tant au niveau du gène que de la protéine. L'effet synergique de la micro-et nano-échelle peut être augmenté par des modifications au niveau chimique dues à l'attaque acide (précipitation de cristaux YF 3 ).

Lors de l'application de techniques de prototypage rapide aux matériaux céramiques, le premier défi rencontré dans le procédé est l'optimisation de l'encre. Une encre composée de zirconium-alumine a été optimisée et utilisée avec succès pour imprimer des structures 3D.

Les premiers essais ont montrés que les ostéoblastes primaires humains peuvent s'attacher aux structures 3D préparées par robocasting. Néanmoins, une optimisation supplémentaire de l'ensemencement cellulaire et des conditions de culture à long terme s'impose.

Le dépistage rapide des interactions entre les cellules et les matériaux peut être un bon outil pour évaluer différentes conditions avant de passer à des études de culture cellulaire plus étendues. L'usinage de multiples motifs ("multipatterning") par laser femtoseconde et l'utilisation de gradients d'attaque chimique ont permis de produire des échantillons pressentant des conditions de rugosité variées. Des analyses morphologiques des cellules souches mésenchymateuses ont été utilisées pour évaluer le potentiel de différenciation ostéogénique. Des changements significatifs ont été apportés par les différentes surfaces de zircone modifiées par laser et par attaque chimique. Les deux approches ont montré des résultats prometteurs pour le dépistage rapide des interactions entre les cellules et les matériaux.

Les différentes stratégies abordées le long de la thèse ont été possibles en raison d'une excellente collaboration au sein du "Biobone" ("Bioceramics for Bone Repair"), un Consortium européen de type ITN (Réseau de Formation Initiale) FP7 Marie Curie, axé sur la formation des ESR (chercheurs débutants, à savoir doctorants) et ER (chercheurs expérimentés, à savoir chercheurs post-doctorants).

Les partenaires universitaires et industriels ont combiné les connaissances et les ressources en

Preface in English

Bone is a highly specialized tissue with high regenerative potential linked to the bone modelling/remodelling process. Yet, bone implants and prostheses are required in cases of severe trauma or degenerative conditions such as osteoporosis. Osseointegration of the implant is required to provide long-term stability and restorative function at the implantation site. Today, the dental and orthopedic implants market is shared between the commercially pure titanium and titanium alloys and more recently introduced ceramics, with zirconia and alumina-zirconia composites being the main alternatives. Currently, bioceramics are combined with metals to guarantee adequate osseointegration. Nonetheless, an all-ceramic solution will open up new possibilities in implant design. For instance, in the case of hip prosthesis, an all-ceramic solution for the acetabular component will free up space for a bigger ball, thus reducing the risk of dislocation and increasing the range of motion of the prosthesis, with great benefit for the patient. Ceramic orthopaedic implants (heads and cups of hip prostheses) have a large share of the market (alumina-zirconia composite -Biolox® since 1974). Concerning the ceramic dental implants market less than 2% of the global dental implants are zirconia-based (the rest being Titanium-based), but it is forecasted that zirconiabased implants will reach 6% of the market by 2020. In order to improve osseointegrative properties, implants surface characteristics are regarded with great interest since surface topography, roughness and chemistry will affect the surface energy, the wettability and further the protein adsorbed, the number and type of attached cells, the type of matrix deposed by the cells and ultimately the implant success. For example, titanium dental (screwtype) implants vary in terms of surface roughness within a S a spectrum of ~ 1-2 µm.

Due to their intrinsic "bio-inertness", ceramics such as zirconia or composites (zirconia toughened alumina ZTA) need surface modifications that will allow changes of surface roughness, chemistry and energy able to guide cell behaviour and further osseointegration.

Perhaps one of the limiting factors for the advancement of new solutions in orthopaedic and dental implants field is a poor surface characterisation. For a better correlation between surface parameters and cell behaviour, 3D characterisation of the surface is preferred to the commonly used R a as surfaces with same R a values can greatly vary in surface specific morphology (height, valleys) with direct consequences on cell response. Therefore, a comprehensive surface characterization is crucial for better understanding the cues modulating cell response and standardization of the surface characterization will facilitate the comparison between studies. Furthermore, in vitro studies with a relevant cell type for the intended application, combining several types of analyses and time points contribute to the understanding and prediction of biological responses that occur in in vivo and further down the line in the clinical scenario. Additionally, testing of a wide range of surface conditions can benefit from rapid screening methods, such as samples with multiple patterns and samples with surface gradients.

This thesis aimed at evaluating the interactions between different cell types and ceramic surfaces. The focus was on zirconia and ZTA composite structures obtained by various techniques as injection moulding, isostatic pressing and robocasting. Different approaches were used for micro-and nano-scale roughness introduction. To sum up we guided the studies along several objectives: (i) identification of an optimal micro-roughness range of ZTA samples obtained by injection moulding into rough moulds and (ii) determination if further selective chemical etching of the micro-rough surface has a positive effect on human primary osteoblast maturation. Furthermore, we focused on (iii) optimizing a ZTA ink suitable for robocasting, (iv) characterizing the robocast 3D-scaffolds and (v) investigating human primary osteoblast behaviour on these ZTA scaffolds in vitro. Due to multiple parameters influencing cell behaviour, determination of a suitable condition requires a high number of specimens and can be time-consuming. Hence, we considered a (vi) rapid screening of cell-material interactions. Two different approaches were selected for zirconia surface modification and mesenchymal stem cells (MSCs) were used to evaluate early morphological changes associated with different differentiation lineages. Femtosecond laser micromachining was used to introduce multi-patterns on a single zirconia sample, while zirconia samples with a roughness gradient were obtained by a continuous control over the chemical etching time.

Concerning the surface roughness effect on hOb behaviour, we found that micro-roughness alone had minor effects and this could be linked to the fabrication method (injection molding with rough moulds) that affects exclusively the roughness parameters. On the other hand, combined micro-/nano-rough ZTA surfaces significantly enhanced human primary osteoblast maturation compared to micro-rough surfaces. Indeed, increased expression of osteogenic differentiation markers such as alkaline phosphatase were seen at both gene and protein levels. The synergic effect of micro-and nano-scale can be enhanced by modifications at the chemical level triggered by the acid etching (deposition of YF 3 crystals).

When applying rapid prototyping techniques to ceramic materials, the challenge firstly encountered in the process is ink optimization. Zirconia-alumina composite ink was optimised and successfully used to robocast 3D structures. Initial screening showed that human primary osteoblasts can attach on robocast 3D structures. Nevertheless, further optimization of cell seeding and long-term culture conditions is required.

Rapid screening of cell-material interactions can be a useful tool to assess different conditions before moving to more extensive cell culture studies. Femtosecond laser multipatterning and chemical etching gradients of zirconia surfaces lead to the production of single specimens with various roughness conditions. Mesenchymal stem cell morphological analyses were used to assess osteogenic differentiation potential. Significant changes on the different patterned laser-modified zirconia surfaces and gradient surface were obtained. Both approaches showed promising results for rapid screening of cell-material interactions.

The different strategies approached along the thesis were possible due to an excellent collaboration within the "Biobone" (standing for "Bioceramics for Bone Repair") European The thesis was part of the WP3 and close collaboration was successfully managed with:

 UPC Barcelona: Quentin Flamant as ESR and his supervisor Marc Anglada,  Ceramtec: Katia Biotteau as ER, and Alessandro Porporati and Meinhard Kuntz as supervisors.

As part of an ITN I had the chance to do a short term secondment at Ceramtec and a six months secondment at MATEIS, INSA Lyon. Both internships gave me the opportunity to learn more about the materials (processing and characterization) from a different perspective.

List of abbreviations

Rapport ALP/DNA sur (a) Différents échantillons d'alumine-zircone microrugueux et (b) Comparaison des échantillons microrugueux reçus, attaqués par acide fluorhydrique (HF) avec ou sans attaque chimique supplémentaire dans l'acide chlorhydrique (HCl): (b) poli, (c) comme fritté, (d) faible et (e) moyen et (f) haute rugosité de surface. A noter que seulement des différences minimales ont été observées parmi les différents groupes d'échantillons microrugueux, tandis que l'attaque chimique a entraîné une augmentation significative en ALP/DNA pour les hOb cultivés sur l'alumine-zircone comme fritté, faible

Index of tables

Alumina

High purity alumina (>99.5%) can be obtained from bauxite (hydrated aluminium oxide) and native corundum (aluminium oxide mineral). Bayer process is commonly used as refining process that yields α-alumina. Commercially available pure alumina normally contains 99.5-99.6% Al 2 O 3 , 0.06-0.12% SiO 2 , 0.03-0.06% Fe 2 O 3 and 0.04-0.20% Na 2 O with a density of 3.65-3.9 g/cm 3 . American Society for Testing Materials (ASTM) standard specification for high-purity dense aluminium oxide for medical application 3 specifies that biomedical alumina should be 99.5% pure with less than 0.1% combined SiO 2 and alkali oxides.

The mechanical properties of alumina are largely dependent on grain size, grain distribution and porosity. Alumina exhibits in general a high hardness, accompanied by low friction and wear, which are the main advantages in using alumina for joint-replacement implants.

Alumina brittleness is one of the main drawbacks that leads to the replacement of alumina on alumina implants.

Zirconia

Zirconium (Zr) belongs to the same subgroup as titanium in the periodic table and share resembling chemical properties. From a commercial perspective, zircon (ZrSiO 4 ) is the main source of zirconium mineral. Zirconia (zirconium oxide, ZrO 2 ) is predominantly found in baddeleyite. In clinics, yttria-stabilized zirconia appeared first in 1985 as alternative to alumina devices and was standardized in 1997 4 .

In terms of structure, zirconia exhibits three allotropes: monoclinic, tetragonal and cubic. The transition between these structural arrangements is temperature-dependent and may occur during the sintering step. In order to avoid the phase transformation and simultaneously maintain the properties obtained through sintering, an approach, first described by Ruff et al. 5 , consists in mixing zirconia with another oxide, which leads to partial or complete stabilization of cubic or tetragonal phase. Zirconia ceramics exhibit a unique strengthening mechanism through phase transformation toughening: in the presence of high stresses, such as the stress concentration occurring at the tip of a propagating crack, the metastable tetragonal phase can transform to the stable, monoclinic allotrope. The (~4%) volume increase accompanying the transformation superimposes a compressive stress field to the existing one, thus efficiently delaying crack propagation and toughening the material.

Yttrium-stabilized zirconia, in the form of 3Y-TZP (tetragonal zirconia polycrystal stabilised by 3 mol. % Y 2 O 3 ) is an interesting material for femoral heads of total hip joint prostheses.

When compared to alumina, two main advantages recommend Y-TZP as a suitable material in arthroplasty: the high fracture strength and toughness and the smaller grain size 6 . The finer grain size and the stable structure with minimum residual porosity determine improved tribological properties.

Despite their success, due to the excellent mechanical resistance, Y-TZP ceramics can also suffer from the tetragonal-to-monoclinic phase transformation when it is activated by the presence of water. For instance in a physiological environment nucleation and propagation mechanisms of the t-m transformation on the surfaces exposed to humid media can lead to micro-cracks where water by interacting with not yet transformed areas will trigger crack propagation. This mechanism described as hydrothermal aging or low temperature degradation (LTD) has as final outcome delayed implant failure 7 . A summary of LTD characteristics can be found in Figure 1.1.

Zirconia toughened alumina (ZTA)

In the late 1990s, Alumina-zirconia composites and nano-composites were proposed as alternative candidates due to improved properties compared to both alumina and zirconia 8 Zirconia-Toughened Alumina (ZTA) composites have been investigated for use in arthroplasty implants (Figure 1.2). ZTA with up to 30% in volume of Y-TZP is most commonly used as it exhibits the best combination of fracture toughness, slow crack growth propagation 9 and reduced aging phenomenon 10 .

The lower sensitivity to LTD of ZTA composites is commonly associated with the alumina matrix that enhances the structure stability and limits the transformation propagation 1 .

Besides, the alumina matrix favours the retention of small zirconia grain size. It is known that a higher stabilizing content or finer grain size raise the resistance to LTD 11 . De Aza et al.

showed increased composites toughness with grain size reduction and homogenous particle distribution 9 . A complementary approach is the introduction of platelets with the role of blocking the crack growth. Zirconia toughened platelet-reinforced alumina (ZPTA) are used by different manufacturers, for instance CeramTec for its Biolox delta material. To conclude, current ZPTA offer a unique combination of strength (up to 1400 MPa), toughness (up to 8

MPa•m 1/2 ), hardness (up to 1600 Hv) and resistance to hydrothermal ageing, that makes them the reference material for orthopaedic ceramic devices.

Open challenges

Despite the considerable progression of advanced ceramics there are no reports that show aging being completely avoided. In 2001, underestimation of the LTD phenomenon resulted in catastrophic in vivo failure of 800 Prozyr® zirconia ball heads shortly (2 years and less) after implantation. The failure was caused by an accelerated aging at the neck level of ball head. A change in the sintering process lead to undesired porosity and micro-cracks growth propagation 7 . This severe event urged the research on the LTD phenomenon and furthermore the ISO 13356 on biocompatibility of ceramic bone-substitutes modification.

Another open challenge in the development of high-tech ceramics is the characteristic bioinertness that does not enable direct integration with the bone tissue. Surface modifications may enable to overcome this challenge. Different approaches have been developed for surface modification in terms of roughness, topography, chemistry, which will be discussed in more detail in the following section.

Surface modifications of bio-inert ceramics

The implant surface represents a critical factor for osseointegration 12 . Specific biological mechanisms (cell attachment, proliferation and differentiation) can be activated by different surface features and further translated into a coordinated tissue response. Surface topography, surface chemistry and surface energy and wettability have a strong impact on cell and tissue response 13,14 . Figure 1.3 illustrates common surface modifications for metallic dental implants. Two of the illustrated methods (acid-etching and grit-blasting) are also commonly applied to ceramic implants.

The characterization of surface topography is not straightforward. Wennerberg and

Albrektsson proposed a three-dimensional characterization of surface topography with filtered measurements of at least one height, one space and one hybrid parameter.

Simultaneously, it is vital to imagine surface topography as a sum of form, waviness and roughness (Fig. 1.4), since different profiles can be exhibited by a surface having the same average height deviation (R a ) (Fig. 1.4). However most articles on the subject focus on the single R a (or S a ) parameter, which makes the results sometimes difficult to compare.

Surface roughness modification techniques

Topography and roughness are known as osseointegration promoters. Gittens et al.

emphasized the importance of the macro-shape of an implant for a favourable primary fixation and the surface roughness at micro-, submicron-and nano-scale level for a long term anchorage to the bone 14 . A large number of surface modifications have been proposed and various are already being used in industry. Machining or grinding in dry or wet conditions can be employed for changes introduction at the micro-roughness level 15 . However, grinding can significantly decrease zirconia device fracture strength 16 etching as an intermediary step for further silica coating 18 . Gradually increased surface roughness was obtained by sandblasting with 25 and 30 µm alumina particles and further more post HF etching. Ito reported an increase in the surface roughness with a R a ~ 400 nm for the etched surfaces. This is in agreement with a more recent study where S a 400 nm was observed for etched polished ZTA surface 19 .

Noro et al. obtained increased zirconia surface roughness with increase in size of the alumina particles used for sandblasting (Fig. 1.5). The combined sandblasted and etched surface showed a larger configuration. Surface modifications were introduced both at the topographical and physicochemical level. Both micro-and nano-topographies resulted with hydrophilicity increase 20 .

In a similar study, Ito reported increased roughness at both micro-and nano-level with additional in vitro studies. Increased MC3T3-E1 cells proliferation was noticed on the sandblasted and etched surfaces with faster ALP gene expression 21 .

Chemical etching is known to introduce nano-topographies on the zirconia or alumina surfaces, while on zirconiaalumina composites a selective acid etching leads to a nanoporous superficial layer with changes in the physicochemical properties as well, as shown recently by Flamant et al. (Fig. 1.6).

It is important to note that surface topography as well as the chemistry modulates the wettability 22 . Surface energy (surface free energy and surface tension) cooperate with surface topography, roughness and chemistry features to modulate the biological response. Figure 1.7

shows the contact angle of water with zirconia surfaces with different modifications.

Surface chemistry modification techniques

Surface chemical treatments have the advantage of not interfering with the bulk mechanical properties of a device. Zirconia surface functionalization with OH groups (by chemical treatment with aqueous solutions of H 3 PO 4 , H 2 SO 4 , HCl or NaOH) was shown to trigger apatite nucleation in vitro and in vivo 24,25 . Coatings of the surface are used for increased interaction with the bone surface, for instance hydroxyapatite coating was used due to the high resemblance to bone mineral 25 . Enzyme immobilization represents another approach for surface functionalization towards a bone biomimetic surface. The three most common enzyme immobilization avenues are the physical adsorption, entrapment and covalent cross-linking. Aminiam et al. reported covalently immobilized (by carbodiimide-mediated chemoligation) alkaline phosphatase on both alumina and zirconia surfaces with beneficial effect on hMSCs and MG-63 osteoblastlike cells adhesion. 27 ALP has an important role in the mineralization by acting locally to increase the phosphate (Pi) concentration via hydrolysis of monophosphate esters (inorganic phyrophophate) at high pH (Harris, 1990), which together with calcium ions form the substrate for hydroxyapatite formation.

Conclusion on surface modification techniques

Surface modification with changes on the hierarchical combination of both micro-and nanoscale roughness showed promising results for increased osseointegration 29,30 . Gittens et al.

stressed the importance of roughness features at different length scales: while nanostructures alone affect osteoblast proliferation, osteoblast differentiation requires roughness at the micron level. However, roughening of the ceramic surface for improved osseointegration has associated risks. The main concern regards the decrease in the mechanical properties. For instance, reduced Weibull modulus was found on post-grinded and sandblasted zirconia surfaces 15 . These factors can contribute to negatively influence reliability of ceramic devices.

A more recent approach for a biomimetic surface proposes covalent grafting of both inorganic and biological active molecules such as bone morphogenetic protein-2 or alkaline phosphatase 31 . Despite the promising outcomes that link these chemical transformations with improved protein adsorption and mineralization by osteoblastic cells, long term evaluation of both mechanical and biological performance is still needed.

Primary bone cells and cell lines 1.4.1. Overview of cell types in living bone

Bone is a vascularised, mineralized connective tissue defined by four types of cells: bone lining cells, osteoblasts, osteocytes and osteoclasts (Fig. ) concentration.

Bone remodelling is a complex process that involves old bone resorption and new bone formation in order to prevent bone micro damage accumulation. The remodelling cycle starts with the (1) activation phase of the osteoclasts, followed by the (2) initiation of bone resorption by osteoclasts, (3) transition (or reversal period) from bone resorption to new bone formation, and the (4) new bone formation by osteoblasts 32 . 33 .

Figure 1.11 Schematic representation of the bone microenvironment with the specific bone cell types: osteocytes, osteoblasts, osteoclasts and the immune component: memory and circulating T cells (T*), memory B cells (B*) and monocytes (M*)

The following subsections will focus on the characteristics and functions of osteoblasts, osteocytes, osteoclasts, bone lining cells (found in the bone tissue), as well as bone marrow derived mesenchymal stem cells, immune cells and blood cells (found in the marrow cavity).

Osteoblasts

Osteoblast cells originate from multipotent mesenchymal stem cells (MSCs), which can generate bone marrow stromal cells, chondrocytes, muscle cells and adipocytes [START_REF] Aubin | Advances in the osteoblast lineage[END_REF] .

Active osteoblasts exhibit a distinctive phenotype, being plump, cuboidal, mononuclear cells lying on the matrix which they have synthesized [START_REF] Kartsogiannis | Cell lines and primary cell cultures in the study of bone cell biology[END_REF] . In vitro, human primary osteoblast cells isolated and expanded in monolayer can exhibit cell elongation or more cuboidal form in dense areas (Fig. 1.12). This morphological heterogeneity is linked to the characteristic of primary cells that supposes a mix of different subpopulations with osteoblasts at different stages, osteoprogenitors, active osteoblasts or mature osteoblasts that are preparing to become osteocytes.

Osteoblast cell differentiation is a complex process with different regulatory mechanisms underlying lineage restriction, commitment and differentiation starting from multipotential stem cells. The model generally accepted for osteoblast lineage development encloses a progressive evolution from multipotential stem cell to immature osteoprogenitor, followed by mature osteoprogenitor, preosteoblast and finally mature osteoblast (Fig. 1.13).

Figure 1.12 Characteristic morphology of cells in monolayer cultures: hMSC (human mesenchymal stem cells) -two distinct subpopulations: small, spindle-shaped RS (self renewing) cells (arrows and large flat FC (flattened) cells (arrows heads) (A), hObstypical polygonal and flattened morphology (B), MG-63 cellsvarious cell morphologies, oval, triangular or spindle-

shaped (C) [START_REF] Docheva | Researching into the cellular shape, volume and elasticity of mesenchymal stem cells, osteoblasts and osteosarcoma cells by atomic force microscopy[END_REF] .

Figure 1.13 Scheme representing the process of osteoblastogenesis : progression from mesenchymal stem cells, commitment towards osteoblast lineage, differentiation and maturation 41 .

For further commitment osteoprogenitors require expression of core-binding factor α1 -Cbfa1 (also called Runx-2) a member of the runt homology domain transcription factor family. At the moment, Cbfa1/Runx2 is the earliest marker for osteoblast differentiation [START_REF] Aubin | Advances in the osteoblast lineage[END_REF] . It is highly expressed in osteoblasts during foetal development and after birth, while in vitro it is upregulated in presence of different osteogenic stimuli (i.e. bone morphogenetic proteins -BMPs) [START_REF] Yamaguchi | Regulation of osteoblast differentiation mediated by bone morphogenetic proteins, hedgehogs, and Cbfa1[END_REF][START_REF] Ikeda | Radiation retards muscle differentiation but does not affect osteoblastic differentiation induced by bone morphogenetic protein-2 in C2C12 myoblasts[END_REF] . Nonetheless Cbfa1/Runx2 is required but not sufficient to support differentiation towards mature osteoblasts [START_REF] Wang | Isolation and characterization of MC3T3-E1 preosteoblast subclones with distinct in vitro and in vivo differentiation/mineralization potential[END_REF][START_REF] Kumar | Identification and cloning of a connective tissue growth factor-like cDNA from human osteoblasts encoding a novel regulator of osteoblast functions[END_REF] .

Elucidation of osteoblast differentiation process has been studied in vitro by using different monolayer cell culture systems. In vitro study of foetal rat calvaria derived osteoblasts lead to one of the most accepted and referenced model for osteoblasts differentiation [START_REF] Lian | Development of the osteoblast phenotype: molecular mechanisms mediating osteoblast growth and differentiation[END_REF] .

A temporal progression of osteoblast markers is characteristic for commitment towards an osteoblast cell phenotype (Fig. 1.14) [START_REF] Lian | Concepts of osteoblast growth and differentiation: basis for modulation of bone cell development and tissue formation[END_REF] . In detail, three stages were described: proliferation, matrix synthesis, matrix maturation and mineralization, each step with defined important molecules that help transition to mature osteoblasts.

Figure 1.14 Scheme representing the osteoblast progression and maturation governed by a temporal expression of genes involved in the fine regulation of the three main phases: proliferation, extracellular matrix development and maturation, mineralisation (adapted from 42 ).

The proliferative phase is associated with genes involved in cell cycle and proliferation regulation: histones, cyclines and proto-oncogenes. Cbfa1/Runx2 plays a crucial role initially in osteoblast progression by influencing the promoter activation of important osteoblastic genes (i.e. osteocalcin (OC), alkaline phosphatase (ALP), bone sialoprotein (BSP), osteopontin (OP) and collagen type I (CollαI)). Osterix (Osx), the zinc-finger transcription factor is downstream of Runx2 with fundamental effects in osteoblast differentiation and subsequent bone formation.

The extracellular matrix (ECM) deposition and maturation phase is a process modulated by cell-cell contact and cell-matrix interactions regulated by hormonal and local factors, in which collagen plays a pivotal role. ALP plays an important role in increasing the phosphate concentration, which with calcium ions are forming the substrate for hydroxyapatite formation. The mineralization of the ECM requires expression of osteopontin, osteonectin (ON) and osteocalcin.

Mature active osteoblasts are cuboidal cells enriched with endoplasmic reticulum due to the main cellular function, large quantities of proteins synthesis [START_REF] Gay | Perspectives on osteoblast and osteoclast function[END_REF] . Proteins constituting the mineralized matrix i.e. osteocalcin, osteopontin, osteonectin and bone sialoprotein (BSP) are produced by osteoblast cells at the highest levels during the mineralization phase [START_REF] Lian | Concepts of osteoblast growth and differentiation: basis for modulation of bone cell development and tissue formation[END_REF] . BSP acts as a nucleation factor for hydroxyapatite formation and deposition [START_REF] Hunter | Nucleation of hydroxyapatite by bone sialoprotein[END_REF] while osteonectin is a promoter of hydroxyapatite crystal formation. Osteoblasts are characterized by a functional asymmetry: secretory cells for collagen and non-collagenous proteins on one side, while their contralateral side is exposed to the extracellular medium, sensitive to signals [START_REF] Robey | Bone matrix proteoglycans and glycoproteins[END_REF] . At the end of the bone forming phase the osteoblasts can follow different pathways: (1) be enclosed in bone matrix as osteocyte, (2) enter an inactive state osteoblasts and become bone-lining cells, (3) undergo apoptosis.

Osteocytes

In humans, 10-30% of osteoblasts differentiate into osteocytes, becoming embedded in the organic matrix of the bone called osteoid [START_REF] Aubin | Advances in the osteoblast lineage[END_REF] . Osteocytes are the most abundant cell type in bone (90-95% of the total bone cells) with a lifespan of up to 25 years [START_REF] Ta Franz-Odendaal | Buried alive: how osteoblasts become osteocytes[END_REF] . When fully embedded in the osteoid, cells reduce their size and lower their activity. The shape and size of the newly formed osteocyte is dictated by the bone type size and activity of the committed osteoblast. For instance, in woven bone with randomly oriented collagen fibres, osteocytes are isodiametric, while in lamellar bone they display an elongated morphology along the longitudinal axis and round or triangular in the transverse plane [START_REF] Holtrop | Light and electron microscopical structure of bone forming cells[END_REF] .

The transition from osteoblast to osteocyte through osteoblast differentiation was described as a four stage process: osteoblastic-osteocyte (type I osteocyte) emerges from a mature osteoblast, followed by an osteoid-osteocyte (type II osteocyte) stage, type III osteocyte, young osteocyte and finally mature osteocyte [START_REF] Ta Franz-Odendaal | Buried alive: how osteoblasts become osteocytes[END_REF] . It was suggested that the osteocyte cells, components of the osteoid, play a crucial role in regulation and control of bone mineralization process being more important than osteoblasts themselves [START_REF] Dallas | Dynamics of the transition from osteoblast to osteocyte[END_REF] .

Osteocytes are synthesizing a number of different proteins involved in cytoskeletal function, comprising CapG, and E11/podoplanin (Fig. 1.15) [START_REF] Dallas | Dynamics of the transition from osteoblast to osteocyte[END_REF] . E11 it is considered the earliest marker for osteocytes, being highly expressed in osteoid-osteocytes, but not in mature osteocytes [START_REF] Zhang | E11/gp38 selective expression in osteocytes: regulation by mechanical strain and role in dendrite elongation[END_REF] .

E11 protein has an important role being linked with the osteocytes development. Dendrites mediate the osteocytes communication and also osteocytes -bone surface cells. It was shown that E11 and dendrites are sensitive to mechanical load and shear stress [START_REF] Zhang | E11/gp38 selective expression in osteocytes: regulation by mechanical strain and role in dendrite elongation[END_REF] . E11 protein appears firstly on the forming dendritic processes of the osteocytes. Osteocytes release nitric oxide (NO), ATP and prostaglandin, as response to shear stress, leading to effects on both osteoblast and osteoclast cells with echo on bone formation [START_REF] Dallas | Dynamics of the transition from osteoblast to osteocyte[END_REF] .

Figure 1.15 Scheme representing the process of osteocytogenesi: transition from osteoblast to

osteocyte with illustration of key osteocyte marker genes [START_REF] Dallas | Dynamics of the transition from osteoblast to osteocyte[END_REF] .

Sclerostin which is known to have negative effects on bone formation, by inhibiting the canonical Wnt/β-catenin signalling pathway is highly expressed in mature osteocytes [START_REF] Dallas | Dynamics of the transition from osteoblast to osteocyte[END_REF] . At the end of their function, upon apoptosis, osteocytes release apoptotic bodies and homing factors (RANKL) that will activate osteoclasts towards bone resorption.

Osteoclasts

Osteoclasts (or osteal macrophages or osteomacs) are bone specific multinucleated macrophages with precursors mononuclear cells of the hematopoietic stem cell lineage.

Figure 1.17 Bone resorption activation: a -Recruitment of polykaryons by CSF-1 and RANKL, b -Acidification of a sealed resorption vacuole results in TRAP and CATK enzymes responsible for the bone mineral degradation, c -TEM micrographs with an activated osteoclast, d -SEM micrograph with an osteoclast generated on a cortical bone slice (red propellers= osteoclasts, black arrows= resorption pit, yellow star= non-resorbed bone surface, blue triangles= mononuclear cells 32 .

Tartrate resistance acid phosphatase (TRAP) is widely used as a marker enzyme of osteoclasts and secreted in Howship's lacunae. Although this enzyme could dephosphorylate osteopontin, its exact role in bone resorption is not clear yet. Bone resorption products are eliminated from the ruffled border via a transcytosolic vesicular pathway to the functional secretory domain on the apical side of the cells into the extracellular space [START_REF] Vaananen | The cell biology of osteoclast function[END_REF] . During osteoclastic degradation of type I collagen network, matrix bound factors, i.e. IGFs are released and they stimulate the osteoblasts in a paracrine manner, leading to new bone formation [START_REF] Gelse | Collagens--structure, function, and biosynthesis[END_REF] . Subsequently, an osteoclast can follow two pathways: repeating once more bone resorption or undergo apoptosis 29 .

Bone marrow niche (MSCs, blood and immune cells)

Bone marrow represents an active milieu that acts as a framework for different micro- MSCs provide structural support and harbour various cell populations [START_REF] Fe Mercier | The bone marrow at the crossroads of blood and immunity[END_REF] . MSCs constitute a class of adult stem cells, characterized by their self-renewal potential as well as their multidifferentiation potential into several cell types of the mesenchymal lineage: bone, cartilage, adipose and muscle [START_REF] Caplan | Why are MSCs therapeutic? New data: new insight[END_REF] . MSCs number is limited and human multipotent adult progenitor cells have been estimated to be present at a density of 1 in 10 7 -1 in 10 8 cells [START_REF] Reyes | Characterization of multipotent adult progenitor cells, a subpopulation of mesenchymal stem cells[END_REF] .

MSCs, typically defined as adherent, fibroblastoid-like cells can vary in frequency depending on the donor. A trend of age-related reduction of osteoprogenitor numbers was reported for rats and mice [START_REF] Tsuji | Effects of donor age on osteogenic cells of rat bone marrow in vitro[END_REF][START_REF] Bergman | Age-related changes in osteogenic stem cells in mice[END_REF] . This is further corroborated with D'Ippolito et al. study that showed in humans a decrease of osteoprogenitors concentration from 66.2 per 10 6 cells in young donors to 14.7 per 10 6 cells in older donors [START_REF] D'ippolito | Age-related osteogenic potential of mesenchymal stromal stem cells from human vertebral bone marrow[END_REF] .

Very important for potential therapies is that MSCs do not elicit an immune response as they express major histocompatibility complex (MHC) class I proteins but not MHC class II proteins [START_REF] Le Blanc | Treatment of severe acute graft-versus-host disease with third party haploidentical mesenchymal stem cells[END_REF] . MSCs were first identified by Friedenstein et al. in the bone marrow [START_REF] Friedenstein | Fibroblast precursors in normal and irradiated mouse hematopoietic organs[END_REF] .

Mesenchymal stem cells can be isolated from different sources, such as bone marrow, adipose tissue, skeletal muscle, periosteum and placenta [START_REF] Barrilleaux | Review: ex vivo engineering of living tissues with adult stem cells[END_REF] . Independent of cell source, MSCs have the potential to give rise to mesenchymal lineages both in vitro and in vivo including bone, cartilage, adipose tissue and muscle. MSCs proliferation rate can be influenced my various cell culturing factors.

It was shown that self-renewal rate is considerably decreased after MSCs first in vitro confluence with effects also on the multipotency capability [START_REF] Digirolamo | Propagation and senescence of human marrow stromal cells in culture: a simple colony-forming assay identifies samples with the greatest potential to propagate and differentiate[END_REF][START_REF] Banfi | Proliferation kinetics and differentiation potential of ex vivo expanded human bone marrow stromal cells: Implications for their use in cell therapy[END_REF] . Nonetheless even after extensive in vitro expansion, BMSCs retain the osteogenic differentiation potential [START_REF] Muraglia | Clonal mesenchymal progenitors from human bone marrow differentiate in vitro according to a hierarchical model[END_REF] .

Different protocols have been developed to direct MSCs in vitro into specific lineages. Haematopoietic stem cells (HSCs) reside in multiple locations (along the blood vessels, vicinity of endosteal surfaces) and express as lineage marker negative SCA1 + KIT + CD41 - CD48 -CD150 +73 . There is an intimate interaction between HSC and blood vessels: during embryonic development HSCs are generated in association with vascular areas and blood flow [START_REF] Adamo | Biomechanical forces promote embryonic haematopoiesis[END_REF] while in adult humans, extramedullary haematopoiesis occurs in perivascular areas.

Bone marrow endothelial cells are haematopoiesis promotors by their secretory activity composed of granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSF), stem cell factor (SCF), interleukin-6 (IL-6) [START_REF] Rafii | Regulation of hematopoiesis by microvascular endothelium[END_REF] . Furthemore, they are supposed to be involved in cell homing by expressing molecules as E-selectin, CXC-chemokine ligand 12 (CXCL12), vascular cell adhesion molecule 1 (VCAM1) [START_REF] Mazo | Hematopoietic progenitor cell rolling in bone marrow microvessels: parallel contributions by endothelial selectins and vascular cell adhesion molecule 1[END_REF] . Similar expression and secretory pattern were found for bone-forming osteoblasts found in bone marrow [START_REF] Calvi | Osteoblastic cells regulate the haematopoietic stem cell niche[END_REF] .

Bone marrow is a primary lymphoid organ by supporting lymphoid system development and by, also hosting different lymphoid cell types. It was found that dendritic cells (DCs) as well as naive recirculating B and T cells colonize bone marrow space along the blood vessels [START_REF] Sapoznikov | Perivascular clusters of dendritic cells provide critical survival signals to B cells in bone marrow niches[END_REF] .

Through complex cell signalling interactions, immune cells situated in perivascular regions in the bone marrow constitute important actors in protection developed against blood-borne pathogens.

Cell lines

Immortalized cell lines show a series of advantages compared to primary cells, such as availability of almost unlimited number of cells without the need for isolation, easy access and maintenance, and good repeatability of results in experiments. Nonetheless, there are reports which link the prolonged cell passaging to progressive phenotypic heterogeneity [START_REF] Wang | Isolation and characterization of MC3T3-E1 preosteoblast subclones with distinct in vitro and in vivo differentiation/mineralization potential[END_REF][START_REF] Leis | Phenotypic heterogeneity of osteoblast-like MC3T3-E1 cells: changes of bradykinin-induced prostaglandin E2 production during osteoblast maturation[END_REF] .

Moreover, both transformed and non-transformed cell lines are cell cycle stage arrested meaning they do not mirror the complete phenotypic features range of normal primary cells.

An overview of cell lines characteristics versus the primary cells can be find in Table 2.

Table 1.2 Comparison between primary osteoblast cells and osteoblast-like cell lines (adapted from Czekanska et al., 2012).

Despite these limitations the use of cell lines is common in basic and applied biology.

Different model systems for osteoblast cell lines have been developed over time:

osteosarcoma cell lines (SaOs-2 [START_REF] Rodan | Characterization of a human osteosarcoma cell line (Saos-2) with osteoblastic properties[END_REF] , MG-63 [START_REF] Billiau | Human interferon: mass production in a newly established cell line, MG-63[END_REF] , U2OS [START_REF] Ponten | Two established in vitro cell lines from human mesenchymal tumours[END_REF] , non-transformed cell lines (MC3T3-E1) [START_REF] Quarles | Distinct proliferative and differentiated stages of murine MC3T3-E1 cells in culture: an in vitro model of osteoblast development[END_REF] and experimentally immortalized cell lines (hFOB 1.19) [START_REF] Harris | Estrogen response in the hFOB 1.19 human fetal osteoblastic cell line stably transfected with the human estrogen receptor gene[END_REF] , RCT-1 [START_REF] Jat | Large T antigens of simian virus 40 and polyomavirus efficiently establish primary fibroblasts[END_REF] .

SaOs-2 is a human osteosarcoma cell line isolated from an 11 year old Caucasian female in 1975, which osteoblastic features were evaluated by Rodan et al. [START_REF] Rodan | Characterization of a human osteosarcoma cell line (Saos-2) with osteoblastic properties[END_REF] . SaOs-2 cells show a mature osteoblast phenotype and high levels of alkaline phosphatase activity (ALP). Murray et al. reported much higher ALP level when compared to other osteosarcoma cell lines (MG-63 and SaOs-1) [START_REF] Murray | Characterization of a human osteoblastic osteosarcoma cell line (SAOS-2) with high bone alkaline phosphatase activity[END_REF] . Plus, SaOs-2 cells versus primary human osteoblast cells showed a similar pattern for ALP activity at early time points, yet a 120-fold increase after 14 days of culturing in the same conditions [START_REF] Saldana | In search of representative models of human bone-forming cells for cytocompatibility studies[END_REF] . Rodan et al. reported calcified matrix formed by SaOs-2 cells in diffusion chambers akin of woven bone [START_REF] Rodan | Characterization of a human osteosarcoma cell line (Saos-2) with osteoblastic properties[END_REF] . Similar collagen structure was found for SaOs-2 and primary human osteoblasts cells, yet higher level of lysyl hydroxylation observed for SaOs-2 cells [START_REF] Fernandes | The post-translational phenotype of collagen synthesized by SAOS-2 osteosarcoma cells[END_REF] . Osteogenic supplements such as dexamethasone [START_REF] Rao | 17 beta-oestradiol enhances the stimulatory effect of 1,25-dihydroxyvitamin D3 on alkaline phosphatase activity in human osteosarcoma SaOS-2 cells in a differentiationdependent manner[END_REF] and β-glycerophosphate substrate [START_REF] Muller | Inorganic polymeric phosphate/polyphosphate as an inducer of alkaline phosphatase and a modulator of intracellular Ca2+ level in osteoblasts (SaOS-2 cells) in vitro[END_REF] can further enhance ALP activity. Concerning the supplementation with βglycerophosphate substrate, timing is another very important aspect; deleterious effects on cell viability can be avoided by addition in the cell culture medium at the post-proliferative stage. In terms of secretory cell pattern, SaOs-2 cells show similar cytokine and growth factor expression to primary human osteoblast [START_REF] Saldana | In search of representative models of human bone-forming cells for cytocompatibility studies[END_REF] . Parathyroid hormone (PTH) and vitamin D receptors are expressed on SaOs-2 cell surface correlating with osteoblast behaviour both in vitro and in vivo [START_REF] Rao | 17 beta-oestradiol enhances the stimulatory effect of 1,25-dihydroxyvitamin D3 on alkaline phosphatase activity in human osteosarcoma SaOS-2 cells in a differentiationdependent manner[END_REF] .

MG-63 cell line was established from human osteosarcoma as well (distal diaphysis of the left femur of a 14 year old male). Interestingly, these cells produce high yields of interferon [START_REF] Billiau | Human interferon: mass production in a newly established cell line, MG-63[END_REF] .

MG-63 osteoblast-like line exhibits rapidly growing cells that lack contact inhibition and therefore lead to aggregate formation [START_REF] Heremans | In vitro cultivation of human tumor tissues. II. Morphological and virological characterization of three cell lines[END_REF] . MG-63 cells manifest an immature osteoblast phenotype but can maturate in long term cultures. Nonetheless, concerning the mineralization potential of MG-63 cells in monolayer, there is not a consensus in literature at the moment.

Kumarasuriyar et al. observed increased ALP activity after 15 days of culture, followed by enhanced collagen type I expression and calcium accumulation following 28 days of culture [START_REF] Kumarasuriyar | Glycosaminoglycan composition changes with MG-63 osteosarcoma osteogenesis in vitro and induces human mesenchymal stem cell aggregation[END_REF] . Saldana et al. showed low ALP activity and no mineralization for MG-63 cells [START_REF] Saldana | In search of representative models of human bone-forming cells for cytocompatibility studies[END_REF] . In agreement with these findings, Czekanska et al. reported 14-fold lower ALP activity in MG-63 cells when compared to primary human osteoblasts and no calcium deposition [START_REF] Czekanska | A phenotypic comparison of osteoblast cell lines versus human primary osteoblasts for biomaterials testing[END_REF] . It is important to note that different surfaces were used in these studies: while Kumarasuriyar and

Czekanska have used cell culture plastic wells, Saldana et al. have evaluated cell behaviour on Ti6AI4V surfaces or in presence of Ti particles. Nonetheless, similar culture media were applied in these studies.

MC3T3E1 cell line was derived from newborn mouse calvaria and presents a preosteoblastic phenotype. Five subclones were reported with mineralizing potential and one of the five (subclone 4) was shown to undergo changes from proliferative state to nodule formation and mineralization similar to intramembranous osteogenesis in vivo [START_REF] Sudo | In vitro differentiation and calcification in a new clonal osteogenic cell line derived from newborn mouse calvaria[END_REF] . It is important to note that osteogenic supplements (ascorbic acid and beta glycerol-phosphate) are prerequisite for matrix mineralisation (Table 1.2). In media lacking these stimulators, ALP activity is low and no mineralisation is registered [START_REF] Hong | Morphological and proteomic analysis of early stage of osteoblast differentiation in osteoblastic progenitor cells[END_REF] . Decreased proliferation was registered for MC3T3E1 cells at high passage number (> 36) and signs of replicative senescence at very high passage number (> 60) [START_REF] Grigoriadis | Subclone heterogeneity in a clonally-derived osteoblast-like cell line[END_REF] .

The human foetal osteoblast cell line (hFOB 1.19) was obtained from transfected primary cultures biopsies (spontaneous miscarriage) [START_REF] Harris | Estrogen response in the hFOB 1.19 human fetal osteoblastic cell line stably transfected with the human estrogen receptor gene[END_REF] . hFOB 1.19 cells are temperature sensitive as they were stably transfected with a gene coding for a temperature -sensitive mutant (tsA58) of the SV40 large T antigen and 1,25-dihydroxyvitamin D3 responsive. For the resultant hFOB cells proliferation occurs at 37˚ C while an increase in the incubation temperature (39˚ C) will determine the differentiation towards mature osteoblasts. hFOB1.19 cells express osteoblast-specific phenotypic markers and mineralize extracellular matrix. High levels of osteopontin, osteonectin, BSP and type I collagen were reported in differentiated hFOB cells.

In terms of osteoclast cell culture systems there still open questions and difficulties encountered in vitro. Stimulated mouse bone marrow cells with 1,25-dihydroxyvitamin D3 or human PTH formed tartrate-resistant acid phosphatase (TRACP)-positive multinucleated cells [START_REF] Takahashi | Osteoclast-like cell formation and its regulation by osteotropic hormones in mouse bone marrow cultures[END_REF] . Other approaches suggest co-culture systems with human peripheral blood mononuclear cells (PBMC) and osteoblast-like cells (SaOs-2) [START_REF] Asai | Osteoclast differentiation by human osteoblastic cell line SaOS-2 primed with bacterial lipid A[END_REF] . At the moment, there are osteoclast-precursors cell lines as 4B12 established from Mac-1(+) cFms (+) RANK(+) cells from calvaria of 14-day-old mouse embryos [START_REF] Adamo | Biomechanical forces promote embryonic haematopoiesis[END_REF] .

Mesenchymal stem cells represent a robust cell culture model that can divide in vitro while maintaining their multipotency. Nevertheless, human mesenchymal stem cell line behaviour is dictated by the single clone used to establish the cell line and companies providing hMSC cell lines do not provide details on each clone, making the choice of the most appropriate hMSC cell line challenging (e.g. with high osteogenic potential for bone studies, with high chondrogenic potential for cartilage studies).

Effect of surface modifications on cellular responses 1.5.1. Roughness effect

Surface roughness and surface topography are key modulators of the cellular response to materials in vitro and in vivo. Many studies highlighted the importance of surface characteristics in influencing cellular processes such as cell attachment and proliferation, metabolism, differentiation or matrix synthesis. The implant fate faith is governed by the chemical, physical, mechanical and topographical properties of the surface. Additional factors that will influence the response triggered by an implant are the design of the implant, the bone status, surgical technique and loading conditions 12 . In a guideline article, Wennerberg and Albrektsson meaningfully noted that bone anchorage components of currently commercially available oral implants differ in surface roughness by a factor of at least six [START_REF] Wennerberg | Suggested guidelines for the topographic evaluation of implant surfaces[END_REF] . Therefore, results between studies are difficult to compare and their outcome should be carefully considered. It was acknowledged that there is a high need for standardized analytical tools and terminology for surface characterization.

Cell behaviour (cell spreading and attachment) are greatly influenced by surface status. In vitro studies observed a positive correlation between increased roughness and high surface energy, with more surface hydroxyl groups, which results in improved osteoblasts adhesion and improved cell activity.

In terms of early cell attachment and mineralization, surface features have a significant effect on cell behaviour. Cells do not ''see'' directly the different implant surfaces but they attach to pre-adsorbed proteins on their surfaces. A rough surface is known to promote adsorption of fibrin that will further guide preosteoblasts migration. The specific ECM will influence cellular processes such as proliferation, differentiation or apoptosis. Cells adapt their behaviour in response to the ECM by integrin dependent activation of intracellular signalling mediating molecules. Signal transduction is done by integrins that associate with proteins part of the focal adhesion complex i.e. vinculin, talin, paxillin. It is important to note that focal adhesion complexes were reported only in in vitro set-ups, not reported in in vivo models.

It is interesting to note that the effects of surface roughness seem to be cell dependent. While osteoblasts, MSCs and macrophages appear to prefer rougher surfaces, fibroblasts display better cell attachment and spreading on smooth surfaces 100 . On rough surfaces the already differentiate osteoblasts will either proliferate more or begin secreting and depositing a noncollagenous mix of proteins called lamina limitans or cement line with role in mineralization initiation 101 . In vitro microrough surfaces with a high density of nano-features enhance osteoblast differentiation 102,103 . Osteocalcin production was increased on rough surfaces while ALP activity decreased, this could be due to the biphasic nature of the enzyme with high activity at early stages of differentiation and a decrease on the mineralization onset 13 .

Boyan et al. states that if the average roughness of a surface is greater than the size of an individual osteoblast, then this surface will be deemed smooth, as long as the distance between two peaks is too big to be perceived by an osteoblast 104 . In the same direction, Hamilton et al. reported that if the peak to peak distance is smaller than the osteoblast body length, they will assume the characteristic cuboidal morphology, while in an opposite situation a fibroblast-like morphology will be adopted 105 All these observations, obtained on both ceramic materials (zirconia-based) and on metallic materials (Ti) are suggesting that osteoblast cultured on rough surfaces could provide a bone healing niche, able to support bone formation and osseointegration.

Chemical effect

As previously stated, surface topography, chemistry and energy are finely orchestrating cellmaterial interactions by determining which and how biological molecules will adsorb to the surface. Surface chemistry plays a key role on protein adsorption. Rappaport's pioneering work in early 1970s focused on understanding surface chemical effects on mammalian cell adhesion 115 . Since then, different approaches were used to modify surface chemistry of materials to direct cell behaviour such as the use of liquid-phase chemical oxidants 116 or gasdischarge treatment 117 that are now regularly used for plastic tissue culture production 118 .

Furthermore, particular surface functional groups influence cell adhesion and proliferation:

for instance, Curtis et al. found that carboxyl groups promote cell attachment 101,119 . However, it is difficult to distinguish between surface chemistry from all other surface properties such as topography and wettability (Fig. 1.21). For example, a hydrophilic surface will allow a close interaction with biological fluid, allowing protein adsorption, while on a hydrophobic surface, air bubbles may be trapped and will interfere with protein and cell receptors adsorption.

Figure 1.21 Interactions between a hydrophilic (A) and a hydrophobic (B) surface. Note the intimate interactions with proteins whithin biological fluids and subsequently with cell receptors for hydrophilic surfaces while for the hydrophobic surfaces the hydrocarbon contamination could lead to air bubbles entrapment and intereference with protein and cell adhesion 120 .

The broadest accepted convention is that anchorage-dependent mammalian cells are favoured by hydrophilic surfaces (contact angle < 60°C) [121][122][123] . The contact angle is defined as the angle between the tangent to the liquid-vapour interface and the actual local surface of the solid.

It is important to note that the concept of optimal contact angle range has evolved, studies Understanding the protein adsorption process could play a pivotal role in further development of material modifications such as grafting proteins or synthesized peptide for increase bioactivity.

1.6. Osseointegration and in vivo performance of bio-inert ceramics 1.6.1. Osseointegration: concept and challenges

The concept of "osseointegration" was first proposed by Brånemark and defined as a direct interface formed between an implant and bone without soft tissue development 12 . Albrektsson has later evidenced that mechanical stability of the implant is a prerequisite for successful integration to bone tissue 129 . This is referred to as "primary stability" and is mainly dictated by the macro-design of the implant and the bone quality. The "secondary stability" improves the primary stability by direct new bone apposition onto the implant. Osteoinduction and osteoconduction are biological processes triggered by the implant properties. Osteoinduction refers to stimulation and recruitment of undifferentiated and pluripotent cells to develop into bone forming cells, osteogenesis being induced. An implant that favours bone growth on its surface and into pores is associated with an osteoconductive material 130 . The implant anchorage to bone is generally assessed by histomorphometry, push-out test and removal torque analysis 131 . More recent studies have evidenced that the osseointegration process will be dependent not only on bone tissue response but also on the immunological response 132 .

Bone tissue regeneration is a temporal sequence of a fine orchestrated process by various cell types via cytokines and growth factors 133 (Fig. 1.22). It is important to note that in the absence of osseointegration, a fibrous capsule can develop around the implant, which in most of the cases leads to aseptic loosening and implant failure. A different path with same outcome is obtained by developing an infection that can compromise the implant success in vivo. With periprosthetic joint infections as the main cause for arthroplasty revision, many studies focus on developing highly biocompatible surfaces for tissue cells and repellent for prokaryotic cells.

Figure 1.22 Scheme representing bone remodelling post-implantation as a succession of different steps: 1 -Excessive torque force, 2 -Osteoclasts and osteoblasts recruitment, 3 -Bone resorption by the osteoclasts, 4 -Debris cleaning by the bone lining cells, 5 -Collagen production by the bone lining cells, 6 -Osteoblasts attachment enhanced by the collagen layer, 7 -Osteoid deposition by the osteoblasts, 8 -Osteoblasts trapped into the osteoid become osteocytes 133

Surface-modified bioinert ceramics: comparison to titanium

A growing market of ceramics in the field of dentistry and orthopaedics has been registered in the last decade and is predicted to continue with same trend. In both fields further market growth partly rely on better osseointegration. In dental, zirconia implants present good esthetics, high resistance to corrosion and the absence of allergic reactions; micro-rough implants also present ver good osseointegration. In orthopaedics, the possibility to replace in total hip arthroplasty the external shell in contact with the acetabulum (that is commonly a metal) by ceramic if sufficient osseointegration would be achieved could allow the use of different designs such as bigger ball sizes with direct impact on the patients life quality. Both fields necessitate the use of bio-inert materials, by definition not fully adequate for good lsseointegration, even though the concept of bio-inertness has evolved together with the biocompatibility and the development of new materials. Today we know that all materials trigger an answer in vivo, however the level of consequences is different depending on the material type. The current opinion is that "bioinert" does not exist, but this term is still used to distinguish ceramic oxide materials such as alumina, zirconia and composites (with weak osseointegration) from calcium phosphate based materials, such as hydroxyapatite and betatricalcium phosphate (with better osseointegration).

Figure 1-23 shows on the one hand histological good implant-bone integration (A) and on the other hand a fibrous capsule developed in between implant and bone (B) that can lead to implant instability and micro-motion and finally implant failure.

Surface modifications technologies should allow modifications of the outer surface of the cup for an improved interface with bone tissue. At the same time, the inner surface of the cup (that is in contact with the ball) needs to be preserved to guarantee low friction and adequate resistance to fracture.

Figure 1.23 Direct osseointegration versus fibrous capsule development of TAN (Titanium-6%aluminium-7% niobium) nails, 12 months post-implantationin sheep tibia : A -Standard microrough TAN in direct contact with bone (i= implant, b= bone); Bpaste polished TAN lead to a fibrous capsule development between the implant and the bone (i= implant, f= fibrous tissue, b= bone) 106 .

The machined (turned) titanium implant first proposed by Brånemark, with a surface roughness of 0.5-1.0 µm, was long-used as gold standard. The most common surfacemodification method at the moment is the surface sandblasting combined with chemical etching. Cochran et al. documented for the first time significantly more bone apposition on SLA-surfaced (sandblasted and acid etched) titanium implants. In long bones of miniature pigs, they showed enhanced effect of coupled sandblasted and acid-etched titanium surfaces when compared to plasma-sprayed ones after 3 months 134 .

It is worth noting that most of the surface topography modifications result in changes at the chemical level as well. The mechanism of fast osseointegration can be linked with interactions at the nanometer level, the SLA surface triggering protein adsorption and cell recruitment at the implant surface. Gittens et al. reviewed recently the synergic effect of combined micro-and nano-scale roughness on cell interaction and protein adsorption 13 .

Zirconia dental implants have rapidly gained a lot of credit due to advantageous mechanical, biological, aesthetic and corrosion properties. On the other hand, the intrinsic bio-inertness leads to the necessity for surface modifications.

Comparable outcomes were found when comparing zirconia and titanium tibial implants in minipigs up to 12 weeks 131,135 (Fig. 1.24). Ceramic composites (such as zirconia-toughened alumina) exhibit increased strength and toughness and superior wear resistance and biocompatibility compared to yttria-stabilised zirconia 136 . In a rat animal model, histological analysis of alumina-zirconia nano-composites shown no major inflammation when compared to reaction induced by metals or polyethylene wear debris 136 . It is important to note that additional factors such as patients (poly)medication might interfere with the immune system and bone turnover leading to bone loss and/or implant failure 131 . Moreover most of the animal studies are up to three months and data on long-term follow up are not yet available.

Correlation between in vitro and in vivo outcomes

The last decades brought promising results in the field of new materials and implants for bone regeneration 137 . Reliable techniques are required for a fast and accurate translation to the clinics. There is a high demand for in vitro methods suitable for predicting in vivo outcomes and reducing the number of animals used.

The majority of in vitro studies are using cell lines with the limitations of not exhibiting a normal cell phenotype and different sensibility to stimulants as growth factors or cytokines.

On the other hand there is a strong inter-donor variability associated with primary cell culture (hMSCs or hObs).

In vivo animal models despite physiological differences specie-and age-dependent, show the advantage of studying complex processes such as osseointegration including interactions at the systemic level.

Poor correlation between in vitro and in vivo outcomes was recently reported by Hulsart et al. The relevance of these observations emphasise the concern and the necessity of protocols standardization.

Additionally as mentioned before, the use of primary cells presents the common issue of donor variation 140 . In an attempt to limit this effect, there are studies with pooled cells from multiple donors 141 . Nevertheless, this in vitro limitation can be beneficial for the in vivo studies, where donor variability mimics the host cell variation.

Conclusions

▲ Bio-inert ceramics are recommended for orthopaedic and dental implants due to their exquisite properties in terms of biocompatibility and bio-inertness. Zirconia-toughened alumina is nowadays used for hip prosthesis due to its superior mechanical properties (namely toughness) compared to alumina or zirconia. The latter is still widely used in dentistry due to its excellent esthetical properties.

▲ The achievement of a primary and secondary stability comparable to metal implants will allow the design of a purely ceramic solution, which in turn will allow the use of different prosthesis designs. For instance, in the case of the hip prosthesis, this will allow a larger ball head, thus reducing the risk of dislocation and increasing range of motion of the prosthesis, with a large benefit for the patient. ▲ Different approaches have been used for bio-inert ceramic surface modifications. Most metal implants are sand-blasted and subsequently acid etched. Due to the high interdependence between the roughness and chemistry of a surface most of the techniques results in changes at both levels. Nevertheless, plasma treatment or molecule grafting (alkaline phosphatase, bone morphogenetic proteins) have been used for specific chemical modifications.

▲ For improving the implant-bone interface a good understanding of both actors is required.

Bone is a highly specialized tissue containing various cell types with specific roles and active communication: osteoblasts (bone formation), osteoclasts (bone resorption), osteocytes (part of bone matrix). Furthermore, it hosts the bone marrow, a complex environment with niches for both mesenchymal stem cells and immune cells. The majority of in vitro evaluation of biomaterials relies on osteoblastic cell lines (murine MG-63 or osteosarcoma SaOs-2), however human primary osteoblasts from osteoarthritic femoral heads of patients undergoing hip replacement surgery have a much higher clinical relevance. ▲ Concerning the in vitro effect of surface properties on cell response, a positive correlation was observed between increased roughness and high surface energy leading to improved osteoblasts adhesion and differentiation. Optimal roughness range between 0.2-2 µm seems to promote osteoblast differentiation.

▲ In vivo, osseointegration is an orchestrated process by both surface roughness and chemistry, in correlation with the complex bone structure and function. The nano-features will influence firstly water adsorption and furthermore the types of protein adsorbed and their conformational state. This biological layer created at the implant surface and the microroughness will influence the cell adhesion, while the implant design (macro-scale characteristics) will play a role in the mechanical interlocking to the bone surface. Surface chemistry with echo in surface energy and wettability will greatly influence the fluid and blood proteins. In conclusion osseointegration is a multi-facet process governed by the surface characteristics and bulk material of an implant as well as status of bone cells and surgical technique.

▲ It has been shown that zirconia performance in vivo (miniature pigs) is comparable to the commercially pure grade 4 titanium implants when similar surface modifications were applied, sand-blasted and acid etching respectively. At the moment there is still a relatively poor correlation between in vitro assays results and the in vivo outcomes of different materials. Standardization at all the steps, along the development of a new material or of a novel processing technique, is highly required for a more accurate comparison between studies with beneficial effects on the advance of new biomaterials.

Chapter 2.

In vitro effects of micro-and micro-/nano-roughness of zirconia-toughened alumina

Abstract

Hip arthroplasty cementless acetabular components require excellent mechanical properties, biocompatibility, low friction and good osseointegration with surrounding bone tissue.

Zirconia-toughened alumina (ZTA) fulfils these demands but requires combination with a rough metal shell for adequate osseointegration. Surface modifications of ZTA could allow a metal-free solution, thus preserving the bone stock for an eventual revision surgery. In this study, selective chemical etching proved to be an innovative method for the introduction of nano-features on micro-rough surfaces obtained by injection moulding. Results suggest that micro-roughness, fluorine enrichment and nano-porosity at the surface of ZTA play a synergistic role on human osteoblast (hOb) maturation. Among the tested groups, hydrofluoric acid etched "medium" roughness (Sa = 330 nm) ZTA showed the highest and/or earliest ALP expression at both the protein and gene level, while micro-roughness alone induced only minor effects on hOb maturation on ZTA.

Keywords: osseointegration, micro-roughness, nano-roughness, human primary osteoblast, selective chemical etching

Introduction

Prostheses with various designs are available for the hip, knee and shoulder joints. In the case of total hip replacements, each device is composed of a femoral stem, a femoral head, and an acetabular cup consisting of an insert (made of ceramic or cross-linked polyethylene) and a metal shell. The first ceramic material exploited in hip arthroplasty was the alumina, afterwards, in the nineties, yttria-stabilised zirconia (Y-TZP) was introduced with the intent to the decrease the revisions for fracture of the ceramic component, because of its high fracture toughness 1 . Nonetheless, Y-TZP is sensitive to low temperature degradation. This issue has been addressed by the latest development of bio-ceramic prosthesis, which is now composed of zirconia-toughened alumina (ZTA), the gold standard ceramic bearing material in hip arthroplasty today. The current ceramic-on-ceramic (CoC) bearings used for cementless hip prostheses need an osseointegrative metal shell on the acetabular side to offer an adequate bone in-growth. In addition, the modularity allows a better positioning of the acetabular cup and a further fixation with the screws; as a downside, the thickness of the acetabular cup cannot be further reduced, otherwise the metal shell would deform, compromising the efficiency of the bearing. As a palliative solution, pre-assembled ceramic cups are offered into the market. However, the thickness of the cup could be further reduced if the ceramic component would be directly implanted without the need of an osseointegrative metal shell. This would allow a metal-free solution with a reduced loss of the bone stock and the possibility to use larger ceramic ball heads, which would decrease the risk of dislocation. At the same time, the low friction arthroplasty guaranteed by CoC with a larger range of motion (ROM) may offer an improvement of the gate, thus enhancing the quality of life of active patients. Furthermore, such solution could be used for a CoC hip resurfacing arthroplasty, which would give the opportunity to have an all-ceramic answer to the hip arthroplasty market. Promising results were obtained on porous ZTA coatings, with a primary stability comparable to metal shells with plasma sprayed titanium coatings 2 .

The concept of "osseointegration" was first introduced by Brånemark in 1952 and was defined as the formation of a direct interface between an implant and bone, without intervening soft tissue. The future generation of ceramic implants targets the need for joint prostheses with increased ROM, which will have a substantial impact on the quality of life, especially for the young and more active patients. If adequate osseointegration could be achieved by solely the bio-ceramic component, the external metal part is no longer required and the overall thickness of the acetabular part of the prostheses could be reduced, thus making space available for a bigger ball with a larger ROM. Ideally, surface modifications technologies should allow changing the outer surface of the cup in contact with bone (for improved osseointegration) without affecting the inner surface of the cup in contact with the ball (preserving low friction) and without affecting the resistance to fracture.

Surface properties are orchestrating the first events after implantation, such as water adsorption, protein adsorption from blood and interstitial fluid, cell attachment, followed by the long-term integration into the native tissue 3 . Surface roughness affects osteoblast proliferation, differentiation, matrix synthesis and local factor production 4,5 . The majority of currently marketed oral implants are moderately rough, with a arithmetic average (R a ) in the range of 1 to 1.5 μm 6 . Surface modifications can be obtained with a broad range of techniques, among which blasting, acidic and alkaline treatment, coating and surface fictionalisation. Both mechanical and chemical surface modifications techniques (such as machining, grinding, polishing, blasting or acidic and alkaline treatment) have been used to improve the integration of alumina and zirconia with bone tissue, especially zirconia implants for the dental field 7,8 .

Chemical etching has been used to increase the roughness and the total surface area of an implant and most commonly utilized acid agents are hydrofluoric, nitric and sulfuric acid, or a combination of different acid solutions. The obtained roughness is a function of different parameters, e.g. solution concentration, temperature and etching time. In vivo studies have shown that osseointegration of acid-etched zirconia is comparable to that of acid-etched titanium 9 . Combinations of sand blasting and chemical etching have been used to produce surfaces with both micro-and nano-scale roughness. In the dentistry field, the majority of commercially available sand-blasted implant surfaces are subsequently acid-etched. In vivo, low-pressure injection moulded acid-etched zirconia showed similar osseointegration as sandblasted and acid-etched titanium 9 . Recently, chemical etching has been applied to microrough ZTA to achieve a selective removal of the zirconia phase from the surface, leading to an increase of the nano-roughness and the generation of surface porosity, without affecting the pre-existing micro-topography 10 .

One of limitations in the surface modification field is represented by the difficulty in modifying only one surface parameter at a time. Indeed, the majority of the roughness modification methods are influencing surface chemistry as well. Injection-moulding has been recently proposed for the fabrication of micro-rough zirconia 11 . With this technique, a wide range of micro-topographies can be obtained without affecting surface chemistry. Moreover, compared to sandblasting or grinding, this technique does not introduce additional surface defects. When implanted in rabbit tibia, micro-rough injection-moulded implants showed a significantly higher removal torque compared to smooth controls 11 . Another limitation is that most studies report on surface roughness focusing on R a , which is a two-dimensional profile parameter. Nonetheless, as roughness is a scale and resolution-dependent measurement, R a is not sufficient to characterize surface roughness. and functional parameter to characterize surface roughness of materials [12][13][14] .

The objectives of this study were: (i) to determine the optimal micro-roughness range of injection moulded ZTA and (ii) to assess whether further selective chemical etching of the micro-rough surface is beneficial for human primary osteoblast proliferation and maturation.

Cell behaviour was investigated on five different micro-rough surfaces without and with chemical etching and surface roughness was assessed using amplitude, spatial, hybrid and functional parameters. Primary human osteoblasts obtained from hip joints undergoing arthroplasty were chosen as a highly clinically-relevant cell source for this study. Cell proliferation, gene expression and alkaline phosphatase activity were followed up to 30 days of culture.

Materials and methods

Micro-rough and selectively-etched micro-rough zirconiatoughened alumina (ZTA)

Zirconia-toughened alumina (ZTA discs -20 mm diameter and 2 mm height) were produced from ceramic slurries by injection moulding as previously described 10 . Samples with same composition but five different grades of surface micro-roughness were obtained (Table 2.1):

mirror-polished after injection ("polished", S a = 13 nm), no treatment following injection in the unmodified mould ("as sintered", -S a = 203 nm) and increasing rough surfaces obtained by mould modification. The different rough surfaces, "low", "medium" and "high" (S a = 176 nm, S a = 330 nm and S a = 410 nm) were obtained by sandblasting the mould surface. The topographies of ZTA discs are reported in Fig. 11 Samples from all groups were either tested as sintered or following further surface modification by selective chemical etching. In the latter case, samples were immerged in a 40% hydrofluoric acid (HF) (QP Pancreac, Spain) bath at room temperature for 4 days (4 mL of acid per sample). Following etching, samples were cleaned twice with fresh DI water in an ultrasonic bath (ten minutes each time). Half of the samples underwent an additional treatment in 37% hydrochloric acid (HCl) for one hour. Therefore, for each roughness, three types of samples were obtained: micro-rough, micro-rough with HF etching, micro-rough with sequential HF and HCl etching. The HF treatment led to the selective removal of zirconia grains on the sample surface, thus creating a superficial zirconia-depleted nanoporous network. Since the alumina matrix was not affected, the nano-scale roughness introduced during the etching process was superposed on the pre-existing micro-roughness. 10 Following chemical attack, yttrium fluoride (YF 3 ) crystals were found on "polished" and "medium" surfaces (Fig. 2.1), while no crystals were observed on "as sintered" and "low" and few on the "high". YF 3 crystal removal could be achieved with HCl (Fig.

Scanning electron microscope micrographs of the zirconia-toughened alumina (ZTA) surfaces. a-e) Comparison between the different micro-rough ZTA surfaces: a) "polished", b) "as sintered", c) "low", d) "medium", e) "high" roughness surface; f-h) comparison of polished surface before and after etching with hydrofluoric acid (HF) or HF followed by hydrochloric acid etching (HF + HCl): f) polished, g) polished + HF and h) polished + HF+HCl. Scale bar = 5 µm.

Following chemical attack, yttrium fluoride (YF 3 ) crystals were found on "polished" and "medium" surfaces (Fig. 2.1), while no crystals were observed on "as sintered" and "low" and few on the "high". YF 3 crystal removal could be achieved with HCl (Fig. 2

Cleaning and sterilisation of ZTA samples

Prior cell culture testing, ZTA discs were cleaned in order to remove organic and inorganic contaminant molecules and sterilized. Firstly, ZTA discs were cleaned by sonication in 10% v/v Alsar25/7 detergent (S3639 Alsa-Chemie, Germany) in deionised water at 56°C for 10 minutes followed by serial washes of 10 minutes each in deionised water. Samples were cleaned with 99% ethanol followed by rinsing in deionised water three times for 10 minutes in a sonication bath. Cleaned discs were placed into 24 well plates and sterilised with ethylene oxide.

A cleaning protocol was established in order to reuse the ZTA discs after in vitro evaluation with each donor. Firstly, the ZTA discs were bleached for 1 hr in a 1% v/v sodium hypochlorite solution in deionised water at room temperature. Secondly an enzymatic detergent, Terg-a-Zyme (Z273287 Sigma-Aldrich) was used for further cleaning in a sonication bath (37, 56 and 100°C, each step for 10 minutes) followed by extensive rinsing in deionised water. In order to remove any organic debris, a calcination at 600°C for 4 hours was performed. After this step, samples followed the preparation steps described above (from cleaning with Alsar25/7 to ethylene oxide sterilisation).

Human primary osteoblast isolation and expansion

Human primary osteoblasts (hObs) were isolated from five different donors (age: 76.0 ± 8.9 years; Ethics Committee Zürich KEK 2010-0444). Mechanical isolation was used to collect three different yields of migrating osteoblasts. 15 Primary outgrowing cells from the second yield were expanded in DMEM 1% glucose (Gibco #11885-084) supplemented with 10% foetal bovine serum (Gibco #10500-064), 1% penicillin-streptomycin (Gibco #15140-122) and 50 μg/mL ascorbic acid (Sigma #A8960) and used at passage 5 for evaluating the ZTA discs.

Human primary osteoblast seeding on ZTA discs

The sterile ZTA discs placed into 24 well plates were incubated in basal medium (DMEM 1% glucose supplemented with 10% foetal bovine serum and 1% penicillin-streptomycin)

and degassed prior to cell seeding. hObs were seeded at a density of 1x10 4 cells/cm 2 (optimised cell suspension volume to cover the whole sample surface: 50 µL for micro rough and 80 µL for combined micro-nano-rough samples). Thermanox TM tissue (TMX) culture coverslips (diameter 13 mm) (Thermo Scientific) were used as control and seeded with the same cell density. Seeded samples were left at 37°C for 30 minutes after which time 0.8 mL of osteogenic differentiation medium (basal medium supplemented with 50 μg/mL ascorbic acid, 10 -7 M dexamethasone -Sigma #D4902 and 5 mM β-glycerol-2-phosphate -Sigma #G9422) was added to each well. Samples were cultured for 30 days at 37°C with time points for analysis at 10, 20 and 30 days. For each time point and analysis, duplicates of each sample type were used. The experiments were repeated with three different hOb donors.

Human primary osteoblast attachment and proliferation

Cell attachment was assessed at 24 hr after seeding by quantifying the DNA content, while hOb proliferation was evaluated at days 10, 20, 30. At each time point, cells were rinsed once with phosphate buffered saline solution (PBS, pH = 7.4) and lysed with 0.5 mL of 0.1%

Triton-X (Sigma #T8787) in 10 mM Tris-HCl pH 7.4 for 2 hours at 4°C on a gyratory shaker and stored at -80°C until further analysis. CyQuant cell proliferation assay (Molecular Probes #C7026) was used to determine the number of cultured cells. 16 The fluorescence of each sample was measured in duplicates with 485 nm excitation and 530 nm emission filters using a plate reader (Multilabel Counter Wallac 1420, Perkin Elmer, US). The amount of DNA in each sample was calculated based on a standard curve using α-DNA.

Alkaline phosphatase activity

The alkaline phosphatase (ALP) activity was quantified from the same lysate as for DNA, as previously reported. 16 Prior to analysis, 4-nitrophenyl phosphate disodium salt hexahydrate (Sigma #N2765) was used as substrate solution and the reaction mix was incubated for 15 minutes at 37°C in an incubator. Serial dilutions of 1 mM p-nitrophenol (Sigma #N7660) were used for the standard curve. The absorbance was measured at 405 nm in duplicates with a plate reader (Multilabel Counter Wallac 1420, Perkin Elmer, US). The enzyme activity was expressed as µmoles of p-nitrophenol liberated per min and normalized to the amount of DNA of each sample.

Gene expression

Total RNA was extracted using TRI-reagent® (MRC) according to the manufacturer instructions. cDNA was obtained by reverse transcription with 500 ng of total RNA per sample using TagMan reverse transcription reagents (Applied Biosystems, CA) with random hexamer primers. Specific oligonucleotide primers and TaqMan probes (Microsynth, Switzerland) and Assays on Demand (Applied Biosystems) were used for gene detection (Table 2.3). PCR was performed on a Quant Studio 6 Flex (Applied Biosystems); the reaction conditions were set at 95°C for 10 minutes, followed by 40 cycles of amplification at 95°C for 15 seconds and 60°C for 1 minute. Ribosomal protein L13a (RPL13A) was used as endogenous control and data were normalized to cells collected on the day of seeding (day 0). Fold-changes in expression were calculated using the 2 -ddCt method.

Alizarin Red staining

In order to assess the calcification ability of hObs on the ZTA surfaces, a qualitative mineralization assay was performed using Alizarin Red S dye (Sigma #A5533). Prior to staining cell layers were washed twice with phosphate buffered saline solution followed by a 15 minute fixation in 4% buffered paraformaldehyde. Samples were washed with deionized water and incubated with 40 mM Alizarin Red S solution on a rotating plate at room temperature for 1 hr. Finally, samples were washed five times with deionized water and stored at 4°C until imaging in transmitted light mode with Axioplan 2 microscope (Zeiss).

Duplicates were used for each analysis and time point. According to the sample distribution, parametric (one-way Anova with Tukey post-hoc) or non-parametric (Kruskall-Wallis test and Dunn post-hoc) analyses were performed (p<0.1 were considered trends, p<0.05 were considered significant differences) using GraphPad Prism 7.01 software (GraphPad). Grubb's test (α=0.2) was used for outlier identification.

Results

Human primary osteoblast proliferation on micro-rough ZTA

and etched micro-rough ZTA

Micro-rough ZTA discs

hOb proliferation on micro-rough ZTA was slightly (but not significantly) lower compared to "polished" ZTA, as attested by the lower DNA content at all time points for the microrough surfaces (Fig. 2.2 a). This trend was observed for 4 out 5 donors (Fig. 2.2).

Etched micro-rough ZTA discs

Overall, selective chemical etching with HF and combined HF+HCl induced minimal change in hObs proliferation compared to the micro-rough control samples and this was the case for all donors and all roughnesses (Figs. 2.2 b-f). At day 10, the DNA content on the chemicallyetched surfaces was similar or lower than on the micro-rough surfaces, but these differences were levelled out at later time points (day 20 and 30). Inter-donor variability was observed in the cell proliferation rate, but similar profiles were observed as a response to etching (Fig. 2.2).

Alkaline phosphatase activity of human primary osteoblasts cultured on micro-rough ZTA and etched micro-rough ZTA

Micro-rough ZTA discs

Our results suggest the existence of an optimal micro-roughness range which stimulates ALP activity in hObs cultured on ZTA. Indeed, the highest ALP/DNA ratio was observed on "low" and "medium" discs at day 30 (Fig. 2.4 a). When comparing "as sintered" and "polished" surfaces, the ALP/DNA ratio was similar or higher on "as sintered" surface for all donors.

Overall, the "low" surface induced an earlier and higher ALP/DNA ratio (4 out of 5 donors).

GraphPad Prism 7.01 software was used for the statistical analyses and post parametric (oneway Anova with Tukey post-hoc) or non-parametric (Kruskall-Wallis test and Dunn posthoc) analyses, p<0.1 were considered trends, p<0.05 were considered significant differences.

Overall when talking about the micro-roughness only induced only trends in hObs behaviour.

Etched micro-rough ZTA discs

The highest ALP/DNA ratios were observed for the chemically-etched "as sintered", "low" and "medium" surfaces. These groups had a higher ALP/DNA ratio than that of the corresponding micro-rough samples (Fig. 2.4 c-e). In addition, statistically significant higher ALP/DNA ratios were found already at day 10 on the etched surfaces for "as sintered" and "medium" compared to the corresponding untreated micro-rough controls (p<0.05). Chemical etching of "polished" and "high" groups induced only minimal changes in ALP/DNA compared to untreated controls (Fig. 2.4 b,f). Within the "medium" surface, for all donors the HF treatment led to the highest ALP/DNA ratio at day 20. Importantly, relatively high ALP/DNA ratios were already observed at day 20 for the HF treated-samples, while similar ALP/DNA values were achieved only at day 30 for the HF+HCl treated surfaces; this was the case for all the surfaces ("polished", "as sintered", "low", "medium", "high"). The expression of RUNX2, an early osteogenic marker, was the highest on the "low" surface at all time points (Fig. 2.5 a). An up-regulation of RUNX2 mRNA levels compared to day 0 was observed for the "low" surface after day 10 for 3 out of 5 donors. Similar RUNX2 expression levels were measured among "polished", "as sintered", "medium" and "high"

surfaces. The investigation of ALP gene expression (Fig. 2.6 a) revealed consistent results

with the ALP protein levels (Fig. 2.4 a), with the highest expression of the ALP gene in the "low" and "medium" surfaces at both day 20 and 30 of culture. ALP expression on "low" surface showed a peak at day 20 (Fig. 2.6 a) and this was observed in for 3 out of 5 donors.

For the other groups ("polished", "as sintered", "high") a lower ALP expression level was found and no peak was observed over the culture period. Collagen-1 (COL1), an extracellular matrix protein, involved in the osteoid maturation, showed a similar gene expression among the "low", "medium" and "high" groups at the different time points (Fig. 2.7). The expression of osteocalcin (OC), a pre-osteoblastic and bone-building protein, showed a slight increase over time for all micro-roughnesses ("low", "medium", "high") (Fig. 2.8).

Etched micro-rough ZTA discs

Overall, chemical etching induced an early increase in RUNX2 gene expression levels in all micro-rough groups compared to not-etched controls, except for the "high" one (Fig. 2.5 b, f).

Significantly higher RUNX2 gene expression was observed at day 10 on HF-etched "as sintered" surfaces compared to the untreated control. For all groups, an earlier increase in ALP expression was observed for the HF treated samples (already at day 10) (Fig. 2.6 b-f).

Indeed, at day 10, ALP gene expression was highest in the HF-treated group, followed by the combined HF+HCl treated group and not-chemically etched ZTA. Statistically significant higher ALP gene expression was seen at day 10 on the HF-etched "medium" group compared to not-etched control. This result supports the ALP activity data, which showed an earlier increase of ALP activity in hObs cultured on the HF-treated surfaces compared to both HF+HCl treated and untreated surfaces (Fig. 2.4). When analysing the individual donor data, for 2 out of 3 donors a clear increase in mRNA levels was seen with the chemical treatment.

The effect of chemical etching on the expression of COL1 was less evident (Fig. 2.7). OC gene expression was similar or higher on the etched surfaces compared to the not-etched controls for the "as sintered", "low" and "medium" surfaces, starting at day 10 (Fig. 2.8).

Figure 2.5 RUNX-2 mRNA expression relative at day 10, 20 and 30 of culture relative to day 0 control: a) Comparison between the different micro-rough ZTA surfaces; b-f) comparison of the surfaces before and after etching HF or HF + HCl. b) Polished, c) As sintered, d) Low, e) Medium, f) High roughness surfaces.

Figure 2.6 ALP mRNA expression relative at day 10, 20 and 30 of culture relative to day 0 control: a) Comparison between the different micro-rough ZTA surfaces; b-f) comparison of the surfaces before and after etching HF or HF + HCl. b) Polished, c) As sintered, d) Low, e) Medium, f) High roughness surfaces.

Figure 2.7 Collagen type I (COL1) mRNA expression relative at day 10, 20 and 30 of culture relative to day 0 control: a) Comparison between the different micro-rough ZTA surfaces; b-f) comparison of the surfaces before and after etching HF or HF + HCl. b) Polished group, c) As sintered group, d) Low roughness group, e) Medium roughness group, f) High roughness group.

Figure 2.8 Osteocalcin (OC) mRNA expression relative at day 10, 20 and 30 of culture relative to day 0 control: a) Comparison between the different micro-rough ZTA surfaces; b-f) comparison of the surfaces before and after etching HF or HF + HCl. b) Polished group, c) As sintered group, d) Low roughness group, e) Medium roughness group, f) High roughness group.

Alizarin red staining of etched micro-rough ZTA

Unfortunately, Alizarin Red S staining was not very informative, as the cell layer had tendency to fold up and/or detach during the culture period and the numerous washing steps of the staining procedure (Fig. 2.9).

Figure 2.9 Alizarin red stained "medium" roughness ZTA discs at day 20 (a-c) and day 30 (d-f) of culture: a,d) untreated, b,d) HF-etched, e,f) HF+HCl-etched "medium" surface. Black arrows indicate cell layer starting to roll, white arrow show cell aggregates. Scale bar = 1 mm.

Discussion

In comparison to sandblasting or grinding, injection-moulding in roughened moulds has several advantages: (i) no additional surface defects are produced, (ii) high flexibility in the choice of the micro-topography pattern and (iii) up-scalability to mass production of components with complex geometry. Additionally, it has been shown that combination of injection-moulding on roughened moulds and selective etching resulted in only moderate changes of mechanical properties. 10 The goal of this study was to determine the optimal micro-roughness range of ZTA obtained by injection-moulding in roughened moulds and to assess whether additional selective etching is beneficial for human primary osteoblast proliferation and maturation.

Our results suggest that micro-scale roughness alone is not sufficient to trigger hOb maturation on ZTA surfaces. DNA content was similar among the different micro-rough samples (Fig. 2.2 a), and a weak trend for higher ALP activity (Fig. 2.4 a), RUNX2 (Fig. 2.5 a) and ALP (Fig. 2.6 a) mRNA expression was observed at both day 20 and 30 on "low" > "medium" > "high"/"polished"/"as sintered" samples. Therefore, among the different microrough samples, the highest mineralization potential was on ZTA with S a ~ 0.2 µm, S ds ~ 45000/mm 2 , S dr ~ 45 % and S ci ~ 1.1 nm. This is in agreement with previous studies on zirconia, whereby an osteoblastic cell line (MC3T3-E1) had increased ALP activity on microrough compared to mirror-polished samples. 17 However, the optimal roughness range is still under debate as the material type, the method used for creating micro-roughness, the cell type and osteogenic supplements in the culture medium, the characterization technique used for surface evaluation will all influence the outcome.

The combination of micro-and nano-scale roughness had the strongest effect on hOb behaviour. This is in agreement with the general concept that each roughness scale contributes to improved osseointegration, with nano-roughness influencing protein absorption, micro-roughness acting on cells and macro-scale roughness contributing to mechanical interlocking of an implant. 4,5 In our study, "as sintered", "low" and "medium" etched groups showed a statistically significant enhancement in ALP/DNA ratio already at day 10 compared to micro-rough control, thus indicating an earlier mineralization commitment. At later time points, a statistically enhanced ALP/DNA ratio was observed for HF-etched "medium" roughness compared to micro-rough only control. These findings suggests that by superimposing nano-roughness (by selective chemical etching) on top of S a = 330 nm ("medium" roughness) an earlier and higher ALP activity can be triggered both at the protein (Fig. 2.4) and gene (Fig. 2.6) levels. A similar behaviour was observed also on yttriastabilised zirconia, whereby an earlier increase in ALP activity in osteoblast-like MC3T3-E1 cells was observed on sand-blasted and etched surfaces compared to sand-blasted control. 17 Furthermore, our results indicate that osteogenic marker mRNA levels were up-regulated on the etched ZTA surfaces compared to the micro-rough controls. Interestingly, the expression of COL1 gene was higher on etched "as sintered" and "medium" roughness surfaces (Fig. 2.6 and Fig. 2.7), possibly indicating a mature osteoblast phenotype since ALP activity has a crucial role in osteoblast maturation and its expression coincides with the synthesis of type I collagen. 18 A decrease in RUNX2 gene expression was seen on the low rough etched surface (Fig. 2.5 d), which could be associated with earlier osteoblastic maturation. Indeed, RUNX2/CBFA1 is an important transcription factor in osteogenesis and is one of first genes to be up-regulated when cells commit to this pathway.

HF treatment not only led to the development of a nano-porous layer, but induced at the same time changes in the surface chemistry, associated to the formation of YF 3 crystals. X-ray photoelectron spectroscopy confirmed that the most notable change following etching was a decrease in zirconium and an increase in fluorine content. 10 The presence of a fluorineenriched nano-porous layer proved to be beneficial for hOb maturation. Indeed, HF etching of "polished" ZTA induced an earlier and higher increase in ALP/DNA ratio (Fig. A three-dimensional characterization of the material surface is essential to obtain a clear overview of the surface topography and help understanding cell response to surface modifications. Considering two surfaces may have the same height variation but differ in spatial distribution, cells will experience a completely different environment. 21 The use of scanning electron micrographs and multi-scale roughness analyses (e.g. with white light interferometry and atomic force microscopy) with adequate filtering procedures in order to exclude form and waviness errors is recommended. We have found that despite the fact that a strong increase in S a , S ds and S dr could be achieved by using micro-rough moulds for injection-moulding of ZTA, micro-scale roughness had only minimal effect on hOb behaviour. Interestingly, the most promising group was the "low" surface, suggesting the existence of an optimal micro-roughness range for hOb maturation on ZTA surfaces, as previously suggested for titanium surfaces. 22 On the other hand, when micro-and nano-scale roughness were combined, the most promising hOb maturation was obtained for ZTA surfaces with the highest S ci (after filtering) at both scales. Indeed, the highest ALP activity at both gene and protein level was detected on the HF-etched "medium" surface. This highlights the importance of performing multi-scale surface characterization and analysis of amplitude, spatial, hybrid and functional parameters. [12][13][14] The importance of including hybrid and functional parameters as S dr and S ci has also been highlighted, among others, by Eliaz et al.,

who have stressed their importance of S dr and S ci when analysing the response of osteoblastlike MBA-15 cells to hydroxyapatite-coated titanium. 23 The choice of the relevant cell type for the in vitro assessment of materials is also of key importance. Indeed, osteoblast response to surface topography varies among cell lines and osteoblast maturation state. 24,25 For hip implants, human primary osteoblasts from joints of patients undergoing hip replacement surgeries are the most clinically-relevant cell type.

Nonetheless, most of the studies assessing the impact of surface modifications on cell behaviour are performed using human or animal cell lines which, due to differences in tissue origin and high passage number, may not fully represent the clinical scenario. 26 One limitation in using human primary osteoblasts compared to cell lines is the high inter-donor variability, which was also observed in the present study. Nonetheless, this better reflects the real setting and can hopefully contribute to bridge the gap between in vitro, in vivo and clinical applications. Surface modifications will impact the overall response of the body to the implant. Osteoblasts represent only one, albeit important, part of the equation and it appears important in addition to assess how surface modifications affect bacterial and immune responses, either separately or as co-cultures. 27,28 Recently, Flamant et al. have

shown that HF-etched ZTA (without antibiotics addition) could reduce E. coli adhesion compared to non-etched ZTA. 10 Therefore, HF-etched micro-rough ZTA may promote osseointegration while reducing bacterial adhesion. The nano-porous layer on ZTA can be further used for drug delivery. For instance, it has been shown that liposome encapsulated gentamicin could be loaded onto HF-etched ZTA and limit E. coli growth on ZTA compared to non-loaded controls. 10

Conclusions

Selective chemical etching proved to be an innovative method for the introduction of nanofeatures on micro-rough surfaces obtained by injection moulding, leading to enhanced osteoblastic markers at both gene and protein level compared to micro-rough controls. Our results suggest that micro-roughness, fluorine enrichment and nano-porosity at the surface of ZTA play a synergistic role on human osteoblast maturation. Among the tested groups, the surface of choice would be the hydrofluoric acid etched "medium" roughness zirconiatoughened alumina, as it showed the highest and/or earliest ALP expression at both the protein and gene level. On the other hand, micro-roughness or nano-roughness alone induced only minor effects on hOb maturation on ZTA. These findings indicate that hObs behaviour is not affected by a single surface characteristic; instead, it can be described as an orchestrated feedback to a sum of different surface features as a whole. Combined micro-/nano-scale roughness is required in order to guide cell behaviour towards a mature osteoblast phenotype. Future work will focus on the evaluation of protein adsorption and early cell response, as well as co-cultures of human primary osteoblasts and osteoclasts, which could better mimic the in vivo response to an implant. Further in vivo studies are required to determine if this surface treatment induces an adequate osseointegration with host bone tissue and contribute toward the long-term goal of producing prostheses with a wider range of motion by using ZTA components in direct contact with bone.

Chapter 3

Robocast zirconia-toughened alumina: can a 3D architecture influence osteoblasts behaviour?

Abstract

Zirconia-toughened alumina (ZTA) is gold standard ceramic in hip arthroplasty. Currently, a metal shell is required for adequate osseointegration of ZTA cementless hip prostheses, but with direct ZTA osseointegration, this component could be omitted with strong impact on patient life quality. Robocasting holds strong potential to produce an architectured ceramic layer on a precise part of an implant, but has not yet been applied to ZTA. This study aimed to robocast ZTA scaffolds (3D-ZTA) and assess their potential. Stable, well-dispersed, high solid loading ink was robocast. X-ray tomography analyses showed regular strut spacing and fully interconnected macro-porosity. Struts were rough and microporous (attested by scanning electron microscopy and mercury intrusion porosimetry). Human primary osteoblasts (hObs) were homogenously distributed inside 3D-ZTA, but cell retention was not complete. Earlier increase in RUNX2 and alkaline phosphatase (ALP) gene expression but lower cell proliferation and ALP activity were observed on 3D-ZTA compared to 2D-ZTA.

Keywords: bimodal pore distribution; robocasting; surface topography; zirconia-toughened alumina.

Introduction

Zirconia-toughened alumina (ZTA) is the gold standard ceramic bearing material in hip arthroplasty today, thanks to its excellent mechanical properties, biocompatibility, and low friction. Currently, ZTA cementless hip prostheses need an osseointegrative metal shell on the acetabular side to offer an adequate osseointegration with the surrounding bone. If an adequate osseointegration could be achieved directly on ZTA, the metal shell could be omitted with a strong impact on the patient quality of life. Indeed, a higher range of motion and lower dislocation rate could be obtained with a purely ceramic solution.

The challenge of implant osseointegration has been widely studied and numbers of studies have shown that surface topography at several scales improves osseointegration, as recently reviewed by Gittens et al. 1 . The current opinion is that three levels of topography participate in implant osseointegration: (i) macroscale topography, having sizes in a range of hundreds of microns to several millimetres, which guarantees an adequate mechanical interlocking with native bone; (ii) Microscale topography with features having sizes starting from hundreds of nanometres to tens of microns, which promote cell attachment, proliferation and differentiation; (iii) nanoscale topography, which facilitates water and protein adsorption (and which in turn affect cell behaviour) 1 . Several studies have shown that multi-scale rough zirconia implants have similar osseointegration to titanium implants with similar topographical features 2,3 .

The development of a multi-scale rough surface on a ZTA acetabular cup of a hip prosthesis is complex since only the outer side of the cup (in contact with bone) should be rough, while the inner side of the cup (in contact with the ball) should be smooth to guarantee low friction motion in the articulation. Therefore, widespread techniques, such as sand-blasting followed by acid etching, may not be easily applicable to the acetabular cup component. Instead, macroporous ZTA coatings have been proposed. Theelke et al. have obtained macroporous ZTA coatings by depositing a ceramic slurry with pore-forming agents onto axially pressed and green-machined bodies; such implants with a macroporous ZTA coating showed a primary stability on artificial bone comparable to plasma-sprayed titanium-coated metal shells [4]. Despite these promising results, further optimisation is required before clinical application of purely-ceramic acetabular cups. Indeed, the surface layer should have an excellent bond to the dense part of the component, not reduce the mechanical performance of the implant below an acceptable level (especially not reduce its strength), and provide good reproducibility and predictability of both mechanical and biological behaviour. Additive manufacturing techniques (AM) hold a strong potential to fulfil these requirements.

Additive manufacturing, also known as solid freeform fabrication (SFF), rapid prototyping (RP) or 3D printing is based on layer-by-layer assembly of different geometries directly generated from a virtual model. These technologies are used to produce 3D objects in their final shape without the need of additional moulds or tools for post-processing. Robocasting is an additive manufacturing technique in which a shear-thinning ink (typically a ceramic slurry) is extruded as a filament from a nozzle and used to form objects layer by layer. In robocasting, a Computer Aided Design (CAD) model of the object is first created, then converted to a Standard Tessellation Language (STL) file which is transferred to the printer to achieve computer-controlled layer-by-layer extrusion of the ink (based on a ceramic slurry) onto the substrate. The main advantage of robocasting technique (also known as direct ink writing, direct-write assembly, 3D deposition, 3D fibre deposition or extrusion free form) is the ability to create specific designs with high accuracy in dimensions and structural features.

This characteristic is fundamental for meeting the clinical needs allowing a precise control of internal morphology, shape, distribution and connectivity, with high reproducibility. In addition, robocasting is a versatile technique which has been used to print several types of ceramics with well-controlled architecture, including β-tricalcium phosphate 5 , hydroxyapatite 6 , combinations of those 7,8 and alumina 9,10 . Furthermore, robocasting technique offers the possibility to use multi-material systems, such as ceramic-ceramic with functional grading, ceramic-plastic or ceramic-metal interfaces. Robocasting technique has the potential of becoming a tool for specific ceramic fabrication that cannot be achieved by other existing methods and it can provide a fast and cheap fabrication method for complex structures. Schlordt et al. reported on alumina structures produced by robocasting, whereby hollow alumina filaments were obtained by robocasting an aqueous alumina gel in an oil bath using relatively big (inner diameter/length ~0.75 mm), circular or quadratic stainless nozzles 9 . In another study, Moon et al. reported micro-and macro-porous alumina scaffolds, which were comprised of hollow microporous filaments obtained by 3-D co-extrusion (3D-CoEx) of alumina/camphene-based slurry. The scaffolds were composed of hollow microporous alumina filaments (500 μm in diameter) separated by interconnected macropores (~250-300 μm x 400-500 μm) 10 .

To the best of our knowledge, robocasting of ZTA has not been reported so far. One of the major challenges in robocasting is the preparation of a suitable ink or slurry with high reproducibility. Therefore, in the present study we aimed to (i) develop a ZTA ink suitable for robocasting, (ii) process and characterise robocast scaffolds and (iii) investigate human primary osteoblasts (hOb) response on robocast ZTA in vitro. The ink contained only minimal organic components, and was in fact an optimised, stable liquid slurry with very good dispersion of the ceramic particles and high solid loading (70 wt.%, i.e. 35.5 vol.%).

Coagulation of this ink only occurred when robocasting was performed in an acidic bath.

Scaffolds were characterised by scanning electron microscopy, X-ray tomography and mercury intrusion porosimetry. Human primary osteoblast adhesion, proliferation and maturation were assessed at day 10, 20 and 30 of culture.

Materials and methods

Zirconia-toughened alumina (ZTA) ink development, scaffold fabrication and characterisation

An alumina (HPA, Sumitomo Chemical, Japan)zirconia (TZ-3YS-E, Tosoh, Japan) (wt.:wt.= 84:16) ink was prepared in 1.5 vol.% Darvan C-N (Darvan® 811, Vanderbilt Minerals, LLC #14255) dispersant and 2 wt./vol.% carboxymethyl methylcellulose (Fluka #21901) in deionised water. Briefly, the carboxy-methylcellulose was dissolved in deionised water on a magnetic stirrer for 1 hr at room temperature. Darvan C-N was added to the 2 wt./vol.% carboxy-methylcellulose solution and subsequently the ceramic powders were incorporated step by step under stirring conditions. The ceramic slurry was left overnight under stirring (ball-milling, with 150 g of 2 mm zirconia balls for 100 ml of ceramic slurry) in order to achieve a homogenous dispersion and prevent aggregation.

Prior robocasting the ceramic ink was degassed in a vacuum desiccator for 2 hr in order to ensure a continuous and uniform flow during printing. Subsequently, the injection syringe was filled with the ink, tapped vigorously for bubbles removal and then fixed on the motion stage controlled by a computer-aided direct write program (Robocad 3.0, 3-D Inks, Tulsa, OK, USA). The ceramic paste deposition was performed with a robocasting machine (3D inks, Tulsa, OK, USA) using a 330 μm diameter nozzle (EFD # 7018260, Nordson, USA) in an acidic water bath, pH=1.5 (deionised water and HCl) over an alumina support for scaffold shape retention and easy detachment respectively. Cylindrical structures (diameter =15 mm) with strut-to-strut distance of 330 µm (in the XY plane) and a dense double ring around were deposited by robocasting (3D-ZTA), each layer being separated from the other by 80% of the strut-to-strut distance (thus 264 µm). The ceramic structures included 7 layers with a 50% shift in the layer to layer geometrical design. Following printing, scaffolds were dried for 48 hr at room temperature, debinded at 600°C (2°C/min heating rate) and sintered at 1350°C for 2 hr (5°C/min heating rate).

As two-dimensional control (2D-ZTA) for cell culture studies, the same ceramic slurry was cast on porous plaster molds, dried, debinded and sintered as above described to achieve 13 mm diameter 2D-ZTA disks. Thermanox TM (TMX) tissue culture coverslips (Thermo Scientific) (diameter = 13 mm) were used as an additional control for cell cultures.

Morphological and structural characterisation

Scanning electron microscopy

Morphological and microstructural analyses were performed using a scanning electron microscope (SEM) (Zeiss Supra55 VP, Germany). A low accelerating voltage of 2 kV was used for the secondary electron (SE) imaging mode in order to avoid sample metallisation.

For backscattered electron (BSE) mode, an AsB detector was used with a higher accelerating voltage (20 kV) and at a 20 Pa pressure. SEM micrographs were used for quantification analysis: grey areas were assigned as ceramic struts, while black areas were assigned to the pores. For both strut and pore size, 20 measurements were performed.

X-ray micro-tomography (CT)

Structural evaluation was performed by X-ray computed tomography (CT) with a Phoenix v/Tome/x (General Electric USA) equipped with a Varian Paxscan detector (1920 x1536 pixels) and an X-ray polychromatic conical beam (focal spot size of 1-4 μm). The experiments were performed at a voltage of 120 kV and a current of 120 µA, with a voxel size of 6 µm 3 . Following 3D reconstruction, the strut and pore sizes (n = 10) were analysed along the two main orthogonal axes. The data segmentation and quantification was performed using ImageJ and 3D Slicer software (http://www.slicer.org) 11 .

X-ray micro-tomography (CT)

Distribution and size of interconnections between pores were determined by mercury intrusion porosimetry (MIP) (Autopore IV, Micrometrics). Pressures ranging from 3.5 kPa to 200 MPa were used and equivalent interconnection diameter was calculated according to Washburn's equation.

Human primary osteoblast (hOb) behaviour on 2D-and 3D-ZTA

Cleaning and sterilisation prior cell cultures

Prior in vitro evaluation 2D-and 3D-ZTA samples underwent cleaning and ethylene oxide sterilisation. Organic and/or inorganic contaminants were removed from the sample surface with a 10 vol.% solution of Alsar25/7 detergent (S3639 Alsa-Chemie, Germany) in deionised water for 10 minutes in an ultrasonic bath set at 56°C, followed by 3 serial washes in deionised water, 70% ethanol, and deionised water again. Samples were sterilised with ethylene oxide.

hOb isolation and expansion

Human primary osteoblasts (hObs) were obtained from femoral heads of patients undergoing total hip arthroplasty (three donors -Table 3

hOb seeding and culture on 2D-and 3D-ZTA

ZTA scaffolds were degassed and incubated in expansion medium without ascorbic acid for 1 hr prior to cell seeding. Following medium removal, passage 5 hObs were seeded on 2D-and 3D-ZTA samples at a density of 1x10 4 cells/cm 2 (~7.7 x10 4 cells/scaffold based on scaffold developed surface area, taking into account the struts but not the micropores). The cell suspension volume was optimised and 30 µL were applied on both sides of the scaffold to ensure a homogenous seeding. After seeding, scaffolds were incubated at 37°C for 30 minutes, after which time 0.8 mL of osteogenic differentiation medium (DMEM 1% glucose, 10% FBS, 1% P/S, 50 μg/mL ascorbic acid and 5 mM β-glycerol-2-phosphate -Sigma #G9422) was added to each well. Samples were cultured in an incubator set at 37˚C and 5% CO 2 for 30 days with medium changes every 3 days. Samples were collected for analysis at 10, 20 and 30 days of culture. Duplicates of each sample type were used for each time point and analysis. The experiment was repeated with hObs from three different donors.

Thermanox (TMX) was used as control surface.

After each experiment, 2D-and 3D-ZTA samples were cleaned, sterilised and used again for the next experiment. The cleaning protocol consisted of 5 steps: (i) a 10 vol.% bleach solution in deionised water for 1 hr at room temperature (as a first disinfection step), (ii) a 10 wt./vol.% Terg-a-Zyme solution (Sigma-Aldrich #MKBS2838V) in deionised water for removal of protein residues (10 minutes each at 37, 56 and 100°C in a sonication bath), (iii) extensive rinsing in deionised water (to remove the Terg-a-Zyme enzymatic detergent), (iv)

burn-out at 650°C for 5 hr (heating at 1°C per minute) to remove any organic residues, and (v) cleaning with Alsar25/7 solution (a detergent used to clean the ceramic surface) as above described.

hOb proliferation by DNA quantification

hOb proliferation was evaluated at days 10, 20, 30 of culture. After rinsing with 1 mL of phosphate buffered saline solution (PBS, pH = 7.4), cells were lysed with 0.5 mL of 0.1 vol.% Triton-X (Sigma #T8787) in 10 mM Tris-HCl pH 7.4 for 2 hr at 4°C on a gyratory shaker and stored at -80°C until further analysis. CyQuant cell proliferation assay (Molecular Probes #C7026) was used to determine the number of cultured cells. Each sample was measured in duplicates with 485 nm excitation and 530 nm emission filters using a plate reader (Multilabel Counter Wallac 1420, Perkin Elmer, US). The amount of DNA in each sample was calculated based on a standard curve using λͅ -DNA.

Alkaline phosphatase activity quantification

The enzyme activity was determined from the same lysate as for DNA, as previously reported 13 . Briefly, 4-nitrophenyl phosphate disodium salt hexahydrate (Sigma #N2765) was used as substrate solution and the reaction mix was incubated for 15 min at 37°C.

Mineralisation assessment by alizarin Red S staining

Qualitative mineralisation assessment was performed by using 40 mM Alizarin Red S dye (Sigma A5533). At each time point, 2D-and 3D-ZTA were washed twice with phosphate buffered saline solution followed by a 15 min fixation with 0.5 mL of 4% buffered paraformaldehyde, and two washes with deionised water (5 min each). Subsequently, samples were incubated with 0.5 mL of 40 mM Alizarin Red S solution on a rotating plate at room temperature for 1 hr. After incubation, ZTA scaffolds were washed five times with deionised water and stored at 4°C until imaging in reflected light mode (Axioplan HRc Zeiss).

Statistical analyses

Three different donors and duplicates were used for each analysis and time point in this study. GraphPad Prism 7.01 software (GraphPad) was used to perform all statistical analyses.

According to the sample distribution, parametric (one-way Anova with Tukey post-hoc) or non-parametric (Kruskall-Wallis test and Dunn post-hoc) analyses were performed and p<0.05 were considered significant differences.

Results

Morphological and structural features of 2D-and 3D-ZTA

Scaffold structural features

The microstructure of 3D-ZTA scaffolds obtained by robotic deposition is shown in Fig. 3.1.

The struts were homogenous in thickness and spacing, although some bending of the robocast filaments was also detected (Fig.

SEM micrographs of 3D-ZTA : a) overview (scale bar = 100 μm); (b) magnified view (scale bar = 10 μm); (c) detail of a strut surface, showing sub-micron-sized pores homogeneously distributed (scale bar = μm); (d) surface composition (in BSE mode, scale bar = μm), showing a homogenous distribution of the alumina (grey spots) and zirconia (brighter spots) and the porosity

at the grain boundaries.

CT analysis

The CT scans revealed some irregularities of the 3D-ZTA structures, mainly located on their upper surface and not affecting their overall homogeneity (Fig.

a) region of interest (ROI) on which density, struts and pores dimensions were measured; b,c) 3D views of the ROI (respectively XY and YZ planes); d) cross section of the ROI (X,Y plane, scale bar = 500 μm).

Porosity evaluation

Mercury intrusion porosimetry measurements revealed a bimodal pore size distribution for the robocast structures (Fig. 3.3). Interconnected pores with a distribution of equivalent diameters centred on 163 µm were observed for the 3D-ZTA structures. A second pore population (pore access diameters around ~100 nm) was observed for both the 3D-ZTA and the 2D-ZTA structures and is related to the partial sintering conditions. Since the whole sample was analysed, the dense exterior ring influenced the percentage of porosity measured in 3D-ZTA.

Table 3.3 shows that micro-CT results correlate well with data obtained with by scanning electron microscopy, and both strut and pore diameter sizes are comparable between these two techniques. Since the macropores of 3D-ZTA are not cylindrical, Washburn equation (used after MIP to transform the intrusion pressure into pore diameter) will only give access to an "equivalent pore access diameter" related to the pore size but also to the pore shape. As the pore shapes are very complex, pores contain many zones with small curvature radius and this lowers the equivalent pore access diameter as compared to the pore size directly measured by CT or SEM.

Table 3.3 Comparison of the pore size, strut size and porosity measured by scanning electron microscopy (SEM), X-ray tomography (CT) and mercury intrusion porosimetry (MIP). * porosity of a region inside the samples without the external rings; ** porosity of the whole sample, with the external rings.

Human primary osteoblast behaviour on 2D-and 3D-ZTA

Cell proliferation

hOb proliferation was highest on TMX, followed by 2D-ZTA and 3D-ZTA (Fig. 3.4 a). In terms of cell proliferation, it is important to note that cell number normalised to surface (cm 2 )

did not considerably increase during the culture time on the 3D-ZTA.

Alkaline phosphatase activity

A much higher ALP activity normalised to DNA was found on 2D-ZTA compared to TMX and 3D-ZTA (Fig. 3.4 b). On 2D-ZTA a peak in ALP activity was observed at day 20 of culture, while on TMX and 3D-ZTA the ALP activity showed minimal differences over the culture period.

Expression of osteogenic markers

RUNX2, an important transcription factor in osteogenesis, was up-regulated on all samples after day 10 (Fig. 3.5 a). A higher expression of COL1 mRNA, an early marker of osteogenesis, was seen for both the 2D-and 3D-ZTA (Fig. 3.5 b) compared to the standard plastic control TMX, with minimal differences between 2D-and 3D-ZTA. In terms of ALP, an intermediate marker of osteogenesis, a peak was observed at day 20 on 3D-ZTA, while ALP mRNA levels are increased over the culture time on TMX and 2D-ZTA (Fig. 3.5 c).

Interestingly, the expression of OC, a late marker of osteogenesis, was highest on 3D-ZTA among all groups at day 30 (Fig. 3.5 d), although differences were not statistically significant.

Figure 3.5 mRNA expression of osteogenic markers in human primary osteoblasts cultured on 3D-ZTA, 2D-ZTA and TMX at day 10, 20 and 30 of culture: a) runx2, b) type I collagen (COL1), c) ALP, d) osteocalcin (OC). Data are expressed as 2 -ddCt relative to the mRNA expression of cells collected on the day of seeding (day 0); data are represented as mean and standard error of mean (n=6).

Mineralisation potential

An increase in alizarin red staining was observed over the culture time on both 2D-and 3D-ZTA (Fig. 3.6). A stronger but less homogenous staining was observed on 3D-ZTA compared to 2D-ZTA, starting from day 20. On TMX, positive alizarin red staining was observed only at a later time point (day 30). Quantification of the mineralized area was possible by using Image J software and allowed to support the stronger mineralization induced by the 3D samples with 16.41% of stained areas relative to the whole sample at day 30. This result is coherent with the alkaline phosphatase activity (Fig. 3.4 b).

Discussion

We report for the first time the processing and in vitro evaluation of robocast aluminazirconia composites, thus opening the way for ZTA implants with improved osseointegrative surface properties. Robotic controlled deposition of composite ZTA slurry allowed good control over the three-dimensional structures. The preparation of a suitable ink for robocasting was one of the biggest challenges in our approach, as reported previously in other studies 6,8,9,14 . A slurry pertinent for robotic deposition must fulfil specific criteria: (i) have a shear-thinning behaviour to allow viscosity decrease under modest shear strain, (ii) retain shape following deposition, (iii) allow for multiple layering to obtain a 3D structure.

Slurries with high solid load (>40 vol.%) have a strong shear-thinning behaviour (thus allowing a good ink flow) and increased viscosity (thus supporting inter-particle interactions leading to increased critical shear stress) 15 . Solid load needs to be high enough to allow good shape retention of the multi-layered structure, but low enough to avoid a too high viscosity that will result in slurry block inside the nozzle. In our study, a 70 wt.% (35.5 vol.%) solid load with minimal organic components and good dispersion of ceramic particles was used to successfully print through a 330 μm nozzle and an acidic bath contributed to post-extrusion shape retention by coagulating the suspension. Macro-and micro-porosity are both required to maximise new bone formation, as a number of in vitro and in vivo studies has evidenced [16][17][18] . The 3D-ZTA scaffolds produced in this study had ~50% fully interconnected macroporosity (Fig. 3.1 a, 3.2 a, 3.2 d and Table 3. Multiple scale topography promotes osseointegration of implants, as recently reviewed by Gittens et al. 1 . The effective sizes of the topographical features is linked to the events that occur at the implant surface, namely water absorption and protein adsorption (nanometric scale), cell attachment, proliferation and differentiation/maturation (R a = 0.2-2 μm scale) [20][21][22][23] .

In our study, both 2D-ZTA and 3D-ZTA promoted an earlier mineralisation onset compared to thermanox control (Fig. 3.6), suggesting that microporous ZTA has potential to support hOb maturation. Interestingly, in 3D-ZTA, the surface of the struts had two levels of topography: one at the nanometric level (Fig. 3.1 c and Fig. 3.3) as a result of the lowtemperature sintering conditions, and another one in the range of tens of microns which was dependent on the nozzle intrinsic surface characteristics (Fig. 3.1 a, b). It was found that 3D-ZTA promoted an earlier RUNX2 and ALP mRNA up-regulation compared to 2D-ZTA (Fig.

a, c), thus indicating an earlier mineralization onset on 3D-ZTA.

A limitation of our in vitro setup was the ability to retain cells inside the 3D-ZTA structures on long term (up to 30 days of culture). Both cell density and mineralisation on 3D-ZTA could be further improved by optimising cell retention inside the 3D structure, for instance by culturing the scaffolds in perfusion bioreactors 24 , modifying the ZTA surface with acid etching 25,26 , silanisation 27 or coating 28 and/or optimising the scaffold design.

Conclusions

This study provides a first proof of concept of the possibility to robocast 3D-ZTA and to prepare multilayer structures that can serve as a substrate for bone cells ingrowth, which represents a first step toward ZTA implants with improved osseointegrative surface properties. A stable liquid slurry with very good dispersion of the ceramic particles and high solid loading (70 wt.%) was obtained and used as an ink for robocasting and an acidic bath was required for ink coagulation. As the liquid ink flows easily, successful robocasting through nozzles much smaller than the ones used here can be foreseen. Moreover, thanks to the high solid loading and good dispersion, it is foreseeable that the obtained struts will present good mechanical properties. Initial screening of human primary osteoblast response on robocast ZTA proved that hObs were successfully cultured on ZTA scaffolds and were homogenously distributed over the whole scaffold thickness. Further work is required to optimise the scaffold architecture as well as the seeding and culture conditions (e.g. using perfusion bioreactors) to achieve a more homogenous mineralised extracellular matrix deposition. Robocasting of ZTA could be applied to produce multi-scale topography on acetabular cup green bodies and future studies will focus on the characterisation of the porous layer bonding onto the dense substrate, as well as evaluation of reproducibility and predictability of mechanical and biological properties. Parmi les conditions testées, le motif de 30 µm de diamètre et 10 µm de profondeur semble favoriser la différentiation des cellules souches vers la lignée ostéoblastique, néanmoins des analyses à plus long terme sont recommandées pour confirmer ces résultats préliminaires.

Abstract

Yttria-stabilized tetragonal zirconia polycrystals (Y-TZP) bioinert ceramics combine excellent strength and toughness, good aesthetics, high resistance to corrosion and absence of allergic reaction. However, improved osseointegration is needed as higher marginal bone loss was sometimes reported. In the present work, 3Y-TZP multi-patterned samples for rapid screening of cell-surface interactions were fabricated by femtosecond laser micromachining.

Pits with well-defined edges and micrometric precision in pit diameter, depth and spacing were produced, as determined by white light interferometry. Pits showed a nanometric granular texture on the sidewalls and ripples at pit bottom, as attested by scanning electron microscopy. Focused ion beam analyses indicated limited laser-induced damage. Micropatterns impacted human mesenchymal stem cell (hMSC) size and morphology. Cell area and aspect ratio were mainly influenced by pit diameter, while solidity and circularity were affected by both pit diameter and depth. The pattern 30 µm diameter/10 µm depth induced the strongest osteoblastic hMSC commitment.

Keywords: Zirconia, Laser, Surface, Topography, Pattern, Human Mesenchymal Stem Cells

Introduction

Yttria-stabilised tetragonal zirconia polycrystals (Y-TZP, short: zirconia) are biocompatible and exhibit the best combination of strength and toughness of single-phase oxide ceramics.

They were introduced as biomaterials in the end of the 1980s to overcome the limitations of alumina in the field of orthopedics. 1 While monolithic zirconia has been almost abandoned for orthopaedic applications, in the last decade its use in restorative dentistry has been growing fast. 2 In particular, its good aesthetics, high resistance to corrosion and the absence of allergic reaction make zirconia a good candidate to replace titanium for the fabrication of dental implants. 3 However, while some authors have described a similar performance of zirconia and titanium implants, 4,5 others have reported a higher failure rate and a higher marginal bone loss when comparing zirconia to titanium. 6,7 According to the latter, the use of zirconia implants does not appear recommendable at the moment except for specific cases (e.g. allergy to titanium), and there is a need for further research before generalising their clinical use.

The key to solve the problem of bone loss mentioned above is to achieve a good osseointegration, which depends on numerous parameters such as topography and chemistry. 8 In particular, it has been shown that micro-rough implants exhibit a better osseointegration than smooth ones. 9 Nevertheless, what is the optimal topography for a dental implant remains unclear. 10,11 One of the reasons for this lack of information is that, with a classical approach, the investigation of the influence of surface topography on cell response requires experiments on a large series of homogenous, individual specimens for each condition tested. Testing a high number of surface types is thus lengthy and costly.

To overcome this problem, there is a strong interest in developing methods to rapidly screen cell-surface interactions, and one convenient approach is the fabrication of multi-patterned samples: it allows testing several surface types at once, thus drastically reducing the number of samples required. 12 Among the different techniques proposed to modify the surface of zirconia, femtosecond laser micromachining appears as an appealing tool to produce multipatterned samples: it provides a high flexibility and a high precision in the surface design, it reduces the presence of residual elements with respect to other laser techniques, it produces a limited damage to the material and has much potential for automation and therefore reproducibility. 13 Femtosecond laser micromachining has been successfully implemented to produce microgrooved zirconia implants which performed well both in vitro and in vivo. [14][15][16][17] Hallgren et al.

have investigated the effect of laser ablation of commercially pure titanium implants in a rabbit model and found that the patterned implant (pits of 19 µm diameter, 8 µm depth and 20 µm distance) induced a higher peak removal torque and bone-to-implant contact compared to an unpatterned implant. 18 The in vitro response to micro-patterned surfaces has been widely studied, as recently reviewed by Jeon et al. 19 and several reports on biomaterials including bio-inert ceramics have demonstrated that the osteogenic response of human mesenchymal stem cells (hMSCs) could be enhanced by controlling the width, height and spacing of microgrooves. [20][21][22] However, to the best of our knowledge, surface multi-patterning of single specimens represents a novel approach for the study of the interaction between cells and zirconia surfaces. In the present work, zirconia samples with multiple micro-patterns constituted of pits of different depths and diameters were produced by femtosecond laser micromachining, with the objective to perform a rapid screening of the influence of both parameters on hMSC response through a systematic morphometric analysis.

Materials and methods

Samples fabrication

Fabrication of zirconia disks

Commercial 3Y-TZP powder (TZ-3YSB-E Tosoh Co., Japan) was cold isostatically compacted under a pressure of 200 MPa in a cylindrical mould for producing a green body, and then sintered in an alumina tube furnace at 1450 ºC for two hours (3 ºC/min heating and cooling rates). The sintered ceramic cylinder was cut into specimens in the form of disks (2 mm thick, 9 mm diameter), which were ground and polished down to a 3 μm diamond suspension.

Surface patterning

A commercial Ti:Sapphire oscillator (Tsunami, Spectra Physics) and a regenerative amplifier system (Spitfire, Spectra Physics) based on chirped pulsed amplification were used for the surface patterning. The system delivered 120 fs linearly polarised pulses at λ = 795 nm with a repetition rate of 1 kHz. The transverse mode was TEM00, and the beam width was 9 mm

(1/e2 criterion). Pulse energy was controlled by means of neutral filters and a half wave plate plus polariser system for fine adjustment. The laser pulses travelled through air to the focusing system. This one consisted of an achromatic doublet lens and an off-axis imaging system (lens, beam splitters, and CCD Philips LucaVR camera) to help sample positioning and beam focusing. Samples were placed on a motorised platform with three-axis motion, X, Y, and Z, controlled by Micos ES100VR software (Nanotec Electronic GmbH & Co., Munich, Germany), so that laser pulses impinged perpendicularly onto the sample surface. As shown Figure 4.1, six quasi-circular patterned zones (diameter = 1 mm) were machined on each individual sample (three samples in total). The pattern consisted of evenly spaced pits (distance between pits = 1x diameter). Based on the existing literature on micro-grooved bioinert ceramics 17,20,21,23 and the average stem cell size reaching the material surface (10-20 µm range), the following pit diameters were chosen for this study: 10 µm (smaller than the average hMSC size), 20 µm (just above the average hMSC size), 30 µm (above the average hMSC size). These diameters were associated to two different depths: 3 µm and 10 µm.

Patterns will be referred as "diameter, depth" (e.g. "10, 3" standing for the "10 µm diameter, 3 µm depth" pattern). The required dimensions for each pit were obtained by varying the pulse energy, the number of pulses and the focal length of the lens. To ensure the circular shape, the beam was diaphragmed by means of a circular aperture. Unpatterned regions of the samples were used as controls in the cell culture studies.

Characterisation of the micro-patterns

Diameter and depth of the pits were determined from topographical images obtained by white light interferometry (WLI, Veeco Wyko 9300NT) (Fig. 4.2). The measured pit dimensions and the processing parameters associated to each type of pattern are summarised in Tables 4.1 and 4.2. The morphology of the pits was characterised by Scanning Electron Microscopy (SEM), and the near-surface was observed on transversal sections milled with a Focused Ion Beam (FIB, Neon40, Carl Zeiss AG, Germany) in order to detect damage.

Sample surfaces were protected with a thin platinum coating to flatten the surface and minimise ion-beam damage and curtain effect during milling. The final polishing of the cross-sections was performed at 500 pA. 4.2 Processing parameters used to obtain each kind of pit/pattern ( * Energy before going through the optical system; ** For an ideal Gaussian beam; *** After passing through the optical system).

Figure 4.2 a) White light interferometry topographical images of the six different types of pits produced by femtosecond laser micromachining; b) example of extracted depth profiles (first number refers to pit diameter and second number refers to pit depth).

Cell cultures

Isolation and expansion of human bone marrow derived mesenchymal stem cells

Human mesenchymal stem cells (hMSCs) were isolated from a human bone marrow aspirate (44 year, male) obtained from the University Hospital of Bern after approval by the local ethical commission (KEK 188/10) and written consent of the patient. hMSCs were isolated by Ficoll gradient centrifugation and adherence to tissue culture plastic. Cell expansion was performed in α-minimum essential medium (α-MEM) containing penicillin (100 U/mL) and streptomycin sulphate (100 µg/mL) (all products from Gibco, Basel, Switzerland), 10% foetal bovine serum (Sera Plus, Pan-Biotech, Aindenbach, Germany) and 5 ng/mL basic fibroblast growth factor (R&D Systems, Minneapolis, MN, USA) with a medium change twice a week.

Cell seeding, culture and staining on laser-patterned disks

The laser-patterned specimens were cleaned in an ultrasonic bath using an enzymatic detergent (Terg-A-Zyme ® , Sigma-Aldrich), followed by extensive rinses with deionised water, 70 % ethanol and deionised water again. The enzymatic detergent was used for the removal and cleavage of big organic components such as proteins. Samples were then transferred to a 24 well plate and sterilised using ethylene oxide.

hMSCs (passage 3) were seeded on the sample surface at a density of 5000 cells/cm 2 . To ensure a homogenous distribution, cells were seeded by gravity using a diluted cell suspension (10 000 cells/mL). Samples were cultured for 48 hours in 0.5 mL α-MEM supplemented with 10% foetal calf serum, penicillin (100 U/mL) and streptomycin sulphate (100 µg/mL).

Following culture, cells were rinsed once with 1 ml of phosphate buffered saline solution, fixed with 70% ethanol and stained with 0.5 % toluidine blue solution for 5 minutes, followed by three rinses with deionised water. Cells were imaged under reflected light with an optical microscope (Leica MacroFluoTM, Wetzlar, Germany); the acquisition time was kept constant for all samples.

Single cell image analysis

Cell area and shape (solidity, aspect ratio and circularity) were analysed using ImageJ (NIH, version 1.47). Cells from two patterned samples were analysed (10 cells for each patterned region and sample, meaning n=20 cells for each pattern). Solidity was calculated as Area Convex Area 2 , where the convex area is the imaginary convex hull around the object. Aspect ratio is defined as

Major Axis

Minor Axis of the object's fitted ellipse. Circularity was calculated as 4π • Area Perimeter 2 . Circularity ranges from 1.0 (perfect circle) to values approaching 0, which indicate an increasingly elongated shape.

Statistics

Statistical analysis was performed using GraphPad Prism 7.01 software (GraphPad). Data were tested for normal distribution and a two-way ANOVA with Tukey's multiple comparison tests was applied to test differences between experimental groups. Significance level was set at p<0.05. When comparing experimental groups to the unpatterned group, a one-way ANOVA with Bonferroni post-hoc was applied. Data are presented as median and interquartile range.

Results

Characterisation of the micro-patterned zirconia

For each type of pattern, the selected processing parameters allowed achieving pits with welldefined edges (Fig. 4.3) and a micrometric precision regarding diameter and depth (Table 4.1). No damage was detected on the surface situated between pits. The reproducibility was excellent as indicated by the low standard deviations (< 1 µm) (Table 4.1) and the pit spacing was highly regular (Fig. 4.3 a). The shape of the pits corresponded to a cone in the case of the 10,10 pattern or a truncated cone in all the other cases (Fig. 4.2 b, Fig. 4.3 a). The sidewalls of the pits presented a nanometric granular texture (Fig. 3 b, Fig. 4 a). Except for the 10-10 pattern, typical laser-induced periodic surface structures known as ripples were formed at the bottom of the pits (Fig. 4.3 b, c). The formation of such structures is difficult to avoid and is believed to be the result of the interference between the incident laser radiation and scattered or excited surface waves, which leads to inhomogeneous absorption of the laser energy. 24 The average width of the ripples observed was close to the laser wavelength (λ = 795 nm) (Table 4.3). The shape of the ripples was more regular for the 20,10 and 30,10 pits than for the 10,3, 20,3 and 30,3 pits, which may be related to the lower number of pulses associated to the latter (Fig. 4.4 b). The presence of micro-cracks and sub-micrometric pores was detected in all the rippled zones, independently of the type of pit (Figure 4.3c). The observation of the FIBproduced cross-sections evidenced that the laser processing also induced the formation of subsuperficial pores up to a depth of about 5 µm, especially in the sidewalls of the pits (Fig.

Cell cultures on micro-patterned zirconia

Micro-patterns induced changes in cell size and, more pronouncedly, in cell morphology (Fig. 4.5). Cell area was mainly influenced by the pit diameter, with the 20 µm diameter pits inducing a significantly smaller average cell area compared to 10 and 30 µm pits, independently of pit depth (Fig. 4.5 a). Cells cultured on pits with same diameter but different depths had a similar size, except for cells cultured on 30 µm diameter patterned regions for which average cell area was significantly bigger for 10 µm deep pits compared to 3 µm ones.

The solidity parameter is an indirect indicator of the extent of pseudopodia and filopodia of the cells: cells with limited extensions have a higher solidity value than cells with many and/or long extensions. Solidity was influenced by both the diameter and depth of the pits in the patterned regions (Fig. 4.5 b). The group with the largest pattern (30 µm diameter, 10 µm depth) showed the highest solidity value, which was significantly higher compared to the control (unpatterned region). A higher solidity was observed with the deeper pits (10 µm versus 3 µm), independent of the diameter. For an equal pit depth (3 µm or 10 µm), pits with a diameter of 30 µm induced a higher solidity than pits with a diameter of 20 µm.

The aspect ratio is a good indicator of the cell morphology: rounded cells have an aspect ratio close to 1, while elongated cells have a higher aspect ratio. In this study, the aspect ratio was above 2 for all groups. The aspect ratio was mainly influenced by pit diameter with a significant decrease in aspect ratio was found as pit diameter increased (Fig. 4.5 c). hMSCs cultured on 30,3 pattern had a significant lower aspect ratio compared to the unpatterned control.

Circularity is another indicator for cell morphology: rounded cells have a circularity of 1 and this value decreases as cells become more elongated. In our study, circularity was below 0.5 in all groups, thus indicating elongated cell morphology. Circularity followed a similar trend to solidity, with the highest value being observed for the cells cultured on 30 µm diameter, 10 µm deep pits (Fig. 4.5 d). The circularity of this group was significantly higher compared to the unpatterned control. Deeper pits (10 µm versus 3 µm) generally induced a higher circularity, except for the 10 µm diameter pits. For an equal pit depth (3 µm or 10 µm), pits with a diameter of 30 µm induced a higher solidity than pits with a diameter of 20 µm. Cell size, morphology and distribution varied depending on the pattern type (Fig. 4.5 e). In fact, in the 10 µm diameter groups, all hMSCs spanned across several pores. In the case of 20 µm diameter pits, the majority of hMSCs spread across several pores, but a small proportion of cells was also found inside a pore or at a pore border. Finally, in the case of larger pore diameters (30 µm), a similar proportion of cells was found between two pores, inside a pore and at the pore border. Similar results were obtained when fibroblasts were cultured on patterned surface with pits ranging from 7 to 25 µm in diameter, whereby cells spread across pores for the smallest diameter, between pores for intermediate diameter and inside pores for the largest ones. 25 Importantly, we have found that the pattern with 30 µm diameter and 10 µm deep pits induced a characteristic osteoblastic cuboid morphology in 25% of the analysed cells, while cells with a cuboid morphology were only seldom observed in the other groups (< 10%).

Figure 4.5 Mesenchymal stem cell size and shape analysis following 48 hours of culture on the laser-patterned discs: a) cell area, b) solidity, c) aspect ratio, d) circularity and e) representative images of the toluidine blue stained cells. Data represent the analysis of 20 cells/group (n=10/sample, 2 samples) and are plotted as single points, as well as median and interquartile range. N indicates the unpatterned control, Y indicates

In summary, our results indicate that 30 µm diameter pits promoted cell morphology with low aspect ratio, high solidity and relatively high circularity compared to unpatterned zirconia control (Fig. 4.5). On 30 µm diameter pits, higher depth of the pits resulted in higher proportion of polygonal cells and larger cell area. On the other hand, cells seeded on regions with smaller patterns (10 µm diameter, 3 or 10 µm deep pits) had a more elongated cell morphology.

Surface patterning had a limited effect on the number of attached cells compared to the unpatterned control (attached cells in the patterned regions were in the range of 80-120% of the unpatterned control). For 10 µm deep pits, the number of attached cells increased with increasing pit diameter (95 ± 21%, 114 ± 7% and 124 ± 2% of the control for pits with a diameter of 10, 20 and 30 µm respectively); for the 3 µm deep pits, no clear trend was observed. Only minor differences in the number of attached cells were observed when comparing regions with same pit diameter but different depth.

Discussion

To the best of the knowledge of the authors, the surface multi-patterning of single specimens proposed in this work constitutes a novel approach for the study of the interaction between cells and zirconia surface. The fabrication of the patterns by femtosecond laser micromachining allowed a high precision and reproducibility. The formation of ripples at the bottom of the pits was not expected, nevertheless this commonly observed phenomenon is difficult to avoid. 24 Femtosecond laser ablation is known to produce significantly less damage than conventional laser techniques, in particular because of the quasi-absence of thermal effects. 14 In this study, micro-cracks and sub-micrometric pores were observed on the pit surface, as well as some sub-superficial pores up to depth of 5 µm. Considering the typical dimension of the critical defect leading to fracture for dental zirconia (3Y-TZP) which is above 7 µm, 26,27 the small dimension of these defects and the limited extension of the affected zone, the damage produced by the femtosecond laser ablation is not likely to be a concern in terms of strength. Nevertheless, the pits themselves could play the role of stress concentrators. On the other hand, zirconia can suffer from low temperature degradation (LTD) and the kinetics of this phenomenon can be affected by minor surface modifications. 1 It comes out of the scope of this study, but the effect of patterning on LTD kinetics need to be thoroughly assessed prior possible applications of laser-patterning to implants for clinical use.

Stem cells are a heterogeneous population of undifferentiated cells, which have the capacity to differentiate toward different phenotypes depending on the stimuli they receive, with surface topography being one of them. 9 Morphometric analyses provide a useful tool to screen early cell response to topographical changes. Based on morphometric analysis, two hMSC subtypes have been identified: small, spindle rapid self-renewing shaped (RS) cells and the large flat cells (FC). 28 The aspect ratio of RS cells is much higher than the one of FC cells and primary osteoblasts (approximately 4 and 2, respectively). Our data suggest that bigger patterns (30 µm diameter pits) favour a lower aspect ratio and a morphology compatible with FC cells and more similar to human primary osteoblasts, while smaller pit diameter (10 µm) favour the RS phenotype. All patterns promoted an average cell area > 3000 µm 2 and aspect ratio > 2, which suggests that stem cells are unlikely to be committed to an adipogenic phenotype, which is associated with low cell area and circular cell shape (aspect ratio ~ 1). 29 This is in agreement with several long-term in vitro studies on bio-inert ceramics which collectively suggest that the optimal size of micro-patterns to foster hMSC osteogenesis, promote osteoblast mineralisation and reduce osteoclastogenesis may lie in the range of 30 µm. 17,[20][21][22][23]30 Indeed, when investigating hMSC response on micro-patterned alumina produced by green body embossing, Nadeem et al. 20 have observed that small microgrooved pattern (10 µm/10 µm groove/pitch) promoted more elongated cell morphology, while larger micro-grooved pattern (100 µm/50 µm groove/pitch) induced spread and polygonal cell morphologies associated with higher osteoid matrix nodule formation following a 21 day culture period. When studying hMSC behaviour on micro-grooved alumina (from 30 µm/30 µm to 180 µm/180 µm groove/pitch), Kim et al. have found an increased alkaline phosphatase activity and calcium deposition by hMSCs with increasing microgroove size. 21 Zhao and co-workers have reported that patterning of hydroxyapatite with 24 µm wide grooves promoted osteogenic differentiation of hMSCs compared to larger grooves 22 and Delgado-Ruíz et al. have indicated that 30 µm/70 µm groove/pitch microgrooves on zirconia increased mineralisation of human foetal osteoblasts compared to an unpatterned control. 17 Wilkinson et al. have investigated the effect of pit diameter (20, 30 and 40 µm, depth 0.3 µm) on the behaviour human pre-osteoblasts (MG63) on polymethylmethacrylate and polycaprolactone patterned surfaces: 30 µm diameter pits supported the highest nodule formation among the tested groups. 30 Recently, Halai et al. have performed osteoblast/osteoclast co-cultures on zirconia-toughened alumina micro-patterned with pits (30 µm diameter, 1.7 µm depth) and unveiled that micro-patterning increased bone nodule formation and decreased osteoclastogenesis compared to an unpatterned control. 23 It is worth noting that the presence of ripples, micro-craks and micro-pores in the patterned pits could affect hMSC response, which introduces an additional parameter when comparing patterns. However, the similarity of the ripples from one condition to another (in particular regarding their dimension which is related to the laser wavelength) limits the importance of this factor. On the other hand, these additional features could add a positive outcome for the clinical application: for instance, it has been shown that rippled surface can reduce bacterial adhesion. 31

Conclusions

The fabrication of zirconia multi-patterned samples for a rapid screening of cell-surface interaction was successfully achieved by femtosecond laser micromachining. The damage induced by the laser ablation was limited. Regarding cell response, the laser-patterning of zirconia with 30 µm diameter/10 µm deep pits may promote, based on morphological parameters, stem cell commitment toward the osteoblastic phenotype compared to no patterning or patterning with smaller features (10 and 20 µm diameter). Further in vitro investigations will focus on the evaluation of osteogenic commitment of hMSCs over longterm cell cultures. In this case, a modification of the sample design would be recommended to avoid possible interactions of cells from different patterns following cell proliferation.

Another interesting development is the use of patterns that contain pointed concave features (such stars or rectangles) which have been shown to promote osteogenesis of hMSCs. 32 Finally, multi-patterned samples could be used to screen bacterial adhesion.

2-2 µm: A -Sur une surface lisse, les ostéoblastes adopteront une morphologie de type fibroblaste; B -Sur une surface ondulée, si la distance entre les pics est supérieure à la taille de la cellule, les cellules reconnaîtront la surface comme plate et se comporteront en conséquence ; C -Sur une surface microrugueuse ils adopteront la morphologie typique des ostéoblastes; D -Sur une surface avec des topographies mélangées, la réponse cellulaire reflètera une moyenne des microtopographies lisses et

microrugueuse. 6 Par conséquent, un seul paramètre sur le profil 2D ne fournit pas d'informations suffisantes. En fin de compte, il est important de noter que la micro-rugosité ou la nano-rugosité par elles-mêmes ne permettraient pas de guider de manière significative la réponse cellulaire.

Ceci suggère que l'interface de cellule-matériau est un processus complexe finement orchestré par l'ensemble des caractéristiques de surface. Une combinaison de rugosité à l'échelle micro-et nano-métrique est une condition préalable pour guider la réponse cellulaire vers la maturation et la minéralisation, ayant ainsi un fort impact sur la stabilité secondaire de l'implant (qui est due à la régénération osseuse et au remodelage à l'interface os / implant).

L'augmentation de la surface spécifique apportée par la superposition de nano-rugosité sur les surfaces ZTA micro-rugueuses pourrait entraîner des changements dans l'énergie de surface avec un impact sur les protéines adsorbées et aussi sur l'adhésion cellulaire.

General summary in English

The bio-inert ceramic bone prosthesis market proposes both zirconia implants, recommended in dentistry, and zirconia toughened alumina (ZTA) for hip arthroplasty. Current bio-inert ceramics need to be combined with metal (rough titanium or stainless steel) to achieve adequate bonding with the patient's bone (osseointegration). An adequate osseointegration between bio-inert ceramics and bone would improve the prosthesis design and quality of life of the patient. For instance, in hip arthroplasty, direct osseointegration of bio-inert ceramics will allow the use of a thinner acetabular component and allow space for a bigger ball (Fig. In this PhD thesis we assessed the response of human primary osteoblasts to alumina-zirconia dense ceramics with nano-and/or micro-roughness and to alumina-zirconia scaffolds. By using roughened moulds in the injection moulding process, it was possible to obtain a whole range of micro-rough ceramics with same surface chemistry. Furthermore, by using acid etching, it was possible to superimpose nano-roughness on top of the micro-roughness obtained by injection moulding (Fig. 5.9). Finally, highly porous and regular micro-and macro-porous alumina-zirconia scaffolds were successfully obtained by robocasting. As for the cell type, osteoblasts are the cells in the bone tissue that produce new bone. Osteoblasts from femoral heads of patients undergoing hip replacement represent the most relevant cell type for the evaluation of surface modifications of hip prosthesis materials in vitro. Recent trends for material properties screening 1, 2 signal the necessity for an appropriate cell type and assays used for in vitro assessment as well as standardized methods. We concluded that the clinical relevance of the human cells isolated from patients undergoing THA will help to better mimic a clinical setup with improved predictions for the implant fate. For a better understanding of cell behaviour in vitro and an accurate identifications of specific cues that induce a specific cell response it is important to perform a meticulous surface characterization.

As previously stated 3

2-2 µm: A -On a smooth surface, osteoblasts will adopt a fibroblast-like morphology; B -On a wavy surface, if the distance between peaks is more than the cell size, cells will recognize the surface as a flat one and behave accordingly; C -On a micro-rough surface will adopt typical osteoblast morphology; D -On a surface with mixed topographies, cell response will reflect an average of both smooth and microrough micro-topographies 6 .

A large sample number (in the order of hundreds) is generally required for in vitro assessment of materials. This is due to the fact that, first of all, for reliable results, cell response to materials should be assessed using cells derived from several donors (generally, three to six donors are used for this type of studies).

Then, several responses (cell attachment, proliferation, differentiation/maturation at gene and protein level) are generally tested at regular time points (generally three to six time points) to have a more comprehensive view of cell behaviour. Moreover, for each analysis and time point, more than one replica is generally used to get more accurate values (generally two to four samples per condition). Even when several analyses can be performed on a single sample (for instance, alkaline phosphatase and DNA quantification from the same cell lysate), the number of samples needed is high. Last but not least, appropriate controls are needed (for instance, polished and "as sintered" controls in the study of ceramics prepared by injection into roughened moulds).

To limit the number of samples required for the in vitro study, a novel cleaning protocol was developed to allow the reuse of the samples after each cell culture experiment. A burn-out of the organic residues that might be trapped in the superficial porous layer was performed.

Samples were then cleaned according to the manufacturer's instructions for new batches of samples. Finally, samples were sterilised by ethylene oxide. From our experiments, it appears that the cleaning was effective (two series of experiments using the same samples (before and after cleaning) and cells from the same donor gave similar results), but further detailed surface analyses would be required to confirm this.

In our experimental setup, micro-roughness alone did not trigger significant differences in hOb behaviour compared to polished control, while combined micro-/nano-roughness had a strong effect on hOb behaviour with significant differences at protein expression level.

Indeed, ALP activity normalized to the DNA content was statistically significantly increased at day 10 by the surfaces "as sintered", "low" and "medium" etched micro-rough surfaces when compared to the micro-rough only control (Fig. 5.11). Among the different microroughnesses tested, the "low" surface with R a ~ 0.13 μm, S a ~ 0.2 μm, S ds ~ 45000/mm 2 , S dr ~ 45% and S ci ~ 1.1 nm expressed the highest mineralization potential. A trend was observed in terms of enhanced alkaline phosphatase activity on this surface accompanied by higher mRNA levels for ALP and RUNX2. The latter is an early marker for osteoblast lineage commitment and the former is a marker for mineralization onset. Our findings are in agreement with previous studies on micro-and nano-rough zirconia prepared by sand blasting and acid etching. For instance, Bergemann et al. reported increased OC production on sandblasted and etched zirconia surfaces (R a = 1.32 µm) obtained by sandblasting and etching 7 . Similarly enhanced ALP production was seen when culturing MC3T3-E1 osteoblast-like cells on micro-and nano-rough (R a ~ 0.4 µm) zirconia surfaces (prepared by sand blasting and acid etching) versus polished control 8 . Ultimately, it is important to note that micro-roughness or nano-roughness by itself did not significantly guide cell response.

This suggests cell-surface interface it is a complex process finely orchestrated by a sum of surface features. A combination of micro-and nano-scale roughness is prerequisite for guiding cell response towards maturation and mineralization, thus having a strong impact on implant secondary stability (which is due to bone regeneration and remodelling at the bone/implant interface). The increase in the surface area brought by the nano-roughness superimposition on the micro-rough ZTA surfaces could lead to changes in the surface energy with impact on the protein adsorbed and further cell adhesion.

The primary stability of an implant is associated with its mechanical interlocking with the surrounding bone. By using a macro-porous ZTA coating of the external surface of the hip implant cup, Theelke et al. have achieved a primary stability on an artificial bone comparable to metal shells with plasma sprayed titanium coatings 9 . Here, robocasting was used to manufacture porous alumina-zirconia scaffolds as a way to produce highly reproducible, well-controlled porous structures (Fig. 5.12). Hence, we processed and evaluated biological properties of robocast alumina-zirconia composites. The most important point was the formulation of a stable ink. We have successfully prepared and printed a 35.5 vol% solid load ceramic slurry with minimal organic content through a 330 μm nozzle in an acidic water bath.

The macro-porosity (~ 50%) obtained for the robocast scaffolds proved to be fully interconnected and the ceramic struts were micro-porous (due to the relatively low sintering conditions). As previously shown, both macro-and micro-porosity are necessary conditions for osseoinduction and osseoconduction 10 . Moreover, it is interesting to note that the ceramic struts showed developed two levels of topography: one obtained following low-temperature sintering conditions (in the sub-micron level) and a second one due to the intrinsic inner morphology of the nozzle used for extrusion (in the range of tens of microns). Based on existing literature, it is hypothesized that these two types of porosity are expected to induce beneficial effects on protein adsorption and further cell adhesion, proliferation and differentiation. Regarding the biological properties, both ZTA structures used in our study, the robocast 3D-ZTA scaffolds and especially the 2D-ZTA control structures obtained by casting, improved hOb maturation when compared to thermanox control surfaces. ALP and collagen type I gene expression was enhanced on the ZTA structures in osteogenic supplemented medium. These findings suggest the development of active mature osteoblasts able to produce extracellular matrix and further mineralization supporting osteogenesis and potentially osseointegration.

Figure 5.11 ALP/DNA ratio on (a) different microrough alumina-zirconia samples and (b) comparison of microrough samples as received, etched with hydrofluoric acid (HF) with or without an additional etching in hydrochloric acid (HCl): (b) polished, (c) as sintered, (d) low and (e) medium and (f) high surface roughness. Note that only minimal differences were observed among the different microrough only groups, while etching induced a significant increase in ALP/DNA in hOb cultured on as sintered, low and medium surface alumina-zirconia samples.

Nevertheless we identified also some limitations in our in vitro system concerning the ability to retain cells inside the 3D-ZTA structures on long term. Different approaches could be envisaged such as pre-coating the culture wells or culture in perfusion bioreactors (that could promote cell retention inside the ceramic scaffold). In agreement with our previous findings, an alternative could be represented by further modification after the robocasting. Selective chemical etching developed by Flamant et al. and used in our study could be an option for developing a nano-porous layer with beneficial effects for cell adhesion. In addition the selective chemical etching could be used as a drug delivery system. Indeed, Flamant et al. Rapid screening of cell-material interactions was another part of this PhD project. Two different strategies were used to produce zirconia samples. In the first one, femtosecond laser micromachining was used to fabrication of zirconia multi-patterned surfaces. Femtosecond laser micromachining provides a high flexibility and a high precision in the surface design, reduces the presence of residual elements with respect to other laser techniques, produces limited damage to the material and has much potential for automation and reproducibility.

Femtosecond laser micromachining is a novel approach to multi-pattern single specimens for evaluating the interaction between cells and zirconia surfaces (Fig. 5.13). We could obtain pits with well-defined edges and micrometric precision in pit diameter, depth and spacing.

Pits showed a nanometric granular texture on the sidewalls with limited laser-induced damage.

Figure 5.13 Multi-patterned disk design (top), overview of the patterned regions (mid), human mesenchymal stem cell morphology on micro-patterned zirconia following 48 h of culture (bottom). The first number indicates pit diameter, second number indicates pit depth.

As for the cells, human bone marrow-derived mesenchymal stem cells (MSCs) are another type of cells prone to come in contact with an implant. For short term evaluation naive MSCs provided a good tool to evaluate early cell morphological changes that underline cell commitment towards a specifique cell lineage. Micro-patterns impacted human mesenchymal stem cell (hMSC) size and morphology. Cell area and aspect ratio were mainly influenced by pit diameter, while solidity and circularity were affected by both pit diameter and depth.

Morphological parameters analysis such as cell circularity suggested the pattern 30 μm diameter/10 μm depth (with a space between pits equal to their diameter) induced the strongest osteoblastic hMSC commitment. From a scientific perspective, with regard to the robocast and injection-moulded ZTA, further in vitro evaluation such as co-cultures of osteoblasts in combination with different cell types such as MSCs, osteoclasts or immune cells could provide more information on the interaction and the niche environment that is formed at the peri-implantation site. An interesting approach could be represented by combining techniques used in separate studies such as robocasting and chemical etching.

Concerning the zirconia surfaces used for rapid screening, identification of a condition with promising results is only a very first step, and further validation at longer time points is required, possibly by confirming the findings on single type specimens.

For all modified surfaces, screening for bacterial adhesion and antibacterial properties could bring additional information for a complete overview of surface properties.

General conclusions and perspectives

The following figure summarizes the conclusions and perspective of this thesis.

Abstract

Roughness is one of the key parameters for successful osseointegration of dental implants.

The understanding of how roughness affects cell response is thus crucial to improve implant performance. Surface gradients, which allow rapid and systematic investigations of cellsurface interactions, have the potential to facilitate this task. In this study, a novel method aiming to produce roughness gradients at the surface of zirconia using hydrofluoric acid etching was implemented. The topography was exhaustively characterized at the micro-and at the nano-scale by white light interferometry and atomic force microscopy, including the analysis of amplitude, spatial, hybrid, functional and fractal parameters. A rapid screening of the influence of roughness on human mesenchymal stem cells morphology was conducted and the potential correlations between roughness parameters and cell morphology were investigated The roughness gradient induced significant changes in cell area (p< 0.001), aspect ratio (p= 0.01) and solidity (p= 0.026). Nano-roughness parameters were linearly correlated to cell solidity (p< 0.005), while micro-roughness parameters appeared nonlinearly correlated to cell area, highlighting the importance of multiscale optimization of implant topography to induce the desired cell response. The gradient method proposed here drastically reduces the efforts and resources necessary to study cell-surface interactions and provides results directly transferable to industry.

Keywords: Y-TZP ceramics, surface modification, hydrofluoric acid etching, topography, human mesenchymal stem cells

Introduction

Yttria-stabilized tetragonal zirconia polycrystals (Y-TZP, short: zirconia) are biocompatible and exhibit the best combination of strength and toughness of single-phase oxide ceramics.

They were introduced as biomaterials at the end of the 1980s to overcome the limitations of alumina in the field of orthopedics 1 . While monolithic zirconia has been almost abandoned for orthopedic applications, in the last decade its use in restorative dentistry has been growing fast 2 . In particular, its good esthetics, high resistance to corrosion and the absence of allergic reaction make zirconia a good candidate to replace titanium for the fabrication of dental implants 3 . However, some authors reported a higher marginal bone loss when comparing zirconia to titanium. According to them, the use of zirconia implants does not appear recommendable at the moment except for specific cases (e.g. allergy to titanium), and there is a need for further research before generalizing their clinical use 4,5 .

The key to solve the problem of bone loss mentioned above is to achieve a good osseointegration, which depends on numerous parameters such as topography and chemistry 6 .

In particular, it has been shown that rough surfaces exhibit a better bone response than smooth ones. Nevertheless, what is the optimal roughness for a dental implant remains unclear 7,8 .

One of the reasons for this lack of information is that, with a classical approach, the investigation of the influence of surface roughness on cell response requires experiments on a large series of homogenous, individual specimens for each condition tested. Testing a high number of surface types is thus lengthy and costly. To overcome this problem, surface gradients appear as a very appealing tool: they allow the high-throughput and systematic investigation of cell response to the variation of surface properties, one single sample containing the whole range of values for the studied parameters 9 . Besides, homogenous experimental conditions are ensured which reduce drastically the number of specimens needed from a statistical point of view.

The gradient approach has been successfully implemented by some authors to investigate the influence of roughness on osteoblast response, osteoclast resorption and stem cell differentiation on several materials (polycaprolactone, epoxy resin, titanium, silicon, hydroxyapatite) [10][11][12][13][14][15][16][17][18][19] . However, to the best of the knowledge of the authors, it has never been applied to zirconia. Besides, the aforementioned studies often limit the roughness analysis to the average roughness parameter (R a ), which is not sufficient to fully characterize surface topography 20 .

Among the numerous existing methods to fabricate morphology gradients (for a review see 21 ), few are easily transposable to zirconia. One possible approach would be to use the route proposed by Kunzler et al., which is material independent 22 : an aluminum sheet is sandblasted and progressively eroded by chemical polishing, the surface is then replicated in epoxy and sputter-coated with the desired material. Nevertheless, this process presents some inconvenient: it is not directly transposable to the fabrication of a real component such as an implant and the coating process might affect the surface properties and consequently the cell response. Another possibility would be to use a dip coating process to bind particles to the surface, as proposed for instance by Huwiler et al. 23 . However, this approach is limited to the fabrication of nanomorphology gradients.

It has been suggested that hydrofluoric acid (HF) etching of zirconia implants could enhance bone apposition, and such implants were compared to commercial titanium implants in a biomechanical study 24 and a histological study 25 in pigs. Based on a previous work which showed that HF etching allows controlling zirconia roughness 26 , the present paper proposes therefore a novel method to fabricate zirconia samples with a surface roughness gradient, associated to an exhaustive characterization of the topography involving a multiscale roughness analysis and a fractal analysis. To get an insight of the influence of topography on the early response of human bone marrow derived mesenchymal stem cells (hMSCs) and to validate the method, hMSCs were cultured on the gradient samples, and potential correlations between topographical parameters and cell morphology were investigated.

Materials and methods

Sample fabrication

Fabrication of zirconia bars

Commercial 3Y-TZP powder (TZ-3YSB-E Tosoh Co., Japan) was cold uniaxially pressed at 100 MPa and then sintered in an alumina tube furnace at 1450 ºC for 2 h (3 ºC/min heating and cooling rates), obtaining prismatic bars of 2.5 mm x 4.6 mm x 45 mm which were ground and polished down to a 3 μm diamond suspension. The specimens were then successively cleaned for 5 min with acetone, ethanol and deionized water in an ultrasonic bath in order to remove contaminants.

Surface modification for obtaining roughness gradients

A previous work showed that HF etching allows producing roughness on zirconia and that it is possible to control the topography by monitoring the etching time [26]. Based on these results, in the present study roughness gradients were obtained by subjecting specimens to etching into concentrated HF (Hydrofluoric Acid 40% QP Panreac, Spain) with a continuous variation of etching time along the axis of the bar. As described in Fig. 6.1, the samples were immersed into a volume of 40 mL of HF contained in a polyethylene flask at a speed of 0.6 mm/min by using an automated device (ND-R Rotary Dip Coater, Nadetech Innovations, Spain). The total duration of the process was set to 1 h, which leads to a substantial roughness for the part of the sample immersed the longest without damaging too severely the material 26,27 . After etching, the specimens were rinsed with deionized water in order to stop the reaction. Finally, they were cleaned in an ultrasonic bath (2 x 10 min with fresh deionized water) in order to remove the precipitates formed during the process.

Surface characterization

Measurements

All surface measurements were performed on the x-y surface in the direction transversal to the gradient (y direction, see Fig. 150 µm x 150 µm obtained by stitching of four images acquired at magnification 50x, resolution: 758 x 758 pixels). Additionally, for the t = 0 min data point, three AFM measurements and ten WLI measurements were performed on polished samples (with no gradient). Finally, to get a better understanding of the etching process and to identify possible correlations between the formation of precipitates on the surface and the evolution of the topography, SEM observations were performed on a sample which was not subjected to sonication after etching.

Roughness analysis

The roughness analysis of the data from AFM and WLI was carried out using Veeco's Vision® software. Tilt was corrected and a robust short wavelength pass Gaussian filter was applied to the data in order to separate waviness from roughness. The cut-off wavelength of the filter was chosen to be 10 µm for WLI and 1 µm for AFM. The roughness parameters described in Table 6.1 were then determined. Considering the cut-off wavelengths and the lateral resolutions (micrometric for WLI, nanometric for AFM) associated to each device, the roughness measurements obtained from AFM data analysis will be referred to as "nanoroughness" whereas the measurements obtained from the WLI data will be referred to as "micro-roughness".

Fractal analysis

A scale sensitive fractal analysis of the AFM and WLI data was performed using the software Sfrax (www.surfract.com). Area-scale analysis is based on the principle from fractal geometry that the area of a rough surface is not unique, but depends on the scale of measurement 31,32 . The software uses an iterative tiling algorithm in which the topography of the surface is modeled using triangular tiles to calculate the relative area as a function of the scale of observation. The area of a tile represents the scale, and the relative area is determined from the following formula:

𝐴 𝑟 = 𝑁 * 𝐴 𝑡 𝐴 𝑝
where A r is the relative area, A t is the area of a tile, A p is the projected area and N is the number of tiles.

An example of semi-log plot obtained by scale sensitive fractal analysis is shown Figure 7-a.

This kind of plot can be split into two parts:

-The left part, in which the curve appears to be steep and linear. The slope of the curve is an indication of the complexity, intricacy or roughness of the surface 31 . The fractal dimension (D) can be estimated by adding two to the absolute value of the slope.

-The right part, in which the relative area approaches one.

The scale (i.e. the area of the tiles) corresponding to the limit between the two parts of the graph is called the smooth-rough crossover (SRC). For scales smaller than the SRC, the surface is considered as "rough", whereas for scales higher than SRC, the surface is considered as "smooth" 32 .

Cell cultures

Isolation and expansion of human bone marrow derived mesenchymal stem cells

Human mesenchymal stem cells (hMSCs) were isolated by Ficoll gradient centrifugation and adherence to tissue culture plastic from a human bone marrow aspirate (44 year old, male) obtained from the University Hospital of Bern after approval by the local ethical commission (KEK 188/10) and written consent of the patient.

Cell expansion was performed in α-minimum essential medium (α-MEM) supplemented with penicillin (100 U/mL) and streptomycin sulfate (100 µg/mL) (all products from Gibco, Basel, Switzerland), 10% foetal bovine serum (Sera Plus, Pan-Biotech, Aindenbach, Germany) and 5 ng/mL basic fibroblast growth factor (R&D Systems, Minneapolis, MN, USA) with a medium change twice a week.

Cell seeding, culture and staining on zirconia bars

Prior to cell seeding, specimens were cleaned in an ultrasonic bath using an enzymatic detergent (Terg-A-Zyme ® , Sigma-Aldrich, Buchs, Switzerland), followed by extensive rinses with deionized water, 70 % ethanol and deionized water again. Samples were then transferred to a single-well chamber (Lab-Tek ® , Milian, Vernier, Switzerland) and sterilized using ethylene oxide. hMSCs (passage 3) were seeded on the sample surface at a density of 5000 cells/cm 2 using a diluted cell suspension (10 000 cells/mL, 5 mL) in -MEM supplemented with 10% foetal calf serum, penicillin (100 U/mL) and streptomycin sulfate (100 µg/mL). Samples were cultured for 48 hours in an incubator set at 37°C, 5% CO 2 and 90% humidity. Cells were rinsed once with 5 mL of phosphate buffered saline solution, fixed with 70% ethanol and stained with 0.5 % toluidine blue solution for 5 minutes, followed by three rinses with deionized water. Cells were imaged under transmitted light using a microscope (Leica MacroFluo TM , Wetzlar, Germany). The acquisition time was kept constant for all samples.

Morphometric analysis and cell measurements

Analysis of single cell area and shape (solidity, aspect ratio and circularity) was performed using ImageJ (NIH, version 1.47). Three gradient samples and three polished (control) samples were characterized, and 20 cells for each region and sample were analyzed. Solidity was calculated as 𝐴𝑟𝑒𝑎 𝐶𝑜𝑛𝑣𝑒𝑥 𝐴𝑟𝑒𝑎 2 , where the convex area is the imaginary convex hull around the object. Aspect ratio was defined as

𝑀𝑎𝑗𝑜𝑟 𝐴𝑥𝑖𝑠 𝑀𝑖𝑛𝑜𝑟 𝐴𝑥𝑖𝑠
of the object's fitted ellipse. Circularity was calculated as 4𝜋 • 𝐴𝑟𝑒𝑎 𝑃𝑒𝑟𝑖𝑚𝑒𝑡𝑒𝑟 2 . Circularity ranges from 1.0 (perfect circle) to values approaching 0 (which indicate an increasingly elongated shape).

Statistical analysis

Statistical analysis of the cell measurements was performed using SPSS® software (version 20, SPSS Inc., Chicago, IL, USA). Normality of the data was assessed by a Shapiro-Wilk test and the homogeneity of variance between groups was verified with a Levene test. Area, aspect ratio and circularity were log-transformed prior to analysis to normalize the distributions. A one-way ANOVA with Tukey's multiple comparison tests was carried out with a significance level set at p<0.05.

The existence of correlations between topographical parameters and cell morphology was assessed by computing Pearson product-moment and Kendall's tau correlation coefficients, with a two-tailed test of significance. For the calculation, the mean values of each topographical and morphological parameter were computed. In the case of the nanoroughness measurements, the data was linearly interpolated to match the position of the cell measurements when necessary. Besides, a visual inspection of cell morphological parameter vs. topographical parameter graphs was carried out in order to find potential non-linear relationships which would not be detected by the previous method.

Results

Surface characterization

Surface before sonication

The observation of the surface of a specimen which was not subjected to sonication evidenced the presence of adhered precipitates formed during etching (Fig. 6.2). For distances comprised between 0 mm and 19 mm (corresponding to and etching time between 0 and 30 min), they consisted exclusively in particles with an octahedral shape (octahedrons) whose size increases with distance/time. Beyond 19 mm, an adhered layer composed of needle-like particles (needles) appears. The layer seems then to detach progressively until it totally disappears at a distance of 35 mm (etching time: 55 min). A previous study showed that the octahedrons are YF 3 crystals and that the needles are composed of yttrium, zirconium and fluorine 26 .

Surface after sonication

The observation of the surface morphology and topography of the gradient evidenced a clear evolution from smooth to rough (Fig. 6.3 and 6.4). The appearance of a granular texture and etch pits, whose number, diameter and depth increased with time, provided both a nano-and micro-roughness gradient as it will be discussed later.

Roughness analysis

The induced roughness gradients were found to be homogenous in the y direction (transversally) on a width of 2 mm (see supplementary information).

-Amplitude parameters (Figure 5)

At the micro-scale, the average roughness (S a ) and RMS roughness (S q ) increase approximately proportionally to the distance. The kurtosis (S ku ) decreases strongly in the At the nano-scale, the average roughness (S a ) and RMS roughness (S q ) increase up to the half of the gradient and then decrease slightly. The kurtosis (S ku ) presents a similar behavior than at the micrometric scale. The skewness (S sk ) is first largely positive and then decreases substantially. The positive values indicate that at the nano-scale the peaks are always predominant, and the decreasing tendency shows that this predominance becomes less and less important along the distance. The discrepancy between the nanoand the micro-skewness results can be explained by the fact that the Gaussian filter eliminates features which are larger than the cut-off wavelength, which means that etch pits larger than 1 µm have no influence on the nano-roughness.

-Spatial parameters (Fig. 6.5)

At both the micro-and the nano-scale, the fastest decay length (S al ) tends to decrease at the beginning of the gradient and then increases along the distance. At the micro-scale, the density of summits (S ds ) increases along the distance up to a maximum at approximately 25 mm and then decreases slightly, whereas at the nano-scale increases up to a maximum at about 10 mm and then decreases. This probably indicates that in a first step numerous small peaks are formed and in a second step a part of them gets eroded to form broader peaks.

-Hybrid parameters (Fig. 6.6)

At the micro-scale, the RMS gradient (S dq ), the mean summit curvature (S sc ) and the developed interfacial area ratio (S dr ) increase proportionally to the distance up to a plateau which is reached at about 28 mm.

At the nano-scale, the RMS gradient (S dq ), the mean summit curvature (S sc ) and the developed interfacial area ratio (S dr ) increase up to a maximum which is reached at about half of the gradient (19 mm) and then undergo a moderate decrease.

The increase in the hybrid parameters indicates that the intricacy of the surface and the ratio surface area / projected area increase too.

-Functional parameters (Fig.

Fractal analysis

The scale sensitive fractal analysis of the AFM and the WLI data showed that (Fig.

6.7):

-Regarding the smooth-rough crossover (SRC), the results are quite different for both techniques. The analysis of the AFM data leads to a SRC which is roughly proportional to the distance, whereas the analysis of the WLI data leads to a SRC which remains approximately constant all over the gradient and increases strongly from 31 mm to 38 mm. Besides, the values obtained from WLI are much more elevated.

-Regarding the fractal dimension (D), the results correlate well for both techniques up to half of the gradient, showing an increase proportional to the distance. Then for WLI the fractal dimension keeps increasing whereas for AFM it slightly decreases.

It is important to note that the discrepancies between the results obtained from the fractal analysis of the AFM and the WLI data are not related to errors in the measurements: the results of the calculation performed by the tiling algorithm strongly depend on the resolution and the area of observation, which define the range of scales (i.e. the area of the tiles) over which the relative area can be computed and are different for both techniques.

Cell cultures

The gradient in roughness influenced significantly the area (p<0.001), aspect ratio (p=0.01) and solidity (p=0.026) of stem cells (Fig. The solidity reached a minimum in the region of the bar exposed for 30 minutes to HF (0 min vs 30 min HF treatment: p=0.075 and 10 min vs 30 min HF treatment: p=0.083). Cells in this region also had the highest aspect ratio. Stem cells had a spindle-like morphology over the whole sample range, but a significantly more elongated cell shape was observed in the region of the sample exposed for 30 minutes to HF as compared to both shorter and longer treatment times (Fig. 6.8-e, g). As compared to the polished control, the mean cell area was significantly reduced in all regions of the gradient bar except the one corresponding to 20 minutes of HF exposure (p<0.01 for each pairwise comparison), while the aspect ratio, circularity and solidity were not significantly different.

Significant correlations were found between cell solidity and several topographical parameters. The correlation was particularly pronounced for nano-roughness parameters (S a , S q , S al , S dq , S dr , S sc , S m and S c ) and the AFM fractal dimension (D) (Table 6.2, Fig. 6.9).

Additionally a possible curvilinear correlation between several micro-roughness parameters, the WLI fractal dimension and cell area was identified (Fig. 6.9). In particular, a good fitting could be obtained with respectively a second-order and a third-order polynomial regression for the developed interfacial area ratio (S dr ) and the fractal dimension (D). Fractal SRC r -0,32 0,02 0,34 0,12 -0,51 -0,04 0,05 -0,35 τ -0,59 -0,20 0,49 0,29 -0,14 -0,14 0,05 -0,33 D r -0,34 -0,13 -0,15 -0,56 0,28 -0,16 -0,71 -0,94 τ -0,05 -0,05 -0,05 -0,43 0,33 0,14 -0,43 -0,81

Discussion

The method proposed in this study was successful to produce surface gradients on zirconia both at the micro-and at the nano-scale. In particular a large range of variation was obtained for roughness parameters belonging to different categories such as S a , S q (amplitude), S ds (spatial), S dr , S sc , S dq (hybrid), S m , S c and S v (functional), as well as for fractal parameters (D, SRC) (Figs. 6.5, 6.6, 6.7). Regarding the etching process itself, interestingly the adhered layer of needles appears around 30 min of etching (Fig. 6.2), which corresponds to an inflection point for some curves such as S a , S q , S dr , S sc , S dq vs. distance/etching time (Fig. 6.5, AFM data). The layer might play a role by shielding the material below from the etching solution.

It comes out of the scope of this study, but controlling the formation / dissolution of these precipitates could thus somehow allow controlling the evolution of the nano-roughness.

The gradients were homogenous transversally on a 2 mm wide band, which provides a large area for cell culture analyses and makes the specimens obtained valuable for the highthroughput screening of cell-surface interactions. Besides, their fabrication process is flexible: polished bars were used here but the method has the potential to be applied to any type of surface (for instance: ground, sandblasted). Finally, zirconia etching is a process which is already used in the industry (for instance: CeraRoot implants with ICE™ surface).

The results obtained with this type of gradients present thus the advantage to be directly transposable to the fabrication of commercial dental components.

Regarding cell cultures, the gradient did induce changes in hMSC response. A minimum in the solidity was associated to a maximum in the aspect ratio for an etching time of 30 min (x = 19 mm) (Fig. 6.8), which may indicate a stronger commitment towards the small spindleshaped rapid self-renewing cell type described by Colter et al. 33 which is associated to more rapid proliferation and mineralization as compared to other cells present in the hMSC heterogeneous population. Cell solidity was found to be linearly correlated with some nanoroughness parameters and the AFM fractal dimension, whereas cell area seemed to present a curvilinear correlation with some micro-roughness parameters and the WLI fractal dimension (Fig. 6.9 and Table 6.2). hMSC solidity appears thus to be sensitive to the nano-topography whereas hMSC area seems more influenced by the micro-topography. This highlights the interest of performing a multi-scale analysis, combining both WLI and AFM measurements.

The effects observed in the present study might appear as moderate. This is however counterbalanced by the fact that, given the absence of culture-to-culture variations, the gradient approach provides a much higher statistical power than a classical approach. The method is thus promising for further high-throughput studies of cell interaction with zirconia surface, which could focus on diverse topics such as for instance the influence of roughness on stem cell differentiation, osteoblast mineralization or osteoclast resorption.

Conclusion

Roughness gradients have been successfully produced at the surface of zirconia by controlling the speed of immersion of specimens into hydrofluoric acid. The gradients achieved present a large range of variation of fractal and roughness parameters at both the micro-and nano-scale. The variation of topography influenced hMSC morphology, with significant changes in cell area, aspect ratio and solidity. Nano-roughness parameters and the AFM fractal dimension were correlated to cell solidity, while micro-roughness parameters and WLI fractal dimension appeared to be correlated to cell area. The method proposed in this work has the potential to be transposed to other type of surfaces and cell-surface interaction studies, reducing drastically the amount of samples and resources required. Premièrement on a étudié des surfaces d'alumine-zircone avec différentes micro-rugosités obtenues par moulage par injection. Le comportement d'ostéblastes humains primaires a été étudié sur les surfaces non modifiées ou modifiées par un traitement à l'acide hydrofluorique (attaque chimique). Des ostéoblastes primaires humains obtenus à partir de têtes de fémurs soumis à arthroplastie ont été choisis pour leur grande pertinence clinique. La micro-rugosité a eu seulement un effet mineur sur la réponse des ostéoblastes tandis que la combinaison de micro-et nano-rugosité a conduit à une maturation soutenue des ostéoblastes humains. Les nano-topographies introduites par attaque chimique ont été superposées sur la microrugosité en ayant un effet synergique sur l'expression génique des molécules engagés dans les processus de maturation ostéoblastique.

Cette stratégie de modification de la surface céramique ouvre la voie vers des cupules acétabulaires céramiques monoblocs directement ostéo-intégrées.

Dans un deuxième temps une technique d'impression 3D, et plus précisément le robocasting, a été exploré pour la production de structures macroporeuses en alumine-zircone avec une haute reproductibilité et un bon contrôle architectural. Les structures imprimées ont présenté une topographie à de multiples échelles grâce au design d'une part et aux conditions de frittage d'autre part. Les ostéoblastes ont pu s'attacher sur les structures 3D mais la rétention des cellules à l'intérieur des scaffolds sur le long terme reste à améliorer.

Des techniques de sélection rapide de conditions de modifications de surface ("screening") ont fait l'objet de la dernière partie de cette thèse. Deux différentes stratégies ont été utilisées pour modifier la surface de la zircone: laser femtoseconde pour la production de multiples motifs sur un échantillon unique ("multi-patterning") et échantillons avec un gradient de rugosité via le contrôle du temps de l'attaque chimique. La morphologie des cellules souches humaines a permis d'avoir un indicateur précoce de la lignée de différentiation cellulaire.

En conclusion, les différentes techniques de modification de la surface de zircone et composites alumine-zircone utilisées à travers la thèse peuvent moduler l'interaction des cellules primaires humaines en offrant des signaux pour la différentiation ostéoblastique et la maturation des ostéoblastes.

MOTS-CLÉS

  Préface en français ................................................................................................................. 6.3. Materials and methods ...................................................................................................................... 6.3.1. Sample fabrication .................................................................................................................... 6.3.1.1. Fabrication of zirconia bars .................................................................................................. 6.3.1.2. Surface modification for obtaining roughness gradients ...................................................... 6.3.2. Surface characterization ........................................................................................................... 6.3.2.1. Measurements ...................................................................................................................... 6.3.2.2. Roughness analysis .............................................................................................................. 6.3.2.3. Fractal analysis ..................................................................................................................... 6.3.3. Cell cultures .............................................................................................................................. 6.3.3.1. Isolation and expansion of human bone marrow derived mesenchymal stem cells ............. 6.3.3.2. Cell seeding, culture and staining on zirconia bars .............................................................. 6.3.3.3. Morphometric analysis and cell measurements.................................................................... 6.3.3.4. Statistical analysis ................................................................................................................ 6.4. Results ............................................................................................................................................... 6.4.1. Surface characterization ........................................................................................................... 6.4.1.1. Surface before sonication ..................................................................................................... 6.4.1.2. Surface after sonication ........................................................................................................ 6.4.1.3. Roughness analysis .............................................................................................................. 6.4.1.4. Fractal analysis ..................................................................................................................... 6.4.2. Cell cultures .............................................................................................................................. 6.5. Discussion ......................................................................................................................................... 6.6. Conclusion ........................................................................................................................................ 6.7. References .........................................................................................................................................

  des creux) avec des conséquences directes sur la réponse cellulaire. Par conséquence, une caractérisation complète de la surface est cruciale pour mieux comprendre les signaux modulant la réponse cellulaire et la standardisation de la caractérisation de surface facilitera grandement la comparaison entre les études. En outre, l'utilisation lors des études in vitro d'un type de cellule pertinent pour l'application envisagée, la combinaison de plusieurs types d'analyses et de points temporels contribuent à la compréhension et à la prédiction des réponses biologiques qui se produisent in vivo et plus loin dans le scénario clinique. En plus, l'analyse d'une large gamme de conditions de surface peut bénéficier de méthodes de dépistage rapide, telles que des échantillons à motifs multiples et des échantillons à gradient de surface. Cette thèse a eu pour but d'évaluer les interactions entre différents types de cellules et des surfaces céramiques, et plus particulièrement la zircone et la ZTA obtenues par diverses techniques comme le moulage par injection, le pressage isostatique et l'impression 3D. De différentes approches ont été utilisées pour l'introduction de la micro-et nano-rugosité. Pour résumer, nous avons guidé les études selon plusieurs objectifs: (i) identifier une gamme optimale de microrugosité des échantillons ZTA obtenus par moulage par injection dans des moules rugueux et (ii) déterminer si une attaque chimique sélective supplémentaire de la surface miro-rugueuse a un effet positif sur la maturation des ostéoblastes primaires humains (hOb). Ensuite, nous nous sommes concentrés sur (iii) l'optimisation d'une encre ZTA adaptée au robocasting (coulage assisté par système robotique), (iv) sur la caractérisation des scaffolds (échafaudages 3D) préparés par robocasting et (v) et sur l'étude in vitro du comportement des ostéoblastes primaires sur ces scaffolds ZTA. En raison de plusieurs paramètres influençant le comportement des cellules, la détermination d'une condition appropriée nécessite d'un nombre élevé d'échantillons et peut demander beaucoup de temps.

Europe comme suit:

  Imperial College in London, UK (4 ESR)  INSA Lyon, France (3 ESR)  UPC Barcelona in Spain (1 ESR)  University of Erlangen in Germany (2 ESR)  UMONS in Belgium (1 ESR)  AO Research Institute Davos in Switzerland (1 ESR). Les partenaires industriels (Ceramtec à Plochingen, Allemagne, Lucideon à Stoke on Trent, Royaume-Uni, Noraker à Lyon, France et Keramat à Ames, Espagne) ont participé activement au projet et ont accueilli les ERs. La collaboration au sein du consortium a été promue parmi différents groupes de travail: WP1 -Traitement et nouveaux matériaux, WP2 -Biodégradation et WP3 -Intéractions Cellules-Matériau. La thèse a fait partie du WP3 et une collaboration étroite a été établie avec succès avec:  UPC Barcelona: Quentin Flamant comme ESR et son superviseur Marc Anglada,  Ceramtec: Katia Biotteau comme ER, et Alessandro Porporati et Meinhard Kuntz comme superviseurs. Dans le cadre de l'ITN, j'ai eu la possibilité de faire un détachement à court terme chez Ceramtec et un détachement de six mois à MATEIS, INSA Lyon. Les deux stages m'ont donné l'occasion d'apprendre davantage sur les matériaux (traitement et caractérisation) avec une perspective différente.



  consortium ITN (Initial Training Network) FP7 Marie Curie project focused on training ESRs (Early Stage Researchers, namely PhD students) and ERs (Experienced Researchers, namely post-doctoral researchers). Both academic and industrial partners combined knowledge and resources around Europe as follows: Imperial College in London, UK (4 ESR)  INSA Lyon, France (3 ESR)  UPC Barcelona in Spain (1 ESR)  University of Erlangen in Germany (2 ESR)  UMONS in Belgium (1 ESR)  AO Research Institute Davos in Switzerland (1 ESR). Industrial partners (Ceramtec in Plochingen, Germany, Lucideon in Stoke on Trent, UK, Noraker in Lyon, France and Keramat in Ames, Spain) were actively involved in the project and hosted ERs. Collaboration within the consortium was promoted among different work packages: WP1 -Processing & New materials, WP2 -Biodegradation and WP3 -Cell-Material Interactions.

Figure 3 . 3 Figure 3 . 5 Figure 3 . 6 Figure 3 . 7

 33353637 ) and day 30 (d-f) of culture: a,d) untreated, b,d) HF-etched, e,f) HF+HCl-etched "medium" surface. Black arrows indicate cell layer starting to roll, white arrow show cell aggregates. Scale bar = 1 mm. .............................................................................. 101 Figure 3.1 SEM micrographs of 3D-ZTA : a) overview (scale bar = 100 μm); (b) magnified view (scale bar = 10 μm); (c) detail of a strut surface, showing sub-micronsized pores homogeneously distributed (scale bar = 1 μm); (d) surface composition (in BSE mode, scale bar = 1 μm), showing a homogenous distribution of the alumina (grey spots) and zirconia (brighter spots) and the porosity at the grain boundaries. ............................................................................................................................... 120 Figure 3.2 a) region of interest (ROI) on which density, struts and pores dimensions were measured; b,c) 3D views of the ROI (respectively XY and YZ planes); d) cross section of the ROI (X,Y plane, scale bar = 500 μm). .............................................. 121 Pore size distribution of 3D-ZTA and 2D-ZTA (two-dimensional ZTA control) by mercury intrusion porosimetry. Note the bimodal pore size distribution for 3D-ZTA. Similar distributions of the submicron porosity were found for both 2D-and 3D-ZTA. . 122 Figure 3.4 a) cell proliferation and b) alkaline phosphatase activity (ALP) normalized to DNA of human primary osteoblasts cultured on 3D-ZTA, 2D-ZTA and Thermanox TM (TMX) at day 10, 20 and 30 of culture. Data are represented as mean and standard error of mean (n=6); * denotes p<0.05 versus TMX atthe same time point. .............................. 123 mRNA expression of osteogenic markers in human primary osteoblasts cultured on 3D-ZTA, 2D-ZTA and TMX at day 10, 20 and 30 of culture: a) runx2, b) type I collagen (COL1), c) ALP, d) osteocalcin (OC). Data are expressed as 2 -ddCt relative to the mRNA expression of cells collected on the day of seeding (day0); data are represented as mean and standard error of mean (n=6). ............................................................................ 124 Representative images of alizarin red (ARS) stained 3D-ZTA, 2D-ZTA and TMX at day 10, 20 and 30 of culture. Scale bar = 2 mm. ............................................. 125 Quantification of the stained areas by using Image J software. The numbers represent percentage of stained area relative to the whole sample area. 126

Figure 4 . 1 Figure 4 . 3 Figure 4 . 4 Figure 4 . 5 Figure 5 . 1 .Figure 5 . 2

 414344455152 Figure 4.1 Top: schematic diagram of a multi-patterned zirconia disk (dimensions are in mm). Bottom: patterned zones (pit diameter = 10, 20 and 30 µm) observed by optical microscopy............................................................................................................... 137 Figure 4.2 a) White light interferometry topographical images of the six different types of pits produced by femtosecond laser micromachining; b) example of extracted depth profiles (first number refers to pit diameter and second number refers to pit depth). ................................................................................................. 139 Figure 4.3 Scanning electron microscope micrographs of the different types of patterns: a) multiple pits, b) individual pit, c) bottom of the pit. Note the nanometric granular texture on the sidewalls and the presence of ripples at the bottom of the pits. ......................................................................................................................... 142 Figure 4.4 Focused ion beam induced cross-sections of the different types of pits: a) entire pit, b) bottom of the pit, c) side of the pit. Note the presence of micro-cracks and sub-micrometric pores on the pit surface, as well as sub-superficial pores up to a depth of 5 µm. ........................................................................................................ 143 Figure 4.5 Mesenchymal stem cell size and shape analysis following 48 hours of culture on the laser-patterned discs: a) cell area, b) solidity, c) aspect ratio, d) circularity and e) representative images of the toluidine blue stained cells. Data represent the analysis of 20 cells/group (n=10/sample, 2 samples) and are plotted as single points, as well as median and interquartile range. N indicates the unpatterned control, Y indicates patterned regions. *, **, *** and **** represent p<0.05, <0.01, <0.001 and <0.0001 between the highlighted groups; #, ## and ### represent p<0.05, <0.01, <0.001 versus the unpatterned control. In e), the first number indicates the pit diameter and the second number the pit depth; scale bar = 50 µm. ............................................................................................................ 145 Figure 5.1. La taille de la tête de fémur détermine la plage de mouvement (ROM). Une cupule céramique plus fine (image droite) permet d'accommoder une tête de fémur plus grande (www.ceramtec.com). .............................................................. 152 Figure 5.2 Micrographies SEM des différentes surfaces ZTA micro-rugueuses: surface de rugosité a) "poli", b) "comme fritté ", c) " faible", d) "medium", e) "haute". L'effet de l'attaque chimique sélective: f) poli, g) poli+ HF et h) poli+ HF+HCl (barra d'échelle = 5 µm). ................................................................................................... 153 Figure 5.3 Le spectre de rugosité efficace proposé par Hayes et al. entre 0.2-2 µm: A -Sur une surface lisse, les ostéoblastes adopteront une morphologie de type fibroblaste; B -Sur une surface ondulée, si la distance entre les pics est supérieure à la taille de la cellule, les cellules reconnaîtront la surface comme plate et se comporteront en conséquence ; C -Sur une surface microrugueuse ils adopteront la morphologie typique des ostéoblastes; D -Sur une surface avec des topographies mélangées, la réponse cellulaire reflètera une moyenne des microtopographies lisses et microrugueuse. 6 ..................................................................................... 154 Figure 5.4. Rapport ALP/DNA sur (a) Différents échantillons d'alumine-zircone microrugueux et (b) Comparaison des échantillons microrugueux reçus, attaqués par acide fluorhydrique (HF) avec ou sans attaque chimique supplémentaire dans l'acide chlorhydrique (HCl): (b) poli, (c) comme fritté, (d) faible et (e) moyen et (f) haute rugosité de surface. A noter que seulement des différences minimales ont été observées parmi les différents groupes d'échantillons microrugueux, tandis que l'attaque chimique a entraîné une augmentation significative en ALP/DNA pour les hOb cultivés sur l'alumine-zircone comme fritté, faible et moyennerugosité de surface. ................................................... 156 Figure 5.5 Micrographie SEM de l'échafaudage 3D-ZTA, barre d'échelle = 100 μm (gauche) et ALP / DNA sur la 3D-ZTA et 2D-ZTA par rapport au contrôle thermanox. À noter l'activité de phosphatase alcaline supérieure sur 2D-ZTA. ........................ 158

Figure 1 . 1

 11 Figure 1.1 Summary of aging features of zirconia.

Figure 1 . 2

 12 Figure 1.2 Example of the microstructure of an alumina-zirconia composite. Note the grey alumina phase and the white zirconia phase with quite large aggregates 11 .

Figure 1 . 3

 13 Figure 1.3 Scheme representing surface modifications for dental implants, scale bar = 3 µm 14 .

Figure 1 . 4

 14 Figure 1.4 Left: different examples of surface profiles with same R a (roughness average) but different shapes of the surface features; right: overview of the types of topographical surface features: form, waviness, roughness (adapted after Wennerberg and Albrektsson).

Figure 1 . 5 Figure 1 . 6 A

 1516 Figure 1.5 Scanning electron microscopy micrographs of zirconia surface: MSmirror polished, SB25, SB50, SB150 -sandblasted with 25 μm, 50 μm and 150 μm respectively diameter alumina diameter; SB150+HF received additionally a hydrofluoric acid etching 20 .

Figure 1 . 7

 17 Figure 1.7 Contact angle of zirconia surface with different surface modifications 23 .

Figure 1 . 8

 18 Figure 1.8 Contact angle measurements (distilled water) on zirconia: MS -, SB50 , SB-150 sandblasted with 50 and 150 μm diameter alumina particles, respectively, SB150E -sandblasted and etched zirconia surfaces 21 .

Figure 1 . 9

 19 Figure 1.9 Schematic representation of plasma surface modification: plasma treatment is converting the 1-3 nm superficial layer into zirconium oxyfluoride; the oxyfluoride layer isinteracting with organo-silanes, enabling silicon attachment26 .

  . Piascik et al. used fluorinated plasma treatment to create an oxyfluoride conversion layer on zirconia surface with increased reactivity for silicon attachment (Fig. 1.9). Direct surface silanisation was proposed by Caravaca et al. as a tailored zirconia surface for osseointegration (Fig. 1.10). Additionally, this plasma treatment resulted in increased aging resistance for zirconia 28 .

Figure 1 .

 1 Figure 1.10 MG-63 cells spreading and morphology after 6 days of culture on plasma treated and further silanisation (right side) compared to the polished control (left side)28

  Osteoclasts general feature is their large size (150-200 µm) and multiple nuclei that are the result of multiple(10-20) individual cells fusion. Terminal differentiation occurs under stimulation of two hematopoietic factors that are both necessary and sufficient: RANKL -TNF related cytokine and CSF-1 -Colony-stimulating factor-151 32 . Both factors have a synergic effect at gene level, activating osteoclast lineage specific genes i.e. tartrate-resistant acid phosphatase (TRAP), cathepsin K (CATK), calcitonin receptor and β 3 -integrin. During osteoclastogenesis (Fig.1.16) and activation there are at least five distinct signalling cascades mediated by protein kinases: inhibitor of NF-kB kinase (IKK), c-Jun N-terminal kinase (JNK), p38, extracellular signal-regulated kinase (ERK) and Src pathways32 .

Figure 1 . 16

 116 Figure 1.16 Scheme representing the process of osteoclastogenesis: development from haematopoietic precursor cells to mature osteoclasts 32 .

  environmental niches. Mesenchymal stem stromal cell (MSCs), immune cells and haematopoietic stem cells (HSCs) contribute to cellular functional compartments. All these specific niches are interdependent interacting continuously by signals for cell-renewal, differentiation and quiescence (Fig. 1.18).

Figure 1 . 18

 118 Figure 1.18 Scheme representing the bone marrow niche hosting hematopoietic stem cells and mesenchymal stem cells 60 .

Jaiswal

  et al. established a technique for MSCs in vitro osteogenic differentiation by using βglycerophosphate, ascorbate-2-phosphate and dexamethasone, which became the standard osteogenic supplements[START_REF] Jaiswal | Osteogenic differentiation of purified, culture-expanded human mesenchymal stem cells in vitro[END_REF] . MSCs commitment and development along different lineages is finely orchestrated by specific groups of genes. Transcription factors direct MSC differentiation towards osteoblast lineage: Runx-2 and Osx 69 , chondrocyte lineage: SRY (sex determining region Y)-box 9 (Sox9)[START_REF] Lefebvre | L-Sox5, Sox6 and Sox9 control essential steps of the chondrocyte differentiation pathway[END_REF] . Commitment towards osteoblast lineage is signalled by expression of these specific transcriptional factors in osteoprogenitor cells. The principal transcriptional controller of osteoblast differentiation is Runx-2 while studies showed Runx-2 null mice completely lack osteoblastic differentiation[START_REF] Komori | Targeted disruption of Cbfa1 results in a complete lack of bone formation owing to maturational arrest of osteoblasts[END_REF] . Nevertheless, Runx-2 is required but not sufficient for stable osteogenesis. Osteodifferentiation progresses via a number of progenitor and precursor stages to the mature functional osteoblast.Mesenchymal stem stromal cells isolated from bone marrow can be easily isolated and expanded in vitro by centrifugation gradient and plastic adherence. Nevertheless it is important to note that cells isolated by this method are heterogeneous. More homogenous populations have been isolated using cell surface markers, although a general consensus on what is a "true" stem cell has not been reached so far. One characteristic of these cells is heterogeneity of both the colonies derived from single MSCs and the cells within each individual colony. Cell diversity regards various cellular features such as size, morphology, level of enzyme activity, proliferation and differentiation potential (Fig.1.12 A). Human bone marrow derived MSCs colonies exhibit three morphologically distinct cell types: spindle-shaped colonies, flat colonies composed of very flattened MSCs and intermediate colonies composed of larger MSCs whose shape is intermediate between spindle and flat[START_REF] Kuznetsov | Single-colony derived strains of human marrow stromal fibroblasts form bone after transplantation in vivo[END_REF] . A more concise characterization defines the MSCs morphology as rapidly self-renewing cells (RS) and flat cells (FC) with very low cell proliferation rates[START_REF] Docheva | Researching into the cellular shape, volume and elasticity of mesenchymal stem cells, osteoblasts and osteosarcoma cells by atomic force microscopy[END_REF] .

  . Moreover, Hayes et al. identified a roughness spectrum between 0.2 to 2 µm which is considered to induce the optimal changes in cell behaviour regarding smooth versus rough surfaces 106 . Not only cell morphology but also cell proliferation and differentiation are influenced by surface roughness. Depending on the surface properties MSCs will differentiate towards osteoblasts if the topographical cues will promote preosteoblasts formation. Once primary stability is reached, bone formation can occur at the bone surface in proximity of the implant (distance osteogenesis) or directly on the implant surface (contact osteogenesis) 107 . Gittens et al. underlined that each roughness scale contributes to improved osseointegration, with nano-roughness acting on protein absorption, micro-roughness influencing cell behaviour and macro-scale roughness determining the mechanical interlocking of an implant to native bone (Fig. 1.19).

Figure 1 . 19

 119 Figure 1.19 Scheme representing different topographical scales influencing implant-bone interaction and osseointegration 14 .

Figure 1 . 20

 120 Figure 1.20 Fluorescence intensity of osteopontin immunostaining as a function of microroughness and nanofeature density 113 . Flamant et al. showed recently that ceramic surface chemical modification can potentially increase osteoblast cell attachment while limiting bacterial adhesion. Selective chemical etching of zirconia toughened alumina surfaces led to nano-rough and interconnected porous surface 19 . A vast amount of work on surface roughness effects on in vitro and in vivo performance of implants has been performed on titanium surfaces, as this material has been used since few decades for internal fixations and implants. Boyan et al. showed that transforming growth factor beta-1 (TGFβ1) and prostaglandin E2 (PGE) expression in osteoblast-like cells MG63 exhibit a surface roughness dependent response. In addition, a significant increase of TGFβ1 levels was reported by Kieswetter et al. on osteoblast-like MG-63 cells coarse sandblasted and titanium plasma sprayed surfaces when compared to tissue culture plastic 114 . These growth factors are known to play a key role in MSC recruitment and proliferation simultaneously with osteoclast activity down regulation.

  suggesting that overly hydrophilic surfaces can reduce attachment of anchorage-dependent cells. Moreover, more recent studies have shown that changes in surface wettability, hydrophobicity and surface charge have been shown to influence protein adsorption. Protein adsorption was enhanced on hydrophilic surface when compared to hydrophobic ones and therefore cells adhered and proliferated better on the first ones124 . Liu et al. concluded that hFOB 1.19 cells-adhesion contacts, attachment and spreading are faster on hydrophilic surfaces 125 . Orientation of adhered proteins can be influenced by ions incorporated to the surface. For instance, fluoroapatite incorporated into hydroxyapatite surface changes both attachment and orientation of calcium-binding proteins and consequently cell adhesion 104 . Chemical modifications by grafting different compounds to the surface have been used to promote cell attachment. Covalent conjugation of a monomer or a protein is a common technique to alter surface chemistry by first activating the surface and then grafting the desired functional group, providing relative long-term stability. Different methods can be used: chemical reactions, radiation exposure, plasma or ozone exposure 126 . For instance, collagen type I was immobilized on a (3-aminopropyl) triethoxy silane and cross linker glutaraldehyde treated surface 127 . Collagen functionalized surface improved MSC adhesion, promoting long term attachment and osteogenesis induction. More recently, Caravaca et al.used oxygen plasma to clean zirconia surface and promote hydroxylation so as to covalently bind 3-aminopropyldimethylethoxy silane to zirconia sufaces28 . Improved MG-63 osteoblastlike cells attachment was observed after three days by SEM on modified surfaces and no cytotoxic effects up to day 10. Nano-porous alumina surface was functionalized with bone morphogenetic protein 2 (BMP2) for synergic effect of chemical factors and surface topography on mesenchymal stem cells behaviour 128 . MSCs adhesion was faster on BMP2 immobilized alumina substrates and ALP activity was enhanced when compared to unmodified control.

Figure 1 . 24

 124 Figure 1.24 Direct osseointegration of titanium, alumina/zirconia and zirconia screws following a 4 week implantation in maxilla of minipigs 131 . Top row: overviews of the screws. Bottom row: detailed images of the implant/bone interface. Note the absence of fibrous capsule.

  between various independent research groups138 . Analogy targeted the in vitro and in vivo stages of biomaterials tested by different in vitro techniques, biocompatibility, cell differentiation, gene expression of early and late markers and calcium deposition, performed by different groups with considerable consensus in assays interpretation.Various inherent factors could potentially influence this unexpected reduced correspondence such as cell type or cell line choice, cell number passage, protocol used or reagents variation.It was previously reported byBara et al., contradictory results of two clinical trials due to the variance in isolation (centrifugation steps) and expansion (media and serum) procedures 139 .

2. 1 .

 1 RésuméEn ce qui concerne l'arthroplastie totale de la hanche sans addition (ciment) les composants acétabulaires exigent d'excellentes propriétés mécaniques, une biocompatibilité parfaite, de faibles frottements et une bonne ostéointégration avec le tissu osseux limitrophe. Les composites alumine-zircone remplissent ces conditions mais l'association avec une coque métallique rugueuse est à ce jour nécessaire pour une intégration osseuse suffisante. La modification de la surface alumine-zircone pourrait représenter le moyen d'avoir une solution sans métal. Le composant acétabulaire, monobloc, deviendrait globalement plus fin. Il serait alors possible d'augmenter le diamètre de la tête fémorale. En conséquence, le risque de dislocation serait réduit et l'amplitude de mouvement de l'articulation serait augmentée. Ou bien, en préservant les diamètres actuels de têtes fémorales, l'encombrement réduit du composant acétabulaire permettrait de préserver l'os pour une éventuelle révision.Dans cette étude, une large plage de micro-rugosité (0.013 μm < Sa < 0.416 μm) a été obtenue par moulage par injection, une technique qui modifie uniquement la rugosité (contrairement à la plupart des techniques de modification de surface qui changent à la fois la rugosité et la chimie de surface). Sur certains échantillons, une attaque chimique sélective à l'acide hydrofluorique a permis d'introduire en outre une nano-rugosité. Nos résultats suggèrent que la microrugosité, l'enrichissement par du fluor et la nano-rugosité de la surface composite ont un effet synergique sur la maturation des ostéoblastes primaires humains.Parmi les conditions examinées, la rugosité moyenne (S a = 330 nm) modifiée avec acide hydrofluorique a déterminé une expression supérieure et rapide de la phosphatase alcaline à la fois au niveau génique et protéique, caractéristique d'une minéralisation plus rapide. Par contre la micro-et la nano-rugosités prises séparément ont seulement eu des effets mineurs sur la réponse des ostéoblastes ostéoblastes. L'étude de l'adsorption des protéines ou des cocultures avec des types cellulaires différentes peuvent être envisagé pour compléter l'étude des propriétés biologiques des surfaces céramiques. La porosité créée par l'attaque chimique peut être utilisée comme réservoir pour des antibiotiques ou des facteurs de croissance.

Figure 2 .

 2 Figure 2.1 Scanning electron microscope micrographs of the zirconia-toughened alumina (ZTA) surfaces. a-e) Comparison between the different micro-rough ZTA surfaces: a) "polished", b) "as sintered", c) "low", d) "medium", e) "high" roughness surface; f-h) comparison of polished surface before and after etching with hydrofluoric acid (HF) or HF followed by hydrochloric acid etching (HF + HCl): f) polished, g) polished + HF and h) polished + HF+HCl. Scale bar = 5 µm.

Figure 2 . 2

 22 Figure 2.2 Cell proliferation assessed by DNA quantification and alkaline phosphatase activity (ALP) normalized to DNA content on Medium roughness surfaces before and after HF or HF+HCl etching at day 10, 20 and 30 of culture. Results from single donors are represented as mean and standard error of mean.

Figure 2 . 3

 23 Figure 2.3 Cell proliferation assessed by DNA quantification at day 10, 20 and 30 of culture: a) Comparison between the different micro-rough ZTA surfaces; b-f) comparison of the surfaces before and after etching HF or HF + HCl. b) Polished, c) As sintered, d) Low, e) Medium, f) High roughness surfaces. Depending on the samples distribution, parametric (one-way Anova with Tukey post-hoc) or non-parametric (Kruskall-Wallis test and Dunn post-hoc) analyses were performed (p<0.1 were considered trends, p<0.05 were considered significant differences, p valuesare given on the graphs) using GraphPad Prism 7.01 software.

Figure 2 . 4 2 . 5 . 3 . 2 . 5 . 3 . 1 .

 242532531 Figure 2.4 ALP normalized to DNA content at day 10, 20 and 30 of culture: a) Comparison between the different micro-rough ZTA surfaces; b-f) comparison of the surfaces before and after etching HF or HF + HCl. b) Polished, c) As sintered d) Low, e) Medium, f) High roughness surfaces.

  2.4 b), RUNX2 (Fig. 2.5 b) and ALP expression (Fig. 2.6 b) compared to the polished ZTA control. Moreover, HF treatment generally induced an earlier hOb maturation compared to HF+HCl treatment. A lower DNA content was observed on HF-etched ZTA at day 10, but this was compensated at later time points (Fig. 2.3 b). These findings are in agreement with previous studies by Popat et al., 19 who have found improved human foetal osteoblasts cell line hFOB 1.19 mineralization on nano-porous alumina compared to amorphous alumina controls. Studies on HF-etched titanium by Cooper et al. suggested that fluorine presence at the surface supported osteoblastic differentiation of human mesenchymal stem cells in vitro and interfacial bone formation in rat tibia in vivo.20

3. 1 .

 1 RésuméLes composites alumine-zircone représentent la référence des matériaux céramiques pour la fabrication de prothèses de hanche, grâce à leurs excellentes propriétés mécaniques, leur biocompatibilité et leur faible coefficient de frottement. Une nouvelle stratégie afin de développer une meilleure stabilité primaire (liée au verrouillage mécanique entre implant et os) favorisant par la suite la stabilité secondaire (liée à l'ostéointégration des implants céramiques) a été développé via l'addition d'une couche céramique architecturée par une technique d'impression 3D (robocasting) sur la partie extérieure de la pièce acétabulaire. Le robocasting est une technique de fabrication additive, proche de la microextrusion 3D, présentant des résultats encourageant en ce qui concerne la formation des topographies à des différentes échelles sur une partie définie d'un implant.Au cours de ce travail, préliminaire à la stratégie exposée ci-dessus, nous avons exploré la possibilité de fabriquer des scaffolds (échafaudages) alumine-zircone par robocasting. Après optimisation de l'encre céramique, des disques poreux en alumine-zircone de 1.5 cm diamètre composés de 7 couches ont été imprimés avec une aiguille de 330 μm diamètre. La structure des scaffolds a été caractérisée par microscopie électronique par balayage, tomographie à rayons X et porosimétrie mercure. Une évaluation in vitro du comportement des scaffolds en alumine-zircone a été conduite avec des ostéoblastes primaires humains. L'adhésion cellulaire, la vitesse de prolifération et le potentiel de minéralisation ont été mesurés après 10, 20 et 30 jours de culture cellulaire. Aucune réaction négative n'a été observée vis-à-vis des structures imprimées en 3D, même si la rétention des cellules à l'intérieur des scaffolds reste à améliorer.

Table 3 . 2

 32 A standard curve was prepared by serial dilution of 1 mM p-nitrophenol (Sigma #N7660). Absorbance at 405 nm in duplicates was measured with a plate reader (Multilabel Counter Wallac 1420, Perkin Elmer, US). The enzymatic activity was expressed as µmoles of p-nitrophenol liberated per min and normalised to the amount of DNA of each sample. Human alkaline phosphatase (hALP) Hs00758162_m1 Human osteocalcin (hOC) Forward primer: 5'-AAG AGA CCC AGG CGC TAC CT-3' Reverse primer: 5'-AAC TCG TCA CAG TCC GGA TTG-3' Probe: 5'-ATG GCT GGG AGC CCC AGT CCC-3' Primer and probe sequences used for real-time polymerase chain reaction.

  3.1 a). An average strut thickness of 196 ± 15 μm, an average pore size of 273 ± 15 μm and post-sintering linear shrinkage of ~30% were found by analysing SEM images. The strut surface was rough due to the intrinsic surface features of the tip used for the printing (Fig. 3.1 b). Struts were microporous as a result of the lowtemperature sintering conditions (Fig. 3.1 c). The zirconia phase was homogenously distributed within the alumina phase (Fig. 3.1 d).

Figure 3 .

 3 Figure 3.1 SEM micrographs of 3D-ZTA : a) overview (scale bar = 100 μm); (b) magnified view (scale bar = 10 μm); (c) detail of a strut surface, showing sub-micron-sized pores homogeneously distributed (scale bar = 1 μm); (d) surface composition (in BSE mode, scale bar = 1 μm), showing a homogenous distribution of the alumina (grey spots) and zirconia (brighter spots) and the porosityat the grain boundaries.

  3.2). A certain amount ofbending of the structures related to the drying conditions is obvious on Figs. 3.2 b, c. As a result, the cross section shown in Fig. 3.2 d shows regularly spaced struts that do not seem to cross the entire specimen.Figure 3.2 also reveals the (ABCD) arrangement of the struts, the struts of layers A and C being parallel but shifted by a half strut-to-strut distance, and layers B and D being orthogonal to A and C and also shifted by a half strut-to-strut distance. This arrangement was chosen to avoid a fast sedimentation of cells during hOb culture while preserving the periodic architecture and the overall porosity. An average strut thickness of 190 ± 5 μm, average pore size of 245 ± 20 μm and ~50% porosity were found by analysing CT scans (Fig. 3.2 a).

Figure 3 .

 3 Figure 3.2 a) region of interest (ROI) on which density, struts and pores dimensions were measured; b,c) 3D views of the ROI (respectively XY and YZ planes); d) cross section of the ROI (X,Y plane, scale bar = 500 μm).

Figure 3 . 3

 33 Figure 3.3 Pore size distribution of 3D-ZTA and 2D-ZTA (two-dimensional ZTA control) by mercury intrusion porosimetry. Note the bimodal pore size distribution for 3D-ZTA. Similar distributions of the submicron porosity were found for both 2D-and 3D-ZTA.

Figure 3 .

 3 Figure 3.4 a) cell proliferation and b) alkaline phosphatase activity (ALP) normalized to DNA of human primary osteoblasts cultured on 3D-ZTA, 2D-ZTA and Thermanox TM (TMX) at day 10, 20 and 30 of culture. Data are represented as mean and standard error of mean (n=6); * denotes p<0.05 versus TMX at the same time point.

Figure 3 . 6

 36 Figure 3.6 Representative images of alizarin red (ARS) stained 3D-ZTA, 2D-ZTA and TMX at day 10, 20 and 30 of culture. Scale bar = 2 mm.

Figure 3 . 7

 37 Figure 3.7 Quantification of the stained areas by using Image J software. The numbers represent percentage of stained area relative to the whole sample area.

3 )

 3 and microporous struts (Figs.3.1 c and Fig.3.3) as a result of the low-temperature sintering condition. Compared to other techniques (e.g. foam replication), robocasting allows to obtain open structures where cells can freely migrate, while at the same time maintaining an overall low level of void, thus preserving the mechanical properties of the scaffold. It goes beyond the scope of this manuscript to assess the influence of porosity, pore size and distribution on the mechanical properties of the scaffold. Further information on this topic can be found in studies from Houmard and Marques8,19 .

  Les céramiques 3Y-TZP (polycristaux de zircone quadratique stabilisée à l'yttrium) se distinguent par une série d'atouts: excellente résistance mécanique et dureté, bonne esthétique, haute résistance à la corrosion et absence de réactions allergiques. Cependant, leur ostéointégration reste un point à améliorer. Dans cette étude, des disques de zircone ont été modifiés par un traitement par laser femtoseconde avec la production de multiples motifs (surface patterning) sur leurs surfaces. L'analyse de la surface par interférométrie optique en lumière blanche a permis de conclure que le traitement laser a conduit à la création de puits aux bords bien définis, avec une précision micrométrique en ce qui concerne le diamètre, la profondeur et l'espace entre les puits. D'autre part, la microscopie électronique à balayage a révélé une texture granulaire nanométrique sur les parois latérales et des ondulations au fond des puits. La fiabilité de la zircone suite à la modification de surface a été aussi évaluée par faisceau d'ions focalisé (focused ion beam, ou FIB), ce qui a mis en évidence un faible endommagement, limités à la surface des échantillons. L'évaluation de la réponse cellulaire à des temps courts a été conduite avec des cellules souches humaines dérivées de la moelle osseuse. Les micro-motifs ont influencé à la fois la taille des cellules (via le diamètre des puits) et leur morphologie (influencée à la fois par le diamètre et la profondeur des puits).

Figure 4 . 1

 41 Figure 4.1 Top: schematic diagram of a multi-patterned zirconia disk (dimensions are in mm). Bottom: patterned zones (pit diameter = 10, 20 and 30 µm) observed by optical microscopy.

Figure 4 . 3

 43 Figure 4.3 Scanning electron microscope micrographs of the different types of patterns: a) multiple pits, b) individual pit, c) bottom of the pit. Note the nanometric granular texture on the sidewalls and the presence of ripples at the bottom of the pits.

Figure 4 . 4

 44 Figure 4.4 Focused ion beam induced cross-sections of the different types of pits: a) entire pit, b) bottom of the pit, c) side of the pit. Note the presence of micro-cracks and sub-micrometric pores onthe pit surface, as well as sub-superficial pores up to a depth of 5 µm.

  Figure 4.5 Mesenchymal stem cell size and shape analysis following 48 hours of culture on the laser-patterned discs: a) cell area, b) solidity, c) aspect ratio, d) circularity and e) representative images of the toluidine blue stained cells. Data represent the analysis of 20 cells/group (n=10/sample, 2 samples) and are plotted as single points, as well as median and interquartile range. N indicates the unpatterned control, Y indicatespatterned regions. *, **, *** and **** represent p<0.05, <0.01, <0.001 and <0.0001 between the highlighted groups; #, ## and ### represent p<0.05, <0.01, <0.001 versus the unpatterned control. In e), the first number indicates the pit diameter and the second number the pit depth; scale bar = 50 µm.

1 .

 1 Résumé général en FrançaisLe marché des prothèses osseuses en céramiques bioinertes propose des implants de zircone, recommandés en dentisterie, et alumine-zircone (zirconia-toughened alumina, ou ZTA) pour l'arthroplastie de la hanche. Les céramiques bioinertes actuelles doivent être combinées avec du métal (titane ou acier inoxydable rugueux) afin d'obtenir une liaison adéquate avec l'os du patient (ostéointégration). Une ostéointégration directe des céramiques bioinertes dans l'os améliorerait la conception de la prothèse et la qualité de vie du patient. Par exemple, pour l'arthroplastie de la hanche, l'ostéointégration directe des céramiques bioinertes permettra l'utilisation d'un composant acétabulaire plus fin et permettra également un espace pour tête fémorale plus grande (Fig.5.1), réduisant ainsi le risque de dislocation et augmentant la plage de mouvement de l'articulation, avec un énorme impact sur la qualité de vie du patient.

Figure 5 . 1 .

 51 Figure 5.1. La taille de la tête de fémur détermine la plage de mouvement (ROM). Une cupule céramique plus fine (image droite) permet d'accommoder une tête de fémur plus grande (www.ceramtec.com).Plusieurs paramètres influencent l'ostéointégration: qualité de l'os, géométrie et rigidité de l'implant, chimie et rugosité de la surface de l'implant. Ces aspects ont été largement étudiés sur le titane, mais peu d'études ont été menées sur les céramiques bioinertes et surtout sur les composites alumine-zircone. Étant donné que la qualité des os est plutôt subie que contrôlée et le choix de la géométrie et de la rigidité de l'implant est souvent limité par les spécifications générales de la prothèse, des techniques de modification de surface sont souvent employées afin d'améliorer l'ostéointégration des implants. Les cultures cellulaires in vitro sont souvent utilisées comme première étape dans l'évaluation du potentiel ostéointégratif des implants, car en fin de compte des tests in vivo sont nécessaires pour tenir

Figure 5 . 2

 52 Figure 5.2 Micrographies SEM des différentes surfaces ZTA micro-rugueuses: surface de rugosité a) "poli", b) "comme fritté ", c) " faible", d) "medium", e) "haute". L'effet de l'attaque chimique sélective: f) poli, g) poli+ HF et h) poli+ HF+HCl (barra d'échelle = 5 µm).Enfin, des scaffolds d'alumine et de zircone micro-et macro-poreux hautement poreux et réguliers ont été obtenus avec succès par robocasting, une technique d'impression 3D. En ce qui concerne le type de cellule utilisé pour tester le potentiel ostéointégratif de ces surfaces, les ostéoblastes sont les cellules du tissu osseux qui produisent de l'os nouveau. Les ostéoblastes provenant de têtes fémorales de patients recevant une prothèse de hanche représentent le type cellulaire le plus pertinent pour l'évaluation des modifications de surface des matériaux de prothèse de la hanche in vitro. Des tendances récentes d'investigation des propriétés des matériaux 1,2 signalent la nécessité d'utiliser des types cellulaires et des analyses

Figure 5 . 3

 53 Figure 5.3 Le spectre de rugosité efficace proposé par Hayes et al. entre 0.2-2 µm: A -Sur une surface lisse, les ostéoblastes adopteront une morphologie de type fibroblaste; B -Sur une surface ondulée, si la distance entre les pics est supérieure à la taille de la cellule, les cellules reconnaîtront la surface comme plate et se comporteront en conséquence ; C -Sur une surface microrugueuse ils adopteront la morphologie typique des ostéoblastes; D -Sur une surface avec des topographies mélangées, la réponse cellulaire reflètera une moyenne des microtopographies lisses etmicrorugueuse.6

  qu'à rugosité "faible" et "moyenne" qui ont attaqué chimiquement les surfaces microrugueuses par rapport aux contrôles micro-rugueux (Fig.5.4).

Figure 5 . 4 .

 54 Figure 5.4. Rapport ALP/DNA sur (a) Différents échantillons d'alumine-zircone microrugueux et (b) Comparaison des échantillons microrugueux reçus, attaqués par acide fluorhydrique (HF) avec ou sans attaque chimique supplémentaire dans l'acide chlorhydrique (HCl): (b) poli, (c) comme fritté, (d) faible et (e) moyen et (f) haute rugosité de surface. A noter que seulement des différences minimales ont été observées parmi les différents groupes d'échantillons microrugueux, tandis que l'attaque chimique a entraîné une augmentation significative en ALP/DNA pour les hOb cultivés sur l'alumine-zircone comme fritté, faible et moyenne rugosité de surface.

  La stabilité primaire d'un implant est associée à son verrouillage mécanique avec l'os environnant. En utilisant un revêtement ZTA macroporeux sur la surface externe de la cupule d'implant de hanche Theelke et al. ont atteint une stabilité primaire (sur os artificiel) comparable aux cupules métalliques avec des revêtements de titane pulvérisés par plasma 9 . Il est donc important d'évaluer aussi la stabilité secondaire de ces céramiques bioinertes poreuses, guidée par leur ostéointégration. Afin de le faire sur des structures reproductibles (et non stochastiques comme la plupart des mousses), la technique du robocasting a été utilisée pour fabriquer des scaffolds poreux d'alumine-zircone afin de produire des structures poreuses hautement reproductibles et bien contrôlées (Fig. 5.5) dont nous avons ensuite évalué les propriétés biologiques. Le point le plus important était la formulation d'une encre stable. Nous avons préparé et imprimé avec succès une suspension de céramique à charge solide de 35.5 vol% avec un contenu organique minimal grâce à une buse de 330 μm dans un bain d'eau acide. La macro-porosité obtenue (~ 50%) pour ces scaffolds s'est révélée être totalement interconnectée et les filaments en céramique étaient microporeux (grâce à des conditions de frittage à température relativement basse).Comme indiqué précédemment, la présence de macro-et micro-porosité est une condition nécessaire pour l'ostéoinduction et l'ostéoconduction10 . En outre, il est intéressant de noter que les filaments en céramique présentaient deux niveaux de topographie: l'un obtenu à la suite de conditions de frittage à basse température (au niveau submicronique) et l'autre due à la morphologie interne intrinsèque à la buse utilisée pour l'extrusion (de l'ordre de quelques dizaines de microns). Ayant comme base la littérature existante, on suppose que ces deux types de porosités devraient induire des effets bénéfiques sur l'adsorption des protéines et par la suite sur l'adhésion, la prolifération et la différenciation cellulaire.

Figure 5 . 5

 55 Figure 5.5 Micrographie SEM de l'échafaudage 3D-ZTA, barre d'échelle = 100 μm (gauche) et ALP / DNA sur la 3D-ZTA et 2D-ZTA par rapport au contrôle thermanox. À noter l'activité de phosphatase alcaline supérieure sur 2D-ZTA.

Figure 5 . 6 .

 56 Figure 5.6. Conception de disques à motifs multiples (haut), vue d'ensemble des régions à motifs (milieu), morphologie des cellules souches mésenchymateuses humaines sur la zircone micromotifs après 48 h de culture (bas). Le premier nombre indique le diamètre du puite, le deuxième nombre indique la profondeur du puit.

Figure 5 . 7

 57 Figure 5.7 Effet du temps d'attaque chimique sur la microstructure de la zircone, barre d'échelle = 10 μm (haut) et l'évolution de la morphologie MSC le long du gradient de zircone, barre d'échelle = 100 μm (milieu) et quantification de la zone cellulaire et des paramètres de solidité par analyse d'image (bas).En tant que deuxième stratégie pour l'investigation rapide des interactions cellule-matériau, une attaque chimique a été utilisée pour créer des gradients de rugosité (Fig. 5.7). Les gradients obtenus ont montré une grande variation des paramètres fractales et de rugosité à la fois au niveau micro-et nanométrique. La morphologie des cellules souches a varié le long du gradient de rugosité de surface. La solidité cellulaire était corrélée avec les paramètres de nano-rugosité et de dimension fractale mesurés par microscopie à force atomique, alors que la surface cellulaire semblait affectée par la micro-rugosité et la dimension fractale mesurés par interférométrie en lumière blanche. Cet outil offre des perspectives prometteuses pour l'investigation de différentes rugosités avec une quantité limitée d'échantillons.

5. 8 )

 8 , thus reducing the risk of dislodgement and increasing the range of motion of the articulation, with great impact on the patient's quality of life.

Figure 5 . 8

 58 Figure 5.8 Ball head size determines the range of motion (ROM) (www.ceramtec.com)

Figure 5 . 9

 59 Figure 5.9 SEM micrographs of the different micro-rough ZTA surfaces: a) "polished", b) "as sintered", c) "low", d) "medium", e) "high" roughness surface; the effect of selective chemical etching: f) polished, g) polished + HF and h) polished + HF+HCl (scale bar = 5 µm).

  surface characteristics such as topography, roughness, chemistry and energy dictate the cell response in vitro and the implant fate in vivo. Albrektsson & Wennerberg reviewed the importance of surface roughness parameters and made a clear distinction between the 2D profile and the 3D evaluation. At the moment the majority of the studies use the R a only to define the roughness of a surface. However, surfaces with same R a may have very different roughness morphologies, which in turn trigger completely different cell behaviour (Fig. 5.10). Therefore, a single parameter on the 2D profile is not providing sufficient information. Albrektsson & Wennerberg recommended at least one height, one spatial and one hybrid parameter to be quantified by confocal laser scanning profilometry or contact interferometry. They have also stressed the importance of numerically filtering for the roughness data for a more accurate comparison between studies 4 . More recently, Flamant et al. highlighted the importance to perform multiscale three-dimensional surface analyses 5 .

Figure 5 . 10

 510 Figure 5.10 The effective roughness spectrum suggested by Hayes et al. between 0.2-2 µm: A -On a smooth surface, osteoblasts will adopt a fibroblast-like morphology; B -On a wavy surface, if the distance between peaks is more than the cell size, cells will recognize the surface as a flat one and behave accordingly; C -On a micro-rough surface will adopt typical osteoblast morphology; D -On a surface with mixed topographies, cell response will reflect an average of both smooth andmicrorough micro-topographies 6 .

Figure 5 .

 5 Figure 5.12 SEM micrograph of the 3D-ZTA scaffold, scale bar = 100 μm (left) and ALP/DNA ratio on the 3D-ZTA and 2D-ZTA versus the thermanox control. Note the higher alkaline phosphatase activity on 2D-ZTA.

  already showed the possibility to load antibiotics with good efficiency against bacterial colonization. One of the leading causes for implant failure is the implant infection as ¨the race for the surface¨ is still opened as any type of surface modification will influence both prokaryotic and eukaryotic cells. Engineering implants with cell adhesion properties and bacterial repellent is still an open challenge. The superficial interconnected layer could be used for delivering growth factors or chemokine known to promote bone healing by osteoinduction or by recruiting cells involved in new bone formation at the implant site.

Figure 5 . 14

 514 Figure 5.14 Effect of etching time on zirconia microstructure, scale bar = 10 μm (top) and evolution of MSC morphology along the zirconia gradient, scale bar = 100 μm (mid) and quantification of cell area and solidity parameters by image analysis (bottom).

Figure 6 . 1

 61 Figure 6.1 Experimental procedure for producing a roughness gradient at the surface of zirconia specimens.

  6.1). The evolution of the surface morphology along the axis of the bar was assessed at evenly spaced positions in the x direction (y = 0 mm) by scanning electron microscopy (SEM, Neon 40, Carl Zeiss AG, Germany). Atomic Force Microscopy (AFM Veeco Dimension 3100) in tapping mode and White Light Interferometry (WLI, Veeco Wyko 9300NT) were used in order to characterize the topography at different scales. AFM measurements were performed at five x-positions with y = 0 mm on three specimens (one measurement per position, area: 50 µm x 50 µm, resolution: 512 x 512 pixels). WLI measurements were performed at thirteen evenly spaced x-positions on three specimens (measurements at y = -1 mm, y = 0 mm and y = 1 mm for each x position, area:

Figure 6 . 2

 62 Figure 6.2 Scanning electron microscopy observations at different positions and magnifications of a sample which was not sonicated after etching, evidencing the presence of adhered reaction precipitates. Dimensions of the scale bars: 30 µm (top), 10 µm (middle), 3 µm (bottom).

Figure 6 . 3

 63 Figure 6.3 Scanning electron microscopy observations of the surface of the gradient at different positions and magnifications. Dimensions of the scale bars: 30 µm (top), 10 µm (middle), 5 µm (bottom).

Figure 6 . 4

 64 Figure 6.4 White light interferometry (WLI) and atomic force microscopy (AFM) topographical images of the surface at different positions on the gradient.

first 7

 7 mm and then has moderate variations without showing a clear tendency. Its value remains superior to 3 indicating a narrow height distribution and steep side-walls. The skewness (S sk ) is first close to 0 and then decreases substantially. The decreasing negative values indicate that valleys are becoming more and more predominant on the surface along the gradient direction, which is consistent with the apparition of etch pits reported above.

. 6 )

 6 At the micro-scale, the surface material volume (S m ) increases up to 28 mm and then reaches a plateau. The surface core void volume (S c ) increases all over the gradient but the increase rate tends to decrease along the distance. The surface valley void volume (S v ) increases proportionally to the distance all over the gradient.At the nano-scale, the surface material volume (S m ) increases up to half of the gradient and then decreases slightly. The surface core void volume (S c ) increases up to a maximum at half of the gradient and then decreases, whereas the surface valley void volume (S v ) always increases along the gradient.

Figure 6 . 5

 65 Figure 6.5 Evolution of amplitude and spatial roughness parameters along the gradient. AFM measurements correspond to the nano-roughness whereas WLI measurement correspond to the micro-roughness. Error bars represent the standard deviations.

Figure 6 . 6

 66 Figure 6.6 Evolution of hybrid and functional roughness parameters along the gradient. AFM measurements correspond to the nano-roughness whereas WLI measurements correspond to the micro-roughness. Error bars represent the standard deviations.

Figure 6 .

 6 Figure 6.7 a) Example of scale-sensitive fractal analysis, showing how smooth-rough crossover and fractal dimension are obtained, b) smooth-rough crossover as a function of position and etching time, c) fractal dimension as a function of position and etching time.

  6.8 a,b,d), while the circularity was not significantly affected (p=0.073) (Fig.6.8-c). A significant increase in cell area (20 min vs 50 min HF treatment: p=0.014) and aspect ratio (20 min vs 30 min HF treatment: p=0.009) were observed in the central part of the bar. The aspect ratio was sensitive to the gradient (10 min vs 20 min treatment: p=0.096, 20 min vs 30 min: p=0.009 and 30 min vs 40 min: p=0.069).

Figure 6 . 8

 68 Figure 6.8 Top: evolution of human mesenchymal stem cell morphology along the gradient: a) area, b) aspect ratio, c) circularity and d) solidity. Statistical differences: ** is p<0.001 and * is p<0.01 relative to 0; # is p<0.05 and ~ is p<0.1 between the highlighted groups. Bottom: representative images of the toluidine blue stained human mesenchymal stem cells on gradient zirconia bars after 48 hours of culture: e) polished control, f) 30 and g) 60 minutes of hydrofluoric acid treatment. Scale bar = 100 µm.

Figure 6 . 9

 69 Figure 6.9 Left: hMSC solidity as a function of three representative nano-roughness parameters and the AFM fractal dimension. Data are represented with linear regression lines and associated 95% confidence bands. Right: hMSC area as a function three representative micro-roughness parameters and the WLI fractal dimension. Data are represented with second-order (for S a , S dr and S c ) or third-order (for D) polynomial regression lines and associated 95% confidence bands.

  'UNIVERSITE DE LYON OPEREE AU SEIN DE L'INSA LYON NOM : STANCIUC DATE de SOUTENANCE : 23.06.2017 Prénoms : Ana-Maria TITRE : In vitro evaluation of cell-material interactions on bioinert ceramics with novel surface modifications for enhanced osseointegration NATURE : Doctorat Numéro d'ordre : 2017LYSEI053 Ecole doctorale : Matériaux de Lyon Spécialité : Matériaux RESUME : Cette thèse fait partie du projet BioBone, un projet européen FP7 Marie Curie, Initial Training Network ("réseau de formation initiale") avec des partenaires académiques et industriels. La thèse a été conduite dans le groupe de régénération musculosquelettique (AO Research Institute Davos, Suisse) et au laboratoire MATEIS de l'INSA Lyon. Cette thèse porte sur l'évaluation de la réponse cellulaire in vitro à différentes stratégies de modification de surface pour améliorer la capacité d'ostéointégration de céramiques bioinertes pour implants orthopédiques et dentaires.

sealed resorption vacuole results in TRAP and CATK enzymes responsible for the bone mineral degradation, c -TEM micrographs with an activated osteoclast, d -SEM micrograph with an osteoclast generated on a cortical bone slice (red propellers= osteoclasts, black arrows= resorption pit, yellow star= non-resorbed bone surface, blue triangles= mononuclear cells 32

  Summary of aging features of zirconia. ................................................... Figure 1.2 Example of the microstructure of an alumina-zirconia composite. Note the grey alumina phase and the white zirconia phase with quite large aggregates 11 . ... Figure 1.3 Scheme representing surface modifications for dental implants, scale bar = 3 µm 14 . ................................................................................................................. Figure 1.4 Left: different examples of surface profiles with same R a (roughness average) but different shapes of the surface features; right: overview of the types of topographical surface features: form, waviness, roughness (adapted after Wennerberg and Albrektsson). ................................................................................. . ....................................................... 49 Figure

	R a Index of figures	Roughness average
	RANKL	Receptor activator of nuclear factor kappa-B ligand
	ALP ROM ATP BMP-2 BSE BSP CATK CapG Cbfa1/Runx2 CD44 CoC CollαI RS S a Sca-1 SCF S ci SEM S dr S ds Sox9 SLA Figure 1.1 Figure 1.5 Scanning electron microscopy micrographs of zirconia surface: MS -Range of movement Alkaline phosphatase Adenosin triphosphate Bone Morphogentic Protein-2 Backscattered electron Bone Sialoprotein Cathepsin K Macrophage-Capping Protein G Core-binding factor α1 Cluster of Differentiation 44 Ceramic-on-ceramic bearings Self-renewing cells (type of MSCs) Average roughness (Amplitude parameter) Stem cells antigen-1 Stem cell factor Core fluid retention index (Functional parameter) Scanning Electron Microscopy Developed interfacial area ratio (Hybrid parameter) Density of summits (Spatial parameter) SRY (sex determining region Y) -box 9 Sandblasted and acid etched Collagen type I CSF-1 Src Proto-oncogene tyrosine-protein kinase mirror polished, SB25, SB50, SB150 -sandblasted with 25 μm, 50 μm and 150 μm Colony-stimulating factor-1 CT TEM Transmission Electron Microscopy respectively diameter alumina diameter; SB150+HF received additionally a Computed tomography CXCL12 DMEM DNA ECM E11/podoplanin ERK Extracellular signal-regulated kinase WLI White Light Interferometry transmembrane glycoprotein VCAM1 Vascular cell adhesion molecule 1 extracellular matrix Ti6Al4V Titanium alloy (90% Ti, 6% Al 4% V, wt.%) DeoxyriboNucleic Acid TNF Tumor necrosis factor Dulbecco's modified Eagle medium TRAP Tartrate-resistant acid phosphatase CXC-chemokine ligand 12 TMX Thermanox TM hydrofluoric acid etching
	FBS Wnt/ β-catenin	Foetal bovine serum Canonical signalling pathway with Wnt as ligand as β-catenin as co-
	FC	Flat cells (type of MSCs) activator
	FIB YF 3	Focused Ion Beam yttrium fluoride
	GM-CSF G-CSF Y-TZP HCl ZTA HF ZPTA hMSC 2D-ZTA	Granulocyte-macrophage colony stimulating factor Yttria-stabilized Zirconia Granulocyte colony-stimulating factor Zirconia-toughened alumina Hydrochloric acid Zirconia-platelets toughened alumina Hydrofluoric acid human Mesenchymal Stem Cells Two-dimensional ZTA obtained by casting
	hOb 3D-ZTA	human Osteoblast cells Three dimensional ZTA obtained by robocasting
	HSC	Haematopoietic stem cell
	IGF	Insulin-like Growth Factor
	IKK	Inhibitor of NF-kB kinase
	IL-6	Interleukin-6
	ISO	International Standards Organisation
	JNK	c-Jun N-terminal kinase
	LTD	Low Temperature Degradation
	Mac-1	Macrophage-1 antigen
	M-CSF	Macrophage colony stimulating factor
	MHC	Major Histocompatibility Complex
	MIP	Mercury intrusion porosimetry
	MT-MMP1	Membrane-type matrix metalloproteinase-1
	NANOZR	Ceria stabilized zirconia/alumina nanocomposites
	NO	Nitric Oxide
	OC	Osteocalcin
	OP	Osteopontin
	Osx	Osterix
	PBS	Phosphate buffered saline solution
	PGE	Prostaglandin E2
	PTH	Parathyroid hormone

20 . ...................................................................................... Figure 1.6 A -Zirconia surface HF etched (from left to right: 30 min, 1h, 2h); Bzirconia-toughened alumina (ZTA) surface (from left to right: polished, HF treated, HF+HCl treated) 19 . ................................................................................................... Figure 1.7 Contact angle of zirconia surface with different surface modifications 23 . . Figure 1.8 Contact angle measurements (distilled water) on zirconia: MS -, SB50 , SB-150 sandblasted with 50 and 150 μm diameter alumina particles, respectively, SB150Esandblasted and etched zirconia surfaces 21 . ..........................................

Figure 1.9 Schematic representation of plasma surface modification: plasma treatment is converting the 1-3 nm superficial layer into zirconium oxyfluoride; the oxyfluoride layer is interacting with organo-silanes, enabling silicon attachment 26 . .. Figure 1.10 MG-63 cells spreading and morphology after 6 days of culture on plasma treated and further silanisation (right side) compared to the polished control (left side) 28 ....................................................................................................................... Figure 1.11 Schematic representation of the bone microenvironment with the specific bone cell types: osteocytes, osteoblasts, osteoclasts and the immune component: memory and circulating T cells (T*), memory B cells (B*) and monocytes (M*) 33 . .... Figure 1.

12 Characteristic morphology of cells in monolayer cultures: hMSC (human mesenchymal stem cells)two distinct subpopulations: small, spindleshaped RS (self renewing) cells (arrows and large flat FC (flattened) cells (arrows heads) (A), hObstypical polygonal and flattened morphology (B), MG-63 cellsvarious cell morphologies, oval, triangular or spindle-shaped (C)

[START_REF] Docheva | Researching into the cellular shape, volume and elasticity of mesenchymal stem cells, osteoblasts and osteosarcoma cells by atomic force microscopy[END_REF]

. ........................ Figure 1.13 Scheme representing the process of osteoblastogenesis : progression from mesenchymal stem cells, commitment towards osteoblast lineage, differentiation and maturation 41 . ............................................................................... Figure 1.14 Scheme representing the osteoblast progression and maturation governed by a temporal expression of genes involved in the fine regulation of the three main phases: proliferation, extracellular matrix development and maturation, mineralisation (adapted from 42 ). ...............................................................................

Figure 1.15 Scheme representing the process of osteocytogenesi: transition from osteoblast to osteocyte with illustration of key osteocyte marker genes 49 . ............... Figure 1.16 Scheme representing the process of osteoclastogenesis: development from haematopoietic precursor cells to mature osteoclasts 32 . .................................................

Figure 1.17 Bone resorption activation: a -Recruitment of polykaryons by CSF-1 and RANKL, b -Acidification of a

1.18 Scheme representing the bone marrow niche hosting hematopoietic stem cells and mesenchymal stem cells 60

. ................................................................................... 50 Figure 1.

19 Scheme representing different topographical scales influencing implant-bone interaction and osseointegration 14

. .............................................................................. 57 Figure 1.

20 Fluorescence intensity of osteopontin immunostaining as a function of micro- roughness and nanofeature density 113

. ......................................................................... 59 Figure 1.

21 Interactions between a hydrophilic (A) and a hydrophobic (B) surface. Note the intimate interactions with proteins whithin biological fluids and subsequently with cell receptors for hydrophilic surfaces while for the hydrophobic surfaces the hydrocarbon contamination could lead to air bubbles entrapment and intereference with protein and cell adhesion 120

  . ............................................................................................................... 60 High roughness surfaces. ................................................................... 95 Figure 2.5 RUNX-2 mRNA expression relative at day 10, 20 and 30 of culture relative to day 0 control: a) Comparison between the different micro-rough ZTA surfaces; b-f) comparison of the surfaces before and after etching HF or HF + HCl. b) High roughness group. ............................................................. 99

	Polished, c) As sintered, d) Low, e) Medium, f) High roughness surfaces. .............. 97
	Figure 2.6 ALP mRNA expression relative at day 10, 20 and 30 of culture relative to
	day 0 control: a) Comparison between the different micro-rough ZTA surfaces; b-f)
	comparison of the surfaces before and after etching HF or HF + HCl. b) Polished, c)
	As sintered, d) Low, e) Medium, f) High roughness surfaces. .................................. 98
	Figure 2.7 Collagen type I (COL1) mRNA expression relative at day 10, 20 and 30 of
	culture relative to day 0 control: a) Comparison between the different micro-rough
	ZTA surfaces; b-f) comparison of the surfaces before and after etching HF or HF +
	HCl. b) Polished group, c) As sintered group, d) Low roughness group, e) Medium
	roughness group, f)

Figure 1.22 Scheme representing bone remodelling post-implantation as a succession of different steps: 1 -Excessive torque force, 2 -Osteoclasts and osteoblasts recruitment, 3 -Bone resorption by the osteoclasts, 4 -Debris cleaning by the bone lining cells, 5 -Collagen production by the bone lining cells, 6 -Osteoblasts attachment enhanced by the collagen layer, 7 -Osteoid deposition by the osteoblasts, 8 -Osteoblasts trapped into the osteoid become osteocytes 133 .... 63 Figure 1.23 Direct osseointegration versus fibrous capsule development of TAN (Titanium-6%aluminium-7% niobium) nails, 12 months post-implantationin sheep tibia : A -Standard microrough TAN in direct contact with bone (i= implant, b= bone); Bpaste polished TAN lead to a fibrous capsule development between the implant and the bone (i= implant, f= fibrous tissue, b= bone) 106 . .............................. 64 Figure 1.24 Direct osseointegration of titanium, alumina/zirconia and zirconia screws following a 4 week implantation in maxilla of minipigs 131 . Top row: overviews of the screws. Bottom row: detailed images of the implant/bone interface. Note the absence of fibrous capsule. .................................................................................................... 65 Figure 2.1 Scanning electron microscope micrographs of the zirconia-toughened alumina (ZTA) surfaces. a-e) Comparison between the different micro-rough ZTA surfaces: a) "polished", b) "as sintered", c) "low", d) "medium", e) "high" roughness surface; f-h) comparison of polished surface before and after etching with hydrofluoric acid (HF) or HF followed by hydrochloric acid etching (HF + HCl): f) polished, g) polished + HF and h) polished + HF+HCl. Scale bar = 5 µm. ............... 87 Figure 2.2 Cell proliferation assessed by DNA quantification and alkaline phosphatase activity (ALP) normalized to DNA content on Medium roughness surfaces before and after HF or HF+HCl etching at day 10, 20 and 30 of culture. Results from single donors are represented as mean and standard error of mean. . 92 Figure 2.3 Cell proliferation assessed by DNA quantification at day 10, 20 and 30 of culture: a) Comparison between the different micro-rough ZTA surfaces; b-f) comparison of the surfaces before and after etching HF or HF + HCl. b) Polished, c) As sintered, d) Low, e) Medium, f) High roughness surfaces. Depending on the samples distribution, parametric (one-way Anova with Tukey post-hoc) or nonparametric (Kruskall-Wallis test and Dunn post-hoc) analyses were performed (p<0.1 were considered trends, p<0.05 were considered significant differences, p values are given on the graphs) using GraphPad Prism 7.01 software. .................................... 93 Figure 2.4 ALP normalized to DNA content at day 10, 20 and 30 of culture: a) Comparison between the different micro-rough ZTA surfaces; b-f) comparison of the surfaces before and after etching HF or HF + HCl. b) Polished, c) As sintered d) Low, e) Medium, f) Figure 2.8 Osteocalcin (OC) mRNA expression relative at day 10, 20 and 30 of culture relative to day 0 control: a) Comparison between the different micro-rough ZTA surfaces; b-f) comparison of the surfaces before and after etching HF or HF + HCl. b) Polished group, c) As sintered group, d) Low roughness group, e) Medium roughness group, f) High roughness group. ........................................................... 100 Figure 2.9 Alizarin red stained "medium" roughness ZTA discs at day 20 (a-c

  ................................................................................... 164 Figure 5.10 The effective roughness spectrum suggested by Hayes et al. Evolution of amplitude and spatial roughness parameters along the gradient. AFM measurements correspond to the nano-roughness whereas WLI measurement correspond to the micro-roughness. Error bars represent the standard deviations. .............................................................................................................. 190 Figure 6.6 Evolution of hybrid and functional roughness parameters along the gradient. AFM measurements correspond to the nano-roughness whereas WLI measurements correspond to the micro-roughness. Error bars represent the standard deviations. ............................................................................................... 191 Figure 6.7 a) Example of scale-sensitive fractal analysis, showing how smooth-rough crossover and fractal dimension are obtained, b) smooth-rough crossover as a function of position and etching time, c) fractal dimension as a function of position and etching time. .................................................................................................... 192

	. 163
	Figure 5.9 SEM micrographs of the different micro-rough ZTA surfaces: a)
	"polished", b) "as sintered", c) "low", d) "medium", e) "high" roughness surface; the
	effect of selective chemical etching: f) polished, g) polished + HF and h) polished + HF+HCl (scale bar = 5 µm). Figure 6.8 Top: evolution of human mesenchymal stem cell morphology along the gradient: a) area, b) aspect ratio, c) circularity and d) solidity. Statistical differences:
	** is p<0.001 and * is p<0.01 relative to 0; # is p<0.05 and ~ is p<0.1 between the
	highlighted groups.

et moyenne rugosité de surface. ................................................... 156 Figure 5.5 Micrographie SEM de l'échafaudage 3D-ZTA, barre d'échelle = 100 μm (gauche) et ALP / DNA sur la 3D-ZTA et 2D-ZTA par rapport au contrôle thermanox. À noter l'activité de phosphatase alcaline supérieure sur 2D-ZTA. ........................ 158 Figure 5.6. Conception de disques à motifs multiples (haut), vue d'ensemble des régions à motifs (milieu), morphologie des cellules souches mésenchymateuses humaines sur la zircone micro-motifs après 48 h de culture (bas). Le premier nombre indique le diamètre du puite, le deuxième nombre indique la profondeur du puit. .. 160 Figure 5.7 Effet du temps d'attaque chimique sur la microstructure de la zircone, barre d'échelle = 10 μm (haut) et l'évolution de la morphologie MSC le long du gradient de zircone, barre d'échelle = 100 μm (milieu) et quantification de la zone cellulaire et des paramètres de solidité par analyse d'image (bas). ....................... 161 Figure 5.8 Ball head size determines the range of motion (ROM) (www.ceramtec.com) .............................................................................................between 0.

2-2 µm:

A -On a smooth surface, osteoblasts will adopt a fibroblast-like morphology; B -On a wavy surface, if the distance between peaks is more than the cell size, cells will recognize the surface as a flat one and behave accordingly; C -On a micro-rough surface will adopt typical osteoblast morphology; D -On a surface with mixed topographies, cell response will reflect an average of both smooth and microrough micro-topographies

6

. .......................................................................... 165 Figure 5.11 ALP/DNA ratio on (a) different microrough alumina-zirconia samples and (b) comparison of microrough samples as received, etched with hydrofluoric acid (HF) with or without an additional etching in hydrochloric acid (HCl): (b) polished, (c) as sintered, (d) low and (e) medium and (f) high surface roughness. Note that only minimal differences were observed among the different microrough only groups, while etching induced a significant increase in ALP/DNA in hOb cultured on as sintered, low and medium surface alumina-zirconia samples. ............................... 168 Figure 5.12 SEM micrograph of the 3D-ZTA scaffold, scale bar = 100 μm (left) and ALP/DNA ratio on the 3D-ZTA and 2D-ZTA versus the thermanox control. Note the higher alkaline phosphatase activity on 2D-ZTA. ................................................... 169 Figure 5.13 Multi-patterned disk design (top), overview of the patterned regions (mid), human mesenchymal stem cell morphology on micro-patterned zirconia following 48 h of culture (bottom). The first number indicates pit diameter, second number indicates pit depth. .................................................................................... 170 Figure 5.14 Effect of etching time on zirconia microstructure, scale bar = 10 μm (top) and evolution of MSC morphology along the zirconia gradient, scale bar = 100 μm (mid) and quantification of cell area and solidity parameters by image analysis (bottom). ................................................................................................................. 171 Figure 6.1 Experimental procedure for producing a roughness gradient at the surface of zirconia specimens. ............................................................................................ 181 Figure 6.2 Scanning electron microscopy observations at different positions and magnifications of a sample which was not sonicated after etching, evidencing the presence of adhered reaction precipitates. Dimensions of the scale bars: 30 µm (top), 10 µm (middle), 3 µm (bottom). ..................................................................... 187 Figure 6.3 Scanning electron microscopy observations of the surface of the gradient at different positions and magnifications. Dimensions of the scale bars: 30 µm (top), 10 µm (middle), 5 µm (bottom). .............................................................................. 187 Figure 6.4 White light interferometry (WLI) and atomic force microscopy (AFM) topographical images of the surface at different positions on the gradient. ............ 188 Figure 6.5 Bottom: representative images of the toluidine blue stained human mesenchymal stem cells on gradient zirconia bars after 48 hours of culture: e) polished control, f) 30 and g) 60 minutes of hydrofluoric acid treatment. Scale bar = 100 µm. ............................................................................................................... 194 Figure 6.9 Left: hMSC solidity as a function of three representative nano-roughness parameters and the AFM fractal dimension. Data are represented with linear regression lines and associated 95% confidence bands. Right: hMSC area as a function three representative micro-roughness parameters and the WLI fractal dimension. Data are represented with second-order (for S a , S dr and S c ) or third-order (for D) polynomial regression lines and associated 95% confidence bands. .......... 196

Table 1 . 1

 11 Mechanical properties of bio-inert ceramics (adapted fromChevalier et al., 2009).

.................................................................................................................................................. 31 Table 1.2 Comparison between primary osteoblast cells and osteoblast-like cell lines (adapted from Czekanska et al., 2012). ................................................................................... 53

Table 2 . 1

 21 List of samples used in the study and corresponding three-dimensional parameters obtained by white light interferometry measurements of zirconia-toughened alumina surface topography; both unfiltered and 10 µm filtered data are reported.11 ..................................... 86

Table 2 .

 2

	2 Three-dimensional parameters obtained by atomic force microscopy measurements
	of polished (P) zirconia-toughened alumina before and after etching with hydrofluoric acid
	(P+HF) and sequential hydrofluoric acid and hydrochloric acid (HF+HCl); both unfiltered
	and 1 µm filtered data are reported. 11 ..................................................................................... 87
	Table 2.3 Primer and probe sequences used for real-time polymerase chain reaction.......... 90
	Table 3.1 Data on human primary osteoblast source (donor details). ................................. 116
	Table 3.2 Primer and probe sequences used for real-time polymerase chain reaction........ 118
	Table 3.3 Comparison of the pore size, strut size and porosity measured by scanning electron
	microscopy (SEM), X-ray tomography (CT) and mercury intrusion porosimetry (MIP). *
	porosity of a region inside the samples without the external rings; ** porosity of the whole
	sample, with the external rings. ............................................................................................. 122

Table 4 .1

 4 Dimensions of the pits obtained for each kind of pattern (12 measurements per pattern). .................................................................................................................................. 138

Table 4 .2

 4 Processing parameters used to obtain each kind of pit/pattern ( * Energy before going through the optical system; ** For an ideal Gaussian beam; *** After passing through the optical system).................................................................................................................. 138

Table 6 .

 6

	1 Description of the 3D roughness parameters used in this study 28-30 . ................... 183
	Table 6.2 Pearson product-moment and Kendall's tau correlation coefficients for all the
	combinations of topographical and morphological parameters (Significance of the
	correlation: p < 0.05 ; p < 0.005) ......................................................................................... 195

Chapter 1 General introduction 1. General Introduction 1.1. Résumé de l'introduction générale

	1
	Le chapitre introductif a pour but de donner le contexte du sujet de la thèse et porte sur les
	interactions cellules-matériaux sur des céramiques bioinertes avec des surfaces modifiées.
	Chronologiquement l'utilisation clinique de ces matériaux a commencé par l'alumine, puis la
	zircone a été introduite, et plus récemment les composites alumine-zircone avec des
	propriétés mécaniques augmentées. Notre analyse critique est dirigée vers les défis de
	l'utilisation en clinique et vers l'exploitation de la possibilité de modifier les propriétés de la
	surface (rugosité, chimie et mouillabilité). La possibilité de réaliser des modifications de
	surface ouvre de nombreuses voies pour le développement des surfaces céramiques
	améliorées.
	La comparaison entre les études publiées dans la littérature souffre très souvent d'une
	caractérisation 3D assez limitée (souvent seule la valeur de R a est donnée). Nous montrons
	l'importance d'aller plus loin dans cette caractérisation, le R a seul ne suffisant pas à
	caractériser la morphologie d'une surface.
	Étant donné que toutes les technologies examinées dans cette thèse ont comme but le
	renforcement de l'interface os-implant, une partie de cette section a été consacré à la structure
	cellulaire de l'os. Le tissu osseux est un milieu extrêmement actif, dont le processus de
	remodelage osseux engage des différents types cellulaires (ostéoblastes, ostéocytes,
	ostéoclastes, cellules souches, cellules immunitaires aussi que cellules sanguines) en continue
	interdépendance. En essayant d'imiter le scenario réel, les analyses in vitro doivent compter
	sur un modèle robuste avec des résultats comparable aux études in vivo et clinique. La plupart
	des études s'appuient sur des lignes cellulaires qui parfois ne reflètent pas le spectre clinique
	qui peut être rencontré avec des cultures primaires. Nous montrons qu'il n'existe le plus
	souvent qu'une faible corrélation entre les données obtenues in vitro et celles obtenues in
	vivo.

.2. Bio-inert ceramic orthopaedic implants 1.2.1. Generalities and history of clinical use of bio-inert ceramics

	Ceramics, from Greek keramos are defined as inorganic, non-metallic materials. High
	biocompatibility and bio-inertness in aqueous conditions are the main features that endorse
	bio-inert ceramics for clinical applications.
	Clinical use of ceramics was pioneered in dentistry in late 18th century with controlled
	implantation of porcelain for crowns. Gypsum (calcium sulfate dihydrate) was first used as
	bone filler in the 19th century 1, 2 . For hip joints implants, tough and strong ceramics were
	represented at the beginning by alumina (Al 2 O 3 ). Alumina hip prosthesis showed lower
	debris formation compared to metal and polyethylene solution. Nonetheless, due to its
	brittleness, the fracture rate for alumina implants was up to 13% for some series 1 . Alumina
	was therefore substituted by zirconia, which proved to have a higher toughness, and later by
	zirconia-toughened alumina.
	After a century in clinical practice, ceramics still have open questions and challenges in terms
	of direct-bone adhesion without fibrous soft tissue interlayer or mechanical properties
	suitable for loaded devices 1 . Bio-inert ceramics are biocompatible, sufficiently hard and
	tough, resistant to corrosion and aesthetically pleasing (in dental field) (Table 1.1). On the
	other hand their limitations in terms of lack of direct bone-material interface or stress-
	shielding and the possibility of aseptic loosening underline the need for surface
	modifications. Their reliability is another important aspect that should be considered as
	different surface modifications may cause surface defects affecting ageing sensitivity.

Material Density ρ (g/cm 3 ) Elastic modulus E (GPa) Toughness (K IC , MPam 1/2 ) Bending strength σ r (MPa) Hardness (Hv)

	Alumina	3.15	410	4.2	500	1800
	Zirconia	5.83	219	5.4	1000	1200

Table 1 .1 Mechanical properties of bio-inert ceramics (adapted from Chevalier et al., 2009).

 1

  Hulsart et al. recommendation is a combination of in vitro tests results on cell differentiation, gene expression and mineralization, which strong correlation could indicate the further recommended steps for the biomaterial evaluation. Furthermore the translation from small to large animal and finally from preclinical to clinical trials seems still a winding road with open challenges. Up to date, only a third of highly cited preclinical studies reach later to human trials 142 . There is the need for standardized methods at all steps, including in vitro, in vivo and

clinical assays. As far as in vivo experiments are concerned, Smith et al. have proposed a checklist prior the experimental design: Design and Execution of Protocols for Animal Research and Treatment (DEPART) in addition to the already existing guidelines for "Animals in Research: Reporting in Vivo Experiments (ARRIVE)" 143 .

  143. Smith, M.M., E.C. Clarke, and C.B. Little, Considerations for the design and execution of protocols for animal research and treatment to improve reproducibility and standardization: "DEPART well-prepared and ARRIVE safely". Osteoarthritis Cartilage, 2017. 25(3): p. 354-363.

  Wennerberg et al. and more recently Deltombe et al. and Flamant et al. have suggested using at least an amplitude, spatial, hybrid

Table 2 .1 List of samples used in the study and corresponding three-dimensional parameters obtained by white light interferometry measurements of zirconia-toughened alumina surface topography; both unfiltered and 10 µm filtered data are reported.

 2 2.1 and white-light interferometry analyses (Veeco Wyko 9300NT, area of observation: 150 µm x 150 µm obtained by stitching of four images acquired at magnification 50x, resolution: 758 x 758 pixels) are summarized in Table 2-1 10 .

		R a	S a (nm)	S ds (1/mm 2 )	S dr (%)		S ci (nm)
	Surface Type	(μm)	No	10 μm	No	10 μm	No	10 μm	No	10 μm
			filter	filter	filter	filter	filter	filter	filter	filter
	Polished		16	13	13365 10852	0.35	0.35	1	1
	As Sintered		386	203	36882 36361	50	50	1	0.84
	Low surface	0.13 224	176	44182 46657	45	45	1.20	1.11
	Medium surface	0.77 719	330	44155 50641	83	81	1.56	1.34
	High surface	1.76 1369 416	43675 46426	102	99	1.62	1.20

Table 2 .2 Three-dimensional parameters obtained by atomic force microscopy measurements of polished (P) zirconia-toughened alumina before and after etching with hydrofluoric acid (P+HF) and sequential hydrofluoric acid and hydrochloric acid (HF+HCl); both unfiltered and 1 µm filtered data are reported. 11

 2 .1). AFM analyses (Veeco Dimension 3100; area of observation: 50 µm x 50 µm, resolution: 512 x 512 pixels) are summarized in Table2.2.10

	Surface type	S a (nm)		S ds (1/mm 2 )		S dr (%)		S ci (nm)
		No	1 μm	No	1 μm	No	1 μm	No	1 μm
		filter	filter	filter	filter	filter	filter	filter	filter
	Polished	5	2	324267 235664 0.05	0.05	1	0.9
	Polished + HF	148	64	122976 145226 27	21	2	1.91
	Polished + HF+HCl	24	20	138367 115160 5	5	0.6	0.3

Table 2 .3 Primer and probe sequences used for real-time polymerase chain reaction.

 2

Table 3 .1 Data on human primary osteoblast source (donor details).

 3

	Patient	Sex	Age (years)	Source
	Patient 1	M	83	Femoral head
	Patient 2	F	75	Femoral head
	Patient 3	F	68	Femoral head

.1) with approval from the Ethics Committee Zürich 18/02. hObs were outgrown from cancellous bone chips

12

and hObs from the second yield were subcultured in culture medium composed of Dulbecco's modified Eagle medium (DMEM) containing 1 wt./vol.% glucose (Sigma #31600-083) and supplemented with 10 vol.% foetal bovine serum (FBS) (Gibco #10500-064), 100 U/mL penicillin and 100 µg/mL streptomycin (P/S) (Gibco #15140) and 50 μg/mL L-Ascorbic acid 2-phosphate sesquimagnesium salt hydrate (Sigma #A8960).

Table 4 .1 Dimensions of the pits obtained for each kind of pattern (12 measurements per pattern).

 4

	Name of the pattern	Measured diameter ± standard	Measured depth ± standard
	(Diameter-Depth)		deviation (µm)	deviation (µm)
		10-3			9.6 ± 0.5			2.7 ± 0.2
		10-10			9.6 ± 0.5			9.6 ± 0.7
		20-3			20.7 ± 0.9			3.4 ± 0.5
		20-10			20.9 ± 0.3			9.4 ± 0.3
		30-3			29.3 ± 0.8			3.3 ± 0.3
		30-10			29.3 ± 0.8			8.8 ± 0.4
	Name of the pattern	Lens focal length (mm)	Pulse energy * (mJ)	Number of pulses	Diaphragm aperture (mm)	Theoretical spot size ** (µm)	Fluence *** (J/cm 2 )	Total energy per area (J/cm 2 )	Total energy per pit (mJ)
	10-3	20	0.042	12	2.1	9.6	2.9	35	0.025
	10-10	20	0.025	100	2.5	8.1	3.4	345	0.18
	20-3	100	0.034	12	5.0	20.2	3.0	36	0.12
	20-10	100	0.023	50	5.0	20.2	2.0	101	0.33
	30-3	100	0.150	12	3.4	29.8	2.8	34	0.24
	30-10	100	0.165	60	2.8	36.1	1.4	86	0.88
	Table

  Albrektsson & Wennerberg ont recommandé au moins une hauteur, un paramètre spatial et un paramètre hybride à quantifier par profilométrie confocale à balayage laser ou interférométrie de contact. Ils ont également souligné l'importance de filtrer numériquement les données de rugosité pour une comparaison plus précise entre les différents travaux de recherche4 . Plus récemment, Flamant et al. ont souligné l'importance de réaliser des analyses de surface tridimensionnelles à plusieurs échelles dimensionnelles5 . De plus, pour chaque analyse et temps de mesure, plus de deux réplicats sont généralement utilisé pour obtenir des valeurs plus précises (généralement deux à quatre échantillons par condition). Même lorsque plusieurs analyses peuvent être effectuées sur un seul échantillon (par exemple, la phosphatase alcaline et la quantification d'ADN à partir du même lysat cellulaire) le nombre d'échantillons nécessaires est élevé. Enfin et surtout, des contrôles appropriés sont nécessaires (par exemple, des contrôles polis et "directement issus du frittage" dans l'étude de la céramique préparée par injection dans des moules rugueux).

	Un grand nombre d'échantillons (de l'ordre de centaines) est généralement requis pour
	l'évaluation in vitro des matériaux. Ceci est dû au fait que, avant tout, pour des résultats
	fiables, la réponse cellulaire aux matériaux devrait être évaluée à l'aide de cellules provenant
	de plusieurs donneurs (généralement, trois à six donneurs sont utilisés pour ce type d'études).
	Ensuite, plusieurs réponses (fixation cellulaire, prolifération, différenciation / maturation au
	niveau des gènes et des protéines) sont généralement testées à des moments réguliers
	(généralement trois à six fois) pour en avoir une vision plus complète du comportement
	cellulaire. Pour limiter le nombre d'échantillons requis pour l'étude in vitro, un protocole de nettoyage a
	été développé pour permettre la réutilisation des échantillons après chaque expérience de
	culture cellulaire. Un déliantage des résidus organiques qui pourraient être piégés dans la
	couche superficielle poreuse a été effectué. Les échantillons ont ensuite été nettoyés
	conformément aux instructions du fabricant pour de nouveaux lots d'échantillons. Enfin, les
	échantillons ont été stérilisés par l'oxyde d'éthylène. De nos expériences, il semblerait que le
	nettoyage est efficace (deux séries d'epériences sur les mêmes échatillons (avant et après
	nettoyage) ont donné des résultats similaires), mais des analyses détaillées supplémentaires
	de la surface seraient nécessaires pour le confirmer.
	Dans notre configuration expérimentale, la microrugosité seule n'a pas déclenché de
	différences significatives dans le comportement des hOb par rapport au contrôle poli, tandis
	que la combinaison de micro-et nano-rugosité a eu un fort effet sur le comportement des hOb
	avec des différences significatives au niveau de l'expression des protéines. En effet, l'activité
	de la phosphatase alcaline (ALP) normalisée à la teneur en ADN a été statistiquement
	significativement augmentée au jour 10 par les surfaces "directement issus du frittage", ainsi

Table 6 .2 Pearson product-moment and Kendall's tau correlation coefficients for all the combinations of topographical and morphological parameters (Significance of the correlation: p < 0.05 ; p < 0.005)

 6

  : interactions cellules-matériaux; ostéoblastes humains; zircone; alumine-zircone; micro-et nano-rugosité; impression 3D; robocasting; gradient; laser femtoseconde. Laboratoire (s) de recherche : Matériaux, Ingénierie et Science (MATEIS) et AO Research Institute Davos Directeur de thèse: Laurent Gremillard (MATEIS, INSA Lyon)

Encadrant: Marianna Peroglio (AO Research Institute Davos) Président de jury :

Composition du jury : Christophe Marquette (rapporteur), Milena Fini (rapporteur), Marc Anglada (examinateur), Mauro Alini (examinateur), Laurent Gremillard (directeur de thèse), Marianna Peroglio (encadrant).

Cette thèse est accessible à l'adresse : http://theses.insa-lyon.fr/publication/2017LYSEI053/these.pdf © [A-M. Stanciuc], [2017], INSA Lyon, tous droits réservés

Acknowledgements

Appendix

Cette thèse est accessible à l'adresse : http://theses.insa-lyon.fr/publication/2017LYSEI053/these.pdf © [A-M. Stanciuc], [2017], INSA Lyon, tous droits réservés

Appendix

Roughness gradients on zirconia for rapid screening of cell-surface interactions: Fabrication, characterization and application

Quentin Flamant