Remerciements J'aimerais tout d'abord remercier les membres du jury pour avoir accepté d'évaluer mon travail, Christèle Combes et Jean-Marie Nédélec en tant que rapporteurs et Catherine Debiemme-Chouvy et Franck Bouchart en tant qu'examinateurs. Je remercie Florence Babonneau et Hélène Pernot de m'avoir accueillie au

General introduction

A human body is constituted by 206 bones connected by more than 290 joints. An example of joint is the hip that connects the limb and the pelvis. It involves the femoral head and the cuplike acetabulum formed at the union of three pelvic bones (the ilium, pubis and ischium), as seen on Figure 0.1. Its role is mainly to support the weight of the body, during standing and walking. The joint is freely movable. However, hip pain is common with ageing, caused by osteoarthritis, a disease characterized by the disappearance of the cartilage favoring the sliding of the head of the femur in the acetabulum. To reduce pain and recover the function of the joint with the goal to improve the quality of life of the patients, the defective joint is replaced by an implant during a total hip arthroplasty. It is one of the most common orthopedic surgery, with more than 100 000 people operated each year in France 1 .

For ages, people have tried to replace defective part of the body by implants made by natural or synthetic materials. For hip replacements, the earliest recorded attempts date back from 1891, in Germany, with the use of ivory (a natural material, component of teeth and elephant tusks) to replace the femoral head 2 . Implants used today are very close to the one developed in the 1960's by Sir John Charnley. These implants are constituted in three parts, a metallic femoral stem, a polyethylene acetabular component and acrylic bone cement. The cement improves the prosthetic fixation, acting like a glue. Nowadays, three types of prosthesis are available, metal-on-polyethylene, metal-on-metal and ceramic-on-ceramic, each with advantages and disadvantages. Cementless techniques are also developed to avoid the use of cement that is degraded during locomotion, causing inflammatory response and failure of the implant. A scheme of a classical hip prosthesis is presented in Figure 0.1.

Concerning cementless prostheses, two main issues remain, aseptic loosening and infections, causing an incidence of revision surgeries of about 10 % 3,4 . Firstly, aseptic loosening is due to a poor interaction between the implant and the bone. Metallic implants are biocompatible, meaning that they do not induce an inflammatory response, but they cannot enhance bone growth as such. Modifications of surface state of the metallic substrate by physical or chemical methods are possible to improve the osseointegration. For instance, the use of hydroxyapatite, the mineral constituting of mammalians bones and teeth, as a coating allows a better interaction between bones and implants. The second issue about prostheses is a significant development of infection, despite the preventive treatment by prophylactic antibiotics and aseptic surgeries techniques. A promising strategy is to use the implant as the support of the antimicrobial strategy, with the possibility to achieve higher dosages near the affected site thereby improving efficiency and reducing the treatment duration and side effect 5 . In this context, this thesis aims to develop an antimicrobial coating on metallic implant, by associating hydroxyapatite and antibacterial molecules, as presented in Figure 0.2. We decided to synthesize biomimetic hydroxyapatite to achieve the best interaction between bone and implant. As antimicrobial resistance is one of the main challenges for public health in the 21 st century, our choice of antibiotics inclined towards natural molecules, extracted from plants, known to be antibacterial, but never used in the field. Four molecules were chosen, baicalin, baicalein, rosmarinic acid and chlorogenic acid. The main objective of this work was thus the incorporation of these natural molecules on or in hydroxyapatite. With this goal, we investigated the interaction between molecules and hydroxyapatite at different levels, in solution, with nanoparticles and finally with the coating.

The first chapter of this manuscript is a literature review on the design of coatings on metallic implants with osseointegration and/or antibacterial properties. Techniques to improve osseointegration will be presented, before an important part devoted to hydroxyapatite synthesis and deposition. Finally, a study of the antibacterial strategies available will lead us to present our strategy to develop an antibacterial hydroxyapatite coating.

The second chapter will be devoted to a study of the four selected molecules in solution, their biological activities and their interaction with calcium. On one side, the complexation of rosmarinic acid and chlorogenic acid with calcium was studied by spectroscopic and spectrometric methods. On the other side, we deeply investigated the stability of baicalin and baicalein in aqueous solution at different pHs, before examining their interaction with calcium. The effect of complexation with calcium on the biological activity of the four molecules was studied.

The third chapter focuses on the incorporation of the antibacterial molecules on/within hydroxyapatite nanoparticles by two methods, during synthesis of hydroxyapatite or by adsorption on pre-synthesized hydroxyapatite. The effect of the presence of organic molecules on hydroxyapatite synthesis was investigated. Baicalein being the molecule incorporated with the highest rate by adsorption, biological activities of nanoparticles with adsorbed baicalein were studied.

The last chapter is dedicated to the development of the biomimetic hydroxyapatite coating on a metallic substrate, the Ti6Al4V titanium alloy. After selection of suitable deposition conditions, biologically-active molecules were incorporated in the coating during synthesis of hydroxyapatite or by adsorption on pre-synthesized hydroxyapatite, and the two methods were compared. Finally, biological activities of coatings with baicalein, incorporated by the two methods, were investigated.

A conclusion with our main findings and perspectives will end this manuscript.

Figure 0.2 Strategy developed in this thesis

Literature review

1.1 Total hip arthroplasty: generalities, loosening and infections "Biomaterials are defined as substances, other than drugs, that can be used to treat, augment, replace, or repair any tissue or organ of the body in vivo for any period of time" 1 . It can be either metals, ceramics or polymers. With ageing of population, the demand for biomaterials is constantly increasing, especially in the field of joint replacements (hip, knee). In the field of joint replacements, the requirements for physical properties do not leave many options in the choice of the material, only metals have acceptable stress and fatigue resistance. Due to the necessity of biocompatibility, titanium and its alloys are widely used. Biocompatibility is defined as the ''ability of a biomaterial to perform its desired function with respect to a medical therapy, without eliciting any undesirable local or systemic effects in the recipient or beneficiary of that therapy, but generating the most appropriate beneficial cellular or tissue response to that specific situation, and optimizing the clinically relevant performance of that therapy" by the William dictionary of biomaterials 2 . Other metals and alloys are good candidates, such as chromium, magnesium and nickel alloy. However, toxicity of chromium and nickel ions limits their use in hip and knee prostheses, and magnesium has an in vitro corrosion rate too important to be used. In this thesis, we will focus on the study of the Ti-6Al-4V alloy, composed of titanium, aluminum and vanadium, because there is a consensus nowadays about its use, as its resistance to the corrosion in body fluids at physiological temperature and pH is better than commercially pure Titanium, with comparable physical properties 3 .

Total hip arthroplasty, which is the surgical procedure where the patient's joint is replaced by an implant, affects more than 100 000 people each year in France 4 . Several studies underline the increasing rate of revisions arthoplasties 5 these last 20 years, reaching around 10 % of the total amount of joint arthoplasties each year in Australia 6 , due to the implant failure, from the first days after implantation to two years post-surgery. Two main reasons explain this failure, the aseptic loosening and the development of infections. Aseptic loosening can be separated in two major cases, depending on the type of implant with or without cement. The cement is used to fix the prosthesis in the implantation site, but the repetition of mechanical stress during locomotion degrades the bonecement and cement-implant interfaces, leading to the disintegration of the cement and the production of debris particles which can trigger an inflammatory response 6 . In cementless arthroplasty, the loosening is related to the ability of osseointegration of the implant material into the bone 6 . The term osseointegration refers to the direct contact between living bone and implant 7 , meaning the absence of an interfacial fibrous tissue. The bone grows directly into or onto the implant surface 8 , which implies that the biomaterial surface is osteoconductive. When the material cannot promote bone growth, osseointegration is poor and the risk of loosening increases. This is the case for all untreated metallic surfaces of bone implant. The second reason for failure is the development of infections near the Chapter 1. Literature review implantation site, with a rate of 0.3-0.4 % depending of the study 9,10 , 15 infections over 5060 studied cases in the first study, and 154 infections over 36340 cases in the second. The main bacterial strains encountered in such infections are gram-positive bacteria such as Staphylococcus aureus and S. epidermidis. The rate of infection remains low thanks to the use of prophylactic antibiotics and aseptic surgical procedures as preventive measures, but is not negligible. In the next sections, we will explore different strategies to improve the osseointegration, and then we will see how to develop an antibacterial surface useable for bone implantation.

Osseointegration improvement strategies 1.2.1 Parameters improving osseointegration

Before exploring the critical points to improve the integration of implants in the bone site, it is necessary to understand how the implant interacts with its environment, and how bone is forming on the implant surface. In a healthy body, bone is constantly remodeled through a process involving osteogenic cells: osteoclasts that degrade the old bone, and osteoblasts, which are able to synthesize the newly formed bone. On an implant surface, the formation of a new bone is conditioned by the osteoblasts' activity. Davies 11,12 assumed that the formation of a new bone at the implant surface occurs in a three-stage process, first osteoconduction, secondly "de novo" bone formation and finally bone remodeling. Osteoconduction is the phenomenon where osteogenic cells, i.e. non-fully differentiated bone cells, migrate to the implant surface. The second step consists of their differentiation into osteoblasts that will synthesize new bone. The differentiation is possible at the contact of the implant surface, or the osteogenic cell can differentiate near the implant surface, leading to a distance bone formation. The contact/ distance osteogenesis depends on the surface characteristics, in terms of chemistry, roughness, topology and wettability.

Before colonization of the surface by osteogenic cells, the first interaction between the biomaterial and its environment is through the blood circulation, reservoir of proteins that will adhere to the surface. The interaction between those proteins and the migratory cells through different signal pathways will end by the cell adhesion to the implant surface. It is then necessary to understand the key surface parameters leading to a good protein adsorption. Proteins interact with the surface through hydrophobic interactions, electrostatic interactions, hydrogen bonding and/or Van der Waals forces. The chemistry of the surface, as well as its surface energy and wettability, are then important parameters that affect the protein adsorption. Because proteins are mainly within the nanometer range, the dimension scale of the surface is a relevant parameter, especially through the topography design (roughness, porosity) 13 . Two main strategies can therefore be used to improve the surface osseointegration, one involving only a physical or chemical surface treatment, and the second using Chapter 1. Literature review the deposition of a coating 6 , modifying both physical and chemical properties of the implant. The next two paragraphs will give some examples of these two strategies.

Topological modifications

It has been commonly accepted that a rough hydrophilic surface is better for protein adsorption and cells adhesion than a smooth hydrophobic surface and that the roughness organization is also a critical parameter. To develop surfaces with suitable roughness and wettability, several technics can be used, such as sandblasting, polishing, acid etching as well as laser and thermal treatments. Anselme et al. showed that adhesion and proliferation of osteoblasts after 14 days of culture is better on mechanically-polished Ti6Al4V surface than on sandblasted one 14 (Figure 1.1). In the case of polishing, grooves are all oriented in the same direction, which cannot be obtain through sandblasting.

Moreover, sandblasted surface is enriched with aluminum oxide, explaining the poor adhesion 15 . Al species can be released during the in vivo dissolution of the oxide layer, which can be toxic for the cells. 14 Deligianni et al. further studied the influence of the polishing grain, showing that protein adhesion, as well as cell adhesion and proliferation, is better on the rougher substrates. (Figure 1.2) 16 .

Figure 1.2 Human bone marrow cell proliferation after 8, 11, 14 and 16 days of culture on TiAlV mechanically polished with 1200-, 600-and 180-grit SiC paper, and on tissue culture plate (control) 16 The roughness orientation can be achieved through other technics such as laser interference lithography, which permits the formation of surface with arrays, dots or dimple with controlled roughness and hydrophilicity depending on the irradiation time 17 . Liu et al 17 showed that cell adhesion is not dependent of the surface nanostructuration, while it is critical for proliferation, with the best proliferation rate visible for doted rough surface. It was also suggested that nanopillars obtained with induction heating treatment (IHT) could serve as anchor points for cell adhesion and also promote osteoblast differentiation (Figure 1.3) 18 .

Figure 1.3 A cross sectionnal view of the FE-SEM images of oriented TiO2 nanopillar layer growth on

the TiAlV surfaces after IHT oxidation for 35 s; scale bar = 500 nm 18 When increasing surface roughness from 0.56 to 2.73 µm of average roughness value through laser surface melting method 19 , only effect on proliferation was visible. Besides elimination of cracks, this method allows a homogeneous distribution of Ti and Al at the surface. A similar situation was met after oxidation in air at 750 °C of smooth or acid-etched rough TiAlV substrates 20 . A polycrystalline titanium oxide was obtained at the surface together with an increase in the aluminium content from 2 to 10 %. Despite these phase changes, the wettability of the surface remained the same, which could explain why authors did not see any difference in cell adhesion on both oxidized surfaces. However, a higher production of differentiation markers for etched oxidized surfaces confirmed that osteoblast were maturing and forming new bones. From these data, the microroughness (due to acid etching) appears more critical than nanoscale modifications (oxidation). A thermal treatment under argon atmosphere at 1000 °C can also be used to modify the surface TiO2 oxide phase, increasing its wettability and roughness and, consequently cell adhesion and bone formation in vivo 21 . The thermal treatment may also be performed after hydroxylation in alkali solution, this "alkali heat treatment" being widely used in the field. Shahriyari et al. used this process combined to friction hardening to obtain nanostructured surfaces of titanate, enhancing cell adhesion and proliferation (Figure 1.4) 22 .

However, it is important to note that they studied the effect on adipose-derived stem cells. A two-step plasma treatment was also recently reported for the nanostructuration of the titanium surface 23 . The surface roughness was increased thanks to ultrasonic surface rolling processing, and then the surface wettability was improved with plasma nitriding. The nanocristallisation of a TiN and Ti2N layer at the surface was shown to enhance the viability of cells. 22 To sum up this part, roughness and wettability are key points to increase the cell adhesion and proliferation on the implant surface, and different strategies are available in the scientist's toolbox to achieve the desired surface properties without any coating.

Incorporation of extracellular matrix components

Extracellular matrix (ECM) is the network of macromolecules organized around the cells to give tissue a stability. It is mainly composed of collagens and proteoglycans, and it is a reservoir for bioactive molecules such as growth factors 24 . One way to promote cell adhesion and proliferation on titanium implant is therefore to functionalize its surface with ECM components.

Collagen immobilization has been used for years in the field of orthopedic and dental implant to improve osseointegration. Collagen is the main structural protein in the extracellular matrix, and the main organic component of bones. It is also found in fibrous tissues. The easiest way to immobilize collagen is by adsorption in physiological conditions. Collagen can be adsorbed either in a fibrillar or non-fibrillar structure. Nevertheless, results obtained after functionalization with collagen at a density of 3-6 µg/cm² of collagen are contrasting. Becker et al. 25 did not observe any significant difference between coated and non-coated Ti6Al4V implants on rat calvarial osteoblasts, while Geßler et al. 26 found a better cell spreading on coated surfaces, whereas the number of adhered cells was similar to uncoated surfaces. During an in vivo study, a higher fibroblasts activity was observed 7 days after implantation on coated implants, together with the presence of osteoblasts, while on uncoated implant osteoblasts are visible only 14 days post-implantation 27 . However, no significant difference in osteoblast activity was found after 28 days between the two surfaces, suggesting that the collagen I (one of the most common collagen form in the body) coating improved the integration in its early Chapter 1. Literature review stages. As an example of covalently-grafted collagen coating, Morra et al. described the nanostructuration of a Ti implant by anodization followed by grafting of acid acrylic and collagen I coupling (Figure 1.5 (a) and (b)) 28 . The implant was first nano structured by anodization, before in vitro, They observed a better cell spreading on collagen coated nanostructured titanium in vitro (Figure 1.5 (c) and (d)) and an improved bone-implant interaction in vivo. Overall, collagen I coating may have a slight effect in osseointegration improvement, but not significant when used alone. That is why research focused on the immobilization of other ECM components, like the Arg-Gly-Asp (RGD) peptide or growth factors. The RGD peptide is an amino acid sequence recognized by integrins, i.e. cell adhesion proteins. Its immobilization through covalent grafting or physical adsorption 29 could promote osteoblasts adhesion. The covalent grafting of RGD can be obtained through silanization of the titanium surface 30 , or thio-anchorage on a deposited gold layer 31 . In both cases RGD-coated surfaces enhance osteoblast activity in vitro and bone formation and thickness in vivo. Schueler et al. used a different strategy where the peptide was grafted on a poly(Llysine)-graft-poly(ethylene glycol) co-polymer adsorbed on the titanium surface 32 (see Figure 1.6). The presence of this anti-fouling coating that does not promote cell adhesion allowed to evidence the effect of RGD on cell adhesion and spreading. However, the same authors later showed that in this configuration RGD could not promote osteoblast differentiation, possibly due to an unsuitable orientation of the peptide on the coating 33 . following self-assembly on the titanium SLA surface 33 As previously mentionned, the ECM also contains proteins, termed growth factors, which promote the differentiation of osteoblasts from the mesenchymal stem cells (multipotent stromal cells that can differenciate in various types of cells) and control factors during osteoinduction 34,35 . Among these proteins, the bone morphogenetic protein (BMP) family is widely studied and used in the field of orthopedic implants, especially the BMP-2. Immobilization of the protein on the implant surface can be achieved by physical adsorption, covalent grafting or incorporation into a polymeric or a ceramic coating. One can take advantage of the known ionic interactions between BMP-2 and anionic molecules, heparin for example, to immobilize the protein. After heparin covalent grafting on functionalized Ti surface, adsorption of BMP-2 is performed. This allows a controlled release over 28 days, with cell proliferation and osteogenic differentiation enhanced compare to Ti surfaces alone or with nonspecific adsorption of BMP-2 36,37 (see Figure 1.7). Bae et al. 38 synthesized a nano cluster of BMP-2 and chondroitin sulfate that was then adsorbed on a calcium phosphate layer deposited at the titanium surface. With a similar approach, BMP-2 and heparin were adsorbed at the surface of a hydroxyapatite layer formed by electrochemical deposition 39 . In each case, cell proliferation and osteogenic differentiation were improved when the growth factor is present. It is also possible to load polymeric films with BMP-2, for example using polyelectrolyte membranes 40 . Direct covalent grafting of BMP-2 on a titanium surface can also be achieved by modifying it with amine groups via a plasma treatment with allyl amine 41 . A spacer can be used to keep the protein far from the surface, preserving its conformation and therefore its osteogenic activity.

Calcium phosphate based coatings

Due to their similarity with bone, composed by hydroxyapatite as the mineral phase, calcium phosphates have been widely studied in the field of bone regeneration and osseointegration 42,43 . This mineral family is composed of twelve main compounds presented in Table 1.1, which differ from their Ca/P ratio that strongly influences their chemical and physical properties [42][43][44] .

Dicalcium phosphate dihydrate (DCPD, brushite mineral), and dicalcium phosphate anhydrous (DCPA, monetite mineral) possess a Ca/P ratio of 1.0. DCPA is rarely found in vivo, but DCPD has been proposed as an intermediate in bone mineralization. None of them were studied as coatings for improved osseointegration, in contrast to octacalcium phosphate (OCP, Ca/P = 1.33) and tricalcium phosphate (TCP, Ca/P = 1.5). OCP is a possible precursor of carbonated apatite in bone, with a structure similar to the one of hydroxyapatite. Due to its high solubility in biological fluids, OCP is used in bone cement for filling bone defects, as it induces hydroxyapatite formation. TCP exists under two crystal forms, α-TCP and β-TCP. TCP is a high-temperature phase of calcium phosphate that cannot be precipitated from aqueous solution, and the α-TCP is produced at even higher temperature than β-TCP. Their solubility is rather high but β-TCP being more stable than α-TCP, it is used in the field of bone regeneration and osseointegration. It has been shown that this phase is osteoconductive, as well as osteoinductive.

Hydroxyapatite is the main base component of bone and teeth. This mineral has a Ca/P ratio of 1.67, with a formula of Ca10(PO4)6(OH)2, but it is never found in this stoichiometric form in vivo. The in vivo HAp is calcium deficient, and mainly nanocrystalline (see For the use of calcium phosphate coatings in the field of osseointegration of implants, several parameters have to be taken into account 42,47 . As said before, surface roughness is a key parameter for protein adsorption, and then cell adhesion. However, data on effect of calcium phosphate surface topography on cell adhesion are contradictory 47 . Porosity is also a factor influencing protein adsorption, where pore size ranging from 20 nm to 500 µm have been shown to favor the adsorption.

Moreover, porous ceramics will allow a rapid invasion of fluids, cells and bone tissue growth. The surface charge and ionic environment are also important. Proteins are charged macromolecules; hydrophilic surfaces will induce a higher protein adsorption than hydrophobic surfaces. Protein adsorption is mediated by pH and ionic strength. Dissolution of calcium phosphate coating will induce a change in calcium and phosphate concentration near the surface, modifying the pH, and consequently protein adsorption. Concerning protein adsorption, TCP or BCP would be the preferred calcium phosphates, thanks to their higher dissolution rate compared to hydroxyapatite. Ion release and recrystallization from TCP to HAp plays important roles in cell adhesion and proliferation. The low crystallinity is more favorable for initial cell attachment and proliferation. Upon dissolution of the coating, Ca 2+ in the culture medium induces an increase in alkaline phosphatase (ALP) activity and osteocalcin mRNA expression in mouse primary osteoblasts. However, the presence of amorphous calcium phosphate with high dissolution rate may have an adverse effect on the long-term. In particular, poorly attached coatings will be delaminated from the substrate.

Hydroxyapatite coatings can be a support to integrate bioactive compounds such as growth factors and peptide-mediated adhesion. For example, Xie et al. co-deposited chitosan and Ag nanoparticles with hydroxyapatite by electrochemical deposition on commercially pure titanium 39 .

BMP-2 and heparin were then loaded on the 20 µm thick coating, by adsorption and evaporation at room temperature. Coatings of hydroxyapatite with chitosan favored osteoblasts attachment and spreading, probably due to the biomimetic structure of hydroxyapatite. Incorporated chitosan allowed a long-term release of BMP in vitro. Proliferation of bone mesenchymal stem cells was nearly equal for the HAp-only coating and with chitosan and BMP-2. However, the later coating induced a higher differentiation in osteoblasts and was positively tested in vivo, with bone maturation 12 weeks after implantation. BMP-2 can also be adsorbed on hydroxyapatite synthesized by a biomimetic method (see 2.3.2.2.2), with sustained release over 28 days, and improvement of osteoblast proliferation and activity 38 . An attempt to take advantage of both osteoconductivity of hydroxyapatite and RGD peptide adhesion properties has been explored by Bitschnau et al. 48 Stainless steel wires were coated with hydroxyapatite by electrochemical-assisted deposition and then loaded with a cyclic RGD and bisphosphonates. No difference in bone growth was found between the hydroxyapatite-only coating and in presence of RGD, possibly due to the fact that that the osteocondutive effect of hydroxyapatite "overwhelmed" the effect of RGD.

Hydroxyapatite coatings processes

Synthesis of hydroxyapatite particles

Hydroxyapatite can be synthesized by wet or solid-state methods.

Co-precipitation synthesis

Co-precipitation is the more used synthesis method, because it is simple to perform and several precursors can be used. It consists in adding at once or dropwise one of the precursor in an aqueous solution to a solution of the other one. Solutions are heated at temperature comprised between 25 and 100 °C, with a precise control of pH. It has to be basic, around 8 to 10, to induce hydroxyapatite precipitation. After a mixing phase, an aging phase is performed with duration between several hours to days, with or without heating and stirring. Long aging times under air atmosphere yield to carbonated hydroxyapatite, which can be interesting in bone integration. After drying, the obtained precipitate undergoes a thermal treatment to improve the crystallinity of hydroxyapatite.

Temperature of thermal treatment has to be chosen cautiously as hydroxyapatite decomposes at high temperature (700 °C) 50 . Synthesized nanoparticles are most of the time needle-shaped, but may also have a plate-like morphology (Figure 1.9).

Calcium nitrate Ca(NO3).4H2O is the more commonly used calcium precursor 46,[50][51][52][53] , while ammonium phosphate (NH4)2HPO4 or (NH4)H2PO4 are mainly used as phosphate precursors. Potassium or sodium salts are less used because Na + and K + ions can integrate the hydroxyapatite crystal lattice.

Depending of the precursor choice, the synthesis is referred to a double decomposition, for calcium and phosphate salts, or neutralization if precursors are calcium hydroxide and phosphoric acid 54 .

Residual ammonium and nitrate are removed by heating. Advantage of neutralization method is the absence of residual ions.

L. M. Rodríguez-Lorenzo et al. 51 systematically studied the influence of temperature, reaction time and aging time on the stoichiometry and crystallinity of hydroxyapatite. Temperature during synthesis influences the crystal size at same reaction and aging times: at 25 °C obtained small crystals are similar to bone, while at 90 °C larger crystals are more similar to enamel. For a reaction at 90 °C, two hours are necessary to synthesize stoichiometric HAp, while shorter times or lower temperature yield to calcium-deficient hydroxyapatite. Generally, crystal size was higher when the time of reaction increased, but this parameter also affected stoichiometry and morphology of particles. The aging time allowed an increase of Ca/P ratio and carbonate uptake.

Hydrothermal synthesis

Hydrothermal synthesis can be defined as a method of synthesis of single crystals that depends on the solubility of minerals in hot water under a high pressure 55 . Solutions of calcium and phosphate precursors in aqueous solutions are transferred in a sealed autoclave and heated at temperature between 100 and 200 °C. When organic solvents are used, the method is called solvothermal 56,57 .

Precursors used are mainly the same as for the co-precipitation method (calcium nitrate and ammonium phosphate). pH is adjusted before the hydrothermal treatment by sodium, potassium or ammonium hydroxide [56][57][58][59][60] , or gradually increased during process by urea decomposition 57 (see Figure

1.10).

By hydrothermal and solvothermal synthesis, nanorods and nanoribbons can be formed. With addition of surfactants and additives, like CTAB, oleic acid or glutamic acid, ultralong nanowires (up to hundreds of micrometers) are obtained 58,60 .The hydrothermal or solvothermal phase is relatively long, from several hours to days, so it is now combined with microwave heating 58 . Reaction time is reduced to 15 minutes, allowing to obtain hydroxyapatite with carbonate substitutions.

Sol-gel synthesis

Sol-gel synthesis corresponds to the mixing at molecular level of calcium and phosphorous precursors, which improves the chemical homogeneity of the resulting hydroxyapatite. The chemistry is based on hydrolysis and polycondensation reactions of precursors (see Equation 1.1) . Reactions may be in aqueous phase 61 or in organic solvent [62][63][64][65] , mainly ethanol. In ethanol, phosphate precursors are phosphorous alkoxides, hydrolyzed by a little amount of water (ratio P:H2O 3:1). Liu et al. suggested that triethyl phosphite has higher activity for hydrolysis than triethyl phosphate 61 , reducing the time needed for reaction. Calcium nitrate, or Ca(OEt)2 65 , used as precursors, are added in stoichiometric proportions to the phosphorous alcoxide solution. Solutions are aged for several hours depending on the chosen temperature to obtain a gel. 120 minutes at 45 °C are enough 66 , while 16 hours are necessary at room temperature 61 . However, alkoxides are expensive reagents and research has focused on finding other precursors, such as P2O5 62,63 . that can be reacted with alcohol to form the alkoxide P(O)(OR)3. temperature, but thermal decomposition may occur at temperature higher than 700 °C. HAp synthesized in ethanol was found more stable than synthesized in water towards thermal decomposition 61 . Advantage of this method is the formation of homogeneous materials, with an easy processing by dip or spin coating to obtain films. It is also very convenient to form composite 67

M-OR+

Solid state synthesis

In solid-state synthesis, solid precursors are mixed in stoichiometric quantities and heated at high temperature (1000 °C). Precursors may be tribasic calcium phosphate (TCP) 69 , dicalcium phosphate dihydrate (DCPD) 70,71 , calcium pyrophosphate 72 , calcium hydroxide 69 , and calcium carbonate 71,72 .

Precursors are first sieved and ball milled in water 69,70,72 or organic solvent 71,72 for homogeneous mixing. The solvent chosen for milling is important, as the morphology obtained after milling in acetone or water is different 72 (see In electrochemical cathodic deposition, an electric current goes through two electrodes in an electrolyte solution, containing calcium and phosphates ions. Reduction of water near the electrode produces hydroxyl ions, leading a local increase in pH. The solubility of calcium phosphates decreases with pH increasing, so it precipitates at the electrode surface [START_REF] León | Thin Calcium Phosphate Coatings for Medical Implants[END_REF][START_REF] Mohseni | [END_REF]84,85 . Heterogeneous precipitation being more favorable than homogeneous precipitation in solution, the electrode is coated. Advantages of this technique are numerous, such as possibility to deposit a thin layer with crystallite size close to bone mineral, control of thickness and chemical composition, homogeneity of the coating, duration of process, and its low need for energy, reducing the cost. However, two major problems are faced:

hydroxyl formation affects the composition of the deposited mineral and hydrogen produced during water reduction may interact with the surface, resulting in poor coating adhesion. The deposited mineral is often brushite or monetite, or amorphous calcium phosphate, because pH is not high enough to produce hydroxyapatite. However a subsequent alkaline treatment in sodium hydroxide allows the crystallization of HAp. Deposition temperature affects the deposited amount, crystallinity and crystal size: the higher the temperature, the thicker and crystalline the coating. The composition of the electrolyte is also critical to control the deposited coating. Importantly, the mild conditions used in this technique are compatible with the incorporation of organic components like collagen, growth factors, peptides or small molecules with antibacterial properties 39 .

Another electrochemical method to deposit calcium phosphate mineral from aqueous solution directly on the substrate is micro-arc oxidation coating (MAO). It is a combination of electrochemical oxidation, plasma chemical oxidation and thermal diffusion in an electrolyte containing calcium and phosphates salts 86 . The substrate acts like the anode and is immerged in the aqueous electrolyte.

Process is fast, only 5 to 180 minutes are needed to achieve thickness around 1-100 µm. Concentration and composition of the electrolytes influence the morphological characteristics, porosity, thickness, corrosion resistance and biocompatibility of the coating. The process can also be used to deposit a titanium oxide layer at the surface of Ti to improve the hydroxyapatite forming ability of the surface.

Biomimetic deposition

All techniques presented before lead to crystalline and stoichiometric hydroxyapatite, either during process or after a post treatment. One way to obtain bioactive hydroxyapatite, i.e. non-stoichiometric poorly crystalline hydroxyapatite with calcium deficiency and substitutions is biomimetic deposition.

The methods consists in soaking the substrate in a solution slightly supersaturated in calcium and phosphate whose composition mimicks blood plasma. Deposition occurs at physiological pH and temperature (pH 7.4, 37 °C).

Kokubo was the first to report a solution allowing the deposition of hydroxyapatite on bioactive surface. He called this solution the Simulated Body Fluid (SBF). The initial uses of SBF were the conversion of bioglasses, which are materials composed of a SiO2 matrix with different oxides like MgO, CaO or P2O5, into hydroxyapatite, and the understanding of their mechanism of bioactivity in the body 87 . The solution mimicked the ionic composition and concentration of the blood plasma, except for Cl -and carbonated whose concentrations are lowered to maintain the stability of the solution (see Table 1.4). Subsequently, the ability of SBF to form hydroxyapatite on ceramics, metals and polymers was tested, as criterion for bioactivity evaluation: if hydroxyapatite was formed, then a good in vivo activity to enhance bone formation could be expected. The success of this approach led other researchers to use it directly as a coating process, especially on titanium and titanium alloy implants.

The original SBF missed the sulfate ions in its composition, which was corrected in 1991 by Kokubo.

Mechanism of the hydroxyapatite deposition was further elucidated in 1998 88 . For titanium, a pretreatment was shown to be necessary to make the surface bioactive. As it is well-known that the rutile phase of TiO2 is bioactive, an acidic treatment followed by crystallization at 600 °C was used to favor its formation. An alkaline treatment in NaOH can also be used to form Ti-OH groups at the surface that are useful to deposit hydroxyapatite. After alkali treatment, the substrate is covered by a sodium titanate layer that interacts with calcium ions of SBF, producing a layer of amorphous calcium titanate.

Then, phosphate ions from the solution react with the surface to produce an amorphous calcium phosphate layer, with a Ca/P ratio close to 1.40. This layer is converted into hydroxyapatite during the maturation step to reach an atomic ratio of 1.65, close to the one of bone hydroxyapatite. The coating was shown to also contain magnesium ions 89 . Morphology of the produced coating is quite typical of the biomimetic process (see Figure 1.14): apatite crystals are plate-like, while, in co-precipitation synthesis, needle-shaped crystals are often produced. The dense and homogeneous layer of hydroxyapatite is usually formed of spherical aggregates that increase in size with time of deposition to cover the whole surface. Coating is well-adherent to the substrate surface thanks to the calcium titanate-sticking layer.

Various surface treatments have been investigated to find the best way to rapidly produce hydroxyapatite. Acidic treatment with HCl, piranha solution (H2SO4, H2O2), or mixture of HCl and H2SO4

were tested. Different alkali and alkali heat treatments were used to form a sodium titanate layer at the substrate surface under the form of a gel or crystalline phase. Pattanayak et al. 92 found that there is no difference in hydroxyapatite deposition after acidic or basic treatment, with or without heating at 600 °C, but even after heat treatment, surface soaked in solution with neutral pH did not show any hydroxyapatite deposition (see Figure 1.15). As mentioned before, the chloride and carbonate contents of the original SBF are not exactly equal to the one in blood plasma. Oyane et al. 93 tried to synthesize hydroxyapatite from a solution with the correct concentration in chloride and carbonate but observed the precipitation of calcium carbonate in solution. Takadama et al. 89,90 revised the concentration of chloride while keeping the carbonate concentration similar to SBF. This solution, labelled n-SBF (newly-improved SBF) had the same stability as SBF, with the same ability to form hydroxyapatite. The influence of carbonate concentration on the structure and crystallinity of the deposited hydroxyapatite was studied using SBF buffered with HEPES or by addition of CO2 in the solution 94 . Solutions with HEPES were unstable while CO2 bubbling was convenient, except that the exact concentration of carbonate ions in solution is unknown. It was found that increasing the carbonate concentration make the deposited layer thicker.

Substitution of HEPES by TRIS 91,9596 to buffer the carbonated solution allowed to decrease the extent of homogeneous precipitation. Overall, higher carbonate contents seem to be favorable to hydroxyapatite deposition on titanium or titanium alloy, but a correct stability of the solution requires the addition of a buffer and the chloride concentration has to remain similar to the one in conventional SBF.

Even if the standard SBF solution is already highly metastable, several attempts to modify the concentration of the solution have been made to enhance the hydroxyapatite deposition. Indeed, in order to deposit a relatively thick layer from SBF, a minimum of 14 days of deposition are needed with regular replenishments of the solution. The most popular way to speed up the deposition is to concentrate the SBF solution, and increase the processing temperature. Jalota et al 96 . used 1.5 x concentrated SBF. Deposition lasted 7, 14 or 21 days with replenishments of the solution every 48 hours. They reported homogeneous precipitation of hydroxyapatite in the solution due to too high supersaturation, but without any effect on the coating if substrates are placed vertically in the deposition solution. Deposition of hydroxyapatite from a 2x concentrated SBF was also studied 97 .

Evolution of composition and morphology of coatings after 12 hours and 4, 8 and 14 days of deposition on TiAlV is visible on 97 Li et al. 98 studied the deposition of hydroxyapatite from a 3x concentrated SBF, at 57 °C. As they did not increase the TRIS concentration, pH was lower than expected (pH 6.8). Deposition time varied from 1 to 4 days, with refreshment every day. As seen in Figure 1.17 coatings consisted in spherical aggregates increasing in size with time of deposition, but the coating was not homogeneous and the whole surface was not covered. They found that brushite was formed in the earlier time of deposition and transformed afterwards into hydroxyapatite. In another approach, Habibovic et al. 99 separated the coating procedure in two steps. First they deposited a thin and amorphous calcium phosphate layer on the TiAlV surface in a solution (SBF-A) consisting of SBF concentrated 5 times, without TRIS buffer and sulfate ions. The second step was the transformation of this layer in a thick and crystallized apatite film using a solution (SBF-B) similar to the previous one but with lower concentrations of Mg 2+ and HCO3 -, known as inhibitors of apatite crystallization. The pH of both solutions was adjusted by a CO2 gas flow during the preparation of the solutions. The coating steps lasted 24 h at 37 C° for the first, and 48 h at 50 °C for the second. After soaking the substrate in SBF-A, a carbonated amorphous calcium phosphate was deposited on the surface (Figure 1.18 (a)). In SBF-B, the dissolution of ACP occurred followed by the crystallization of HAp (Figure 1.18 (b)). In these conditions of high supersaturation, nucleation occurred both in the solution and on the substrate surface but it was suggested that formed nuclei are more stable on the preexisting ACP layer. A study on the formulation of revised simulated body fluid showed that the concentration of the solution affects the morphology and the stoichiometry of the deposited calcium phosphate 100 .

When the concentration of the SBF increases, the Ca/P ratio and the amount of incorporated carbonates also increased. The composition of the films was less close to the bone than when deposited in conventional or revised SBF at the usual concentrations. To conclude, it is better to keep the ionic concentrations in SBF close to the ones of blood plasma, in order to prevent precipitation in solution and to obtain a coating composition as close as possible to bone hydroxyapatite. An exception can be made for carbonate content, which can be lowered like in conventional SBF, or the same than in blood plasma like in the revised SBF, if the chloride content remains high.

Other media can be used as solution for biomimetic deposition. wet method consisting in soaking the substrate in the solution. However, deposition lasts several days or weeks, which is the major disadvantage of this method. To accelerate the deposition, it is possible to concentrate the solution, while keeping in mind that it is very likely to induce precipitation in solution and make the deposited hydroxyapatite less "biomimetic".

Antibacterial strategies

Prosthetic joint infections (PJI) are a major concern nowadays as their number is constantly increasing. Indeed, even if their prevalence is lower than before, as the number of patients expands every year, the number of infections goes the same way. The most frequent bacterial strain encountered in PJIs is the gender Staphylococcus, especially S. aureus and S. epidermidis, besides S. The majority of bacteria have a hydrophobic surface, because their membranes are composed by peptidoglycans for Gram + bacteria and phospholipids for Gram -bacteria. To prevent bacterial adhesion, one can play with surface hydrophilicity, with chemical treatments, nanopatterning, or different coatings like polymers or titania. Such surfaces are named antiadherent, in contrast with bactericide surfaces that kill planktonic or adhered bacteria thanks to bactericidal agents like metal ions or nanoparticles or antibiotics (encapsulated or tethered). This part will present these different methods used to achieve antibacterial surfaces. First, we will focused on surface modifications, then we will discuss the use of bactericidal metals such as silver, and finally we will explore the use of antibiotics, with an insight on antibioresistance and the necessity of using new antibacterial agents.

Surface topology tuning

Titanium surface before any treatment is slightly hydrophilic (water contact angle ≈ 70 ° 106,107 ).

To increase the hydrophilicity of the surface, plasma 107 , laser 106 , UV 108 and chemical 109 treatments are used.

A non-thermal ambient atmosphere plasma treatment used on pure titanium surfaces allowed the formation of a TiO2 anatase upper layer, together with the formation of imine and imides functional groups 107 . Treatment duration affected the surface energy and water contact angle of the surface. A 10 minutes treatment lead to more hydrophilic surface than the 2 minutes one, see Recently, research has focused on the nanopatterning of titanium surfaces 110,111 . Producing nanoscale structures, sometimes inspired by Nature, with specific dimensions on the surface could mechanically kill the bacteria, in addition to the anti-adhesion effect. Numerous fabrication process are available, among them anodizing [112][113][114] , chemical etching 109 and hydrothermal are the most popular. Dimension of nanopatterns is the key parameter for the bactericidal property. Most studies report the bactericidal behavior for dimensions ranging between 100 and 1000 nm in height, 10 to 300 nm in diameter/width and spacing lower than 500 nm 110 . Rupture of the cell wall by penetration of high aspect ratio nanopatterns is commonly assumed as the underlying bactericidal mechanism. The control of anodization time of TiAlV substrates at a constant voltage of 20 V in an electrolyte containing H2SO4 and HF can control the nanopattern obtained at the end of the process. 5 min leads to nanoporous TiO2 while after 60 minutes nanotubular oxide is formed 112 . However, S. aureus and S.

epidermidis adhesion is the same on nanoporous and nanotubular surfaces. Chemical etching in acidic oxidant solution (H2SO4 and H2O2 mixture) can form TiO2 nanopores on TiAlV substrates 109 . After 30 minutes of etching, surfaces exhibit a trend to hydrophobic behavior, while after 60 minutes the hydrophilicity is enhanced. Adhesion of fungi (Candida albicans) depends on the time of contact with the surface. After 2 hours of contact, adhesion is reduced on treated surface, while after 6 hours the cell viability is higher on surfaces treated for 60 minutes, because the dimension of created pores is different depending on the treatment duration and pore dimension seems to have an effect on cell viability.

Overall, modification in surface hydrophilicity and nanopatterning are promising ways to reduce bacterial adhesion and activity on implant surface, with easy to handle techniques without an additional coating. In case of nanopatterning, surfaces may also be mechanically bactericidal.

Surface functionalization

With inorganics compounds

Some metals have intrinsic antibacterial activity, like copper, zinc, and silver, which is the most studied. Metals may be added on the implant by incorporation within a coating of titania 115 , titania nanotubes 114,116 or calcium phosphate coatings 117,118 . Metals are interesting bactericide agents because they are active against both Gram-positive and Gram-negative bacteria. Because it is a very potent bactericide, silver is widely used, especially under the form of nanoparticles (NPs). It is commonly accepted that the antibacterial activity of Ag NPs comes from Ag + ions released upon NPs dissolution.

Silver ion is capable to bind to bacterial DNA and RNA to prevent bacterial proliferation 116,118 . Silver NPs were incorporated in hydroxyapatite coating deposited on TiAlV substrates by a Ag mirror reaction (formation of metallic silver as a result of an oxidation-reduction reaction) 118 . Morphology of the hydroxyapatite coating affected the Ag release, as nanorods and nanoplates arrays retained more Ag than smooth surfaces over 70 hours. However, it did not affect antibacterial activity and the Agincorporating coating reduced the number of viable bacteria by 100 %. Toxicity of coatings were evaluated on human bone mesenchymal stem cells. Cells adhered and proliferated on every surfaces, meaning that the presence of silver at these concentrations was not cytotoxic. However cytotoxicity of silver is usually a major concern, requiring the control of silver ion release. For instance, Ag NPs loaded in titania naotubes (TNT) were release in two steps, a first rapid initial burst release and then a sustained one over 14 days 114 . These coatings were very efficient against S. aureus ( One can take advantage to the flexibility of the structure of hydroxyapatite, which is favorable for ionic substitutions and insertions. Numerous studies have been done to dope HAp with various metal ions, such as silver 120 , zinc [121][122][123] , copper 122,124-126 , iron 127-129 , strontium 130,131 , lanthanides 132 , in order to enhance its biological activity. In particular, substitutions -or insertions -of copper and zinc are very promising for their antibacterial properties. Depending on the ionic content and synthesis method, doping ions may substituted calcium, or inserted into the hydroxyl channel, which does not modify crystals lattice parameters 121,128 . Employed as nanopowders, Cu-and Zn-doped HAp showed bacteria reduction against E. coli and in a lesser extent S. aureus 122 . Copper substituted hydroxyapatite reached more than 98 % of activity against S. aureus 125 . Combined doping of bismuth and silver Bi0.5Ag0.5Ca4(PO4)3OH was also very efficient against E. coli and S. aureus 120 . However, dopedhydroxyapatite are mainly used as nanoparticles or cement. To the best of our knowledge, only few very recent promising studies have been conducted on doped-hydroxyapatite coatings. Solution precursor plasma spraying process was used to deposit Zn-or Cu-doped hydroxyapatite on TiAlV 123,126 .

Antibacterial activity of these coatings are not available yet.

Overall, metals are very efficient as bactericide agents, but it is necessary to control their release in body fluids to prevent any cytotoxicity, especially in the case of silver ions. However, promising strategies using additional polymeric coating or other bactericide metals like zinc have to be further investigated, notably due to the broad-spectrum activity of such agents.

With organics compounds

1.4.2.2.

Antibiotics

Local delivery of molecular antibiotics present at the implant surface has been widely studied over the past ten years. Antibiotics may be covalently immobilized or encapsulated in polymers or ceramics matrixes. One main disadvantage of antibiotics compared to metals ions -or NPs -and topological modifications is their narrow spectrum of efficacy. Indeed, antibiotics molecules have been developed against Gram-positive or Gram-negative bacteria, but do not have the same efficiency against both and can also show a strong strain-dependent activity. Nonetheless, some broad-spectrum antibiotics are available, a selection of which (see molecular structures on Figure 1.25) will be studied here for incorporation on titanium surfaces by tethering or encapsulation With the goal of combining antibacterial activity with osseointegration, loading of antibiotics within hydroxyapatite has been widely studied 101,[138][139][140][141][142] . In these studies, drugs may be incorporated after in the coating by adsorption 101,138,140 or lyophilization 141,142 . The synthetic route of the hydroxyapatite coating may affect the antibiotic loading and release. Tobramycin adsorbed on plasmasprayed HAp was released over 2 days in PBS, while release was sustained for 8 days at concentration above MIC (against S. epidermidis) if the drug was adsorbed on biomimetic HAp 128 . This was attributed to the nanoporous structure of biomimetically deposited HAp. By adsorption on such a coating, tobramycin remained active up to 6 days in vitro with S. aureus incubation every day, despite an initial burst release in the first 15 minutes 138 . The large amount of drug loaded allows to deliver tobramycin for 8 days with a concentration above the MIC. Adsorption of tobramycin, cephalotin, amoxicillin and gentamicin was studied on hydroxyapatite 140 . Coated surfaces inhibited S. aureus growth after 24 hours of incubation. In vitro release showed an initial burst release followed by a slow and reproducible release for 22 hours. Release rates indicated that the release of a therapeutically relevant dose could be achieved for about 2 days. This strategy could be useful to avoid initial contamination of the implant.

The same drawback was observed when gentamicin was loaded by lyophylization on carbonated hydroxyapatite coating on TiAlV 142 . Macropores and mesopores of carbonated hydroxyapatite allowed to adsorb a large amount of gentamicin, but the totality of the drug was eluted after 24 hours (Figure Encapsulation of antibiotics follows the same principles than for encapsulation and release of osteogenic agents. For example, thanks to their small size, antibiotics molecules can be incorporated into titania nanotubes (TNTs) formed by anodization on titanium and titanium surfaces 113 . Filling of TNT via lyophylization allowed an uptake of gentamicin up to 600 µg for 0.5 cm x 0.5 cm surfaces. S.

aureus adhesion was reduced by 70 % when TNTs are loaded with gentamicin, after 4 hours of incubation. However, this coating possesses a very short-term effect, as the drug is eluted in 150 min, and would be only useful to prevent initial bacterial colonization, before the adhesion and proliferation of osteoblastic cells. Another way to encapsulate antibiotics is inside a polymeric coating. Vancomycin was successfully incorporated in a PEG-poly(lactic-co-caprolactone) (PEG-PLC) hydrogel covalenty bounded to Ti surface via adhesive polydopamine. Addition of starch in the coating slowed down vancomycin release in vitro, because of slower swelling of PEG-starch gel compared to PEG alone 136 . In vivo, vancomycin was delivered for nearly 4 weeks, with the reduction of inflammation in a rabbit model of S. aureus infection. Drugs can also be loaded within ceramics coatings, like silica or calcium phosphate. Triclosan was incorporated in a silica sol-gel coating on stainless steel fixation pins via solgel synthesis 137 . After an initial burst release, sustained release up to 8 weeks was observed, with elution of 33 % of the original triclosan load. An in vivo study in rabbits showed the ability of the coating to prevent infection and promote tissue healing.

When encapsulated in hydroxyapatite coating, antibiotic during may be added during the coating synthesis, especially for biomimetic deposition. When the molecules are added to the coating solution, they gradually become integrated within the forming calcium phosphate deposit 139 . For this strategy, functional groups of the antibiotic molecule is important for interaction with calcium and phosphate ions. Cephalotin and carbenicillin showed the best incorporation compared to cefamandol, amoxicillin, tobramycin and gentamicin. Looking to the antibiotic molecular structures, the incorporation efficiency seems to be related to the number of carboxylic acids, except for cephalotin that only possesses one carboxylic group. Vancomycin was poorly incorporated in the coating, probably due to its molecular size. As for their release, cephalotin was released at the slowest rate, and no more than 70 % of the loaded drug was release within 16 hours. Drug loading and release were correlated to antibacterial activity of the coating as the cephalotin-loaded deposit was the more efficient against S. aureus.

Covalent immobilization

Tethering antibiotics has several advantages, such as long-term antibiotics effect and local activity. As the drug is in principle immobilized on the surface for the lifetime of the implant, it should be effective in case of late colonization, and also limit its toxicity to the closest tissues 133 . MIC is the lowest concentration at which a compound will inhibit bacterial growth when dosed against planktonic bacteria cells 105 . The group of Shapiro and Hickok successfully tethered different antibiotics like vancomycin and gentamycin on titanium and titanium alloy surfaces with the help of self-assembled coli, a gram negative organism that is not sensitive to vancomycin 135 .

Even if these coatings seem promising, few drawbacks remain. Physical and chemical stability of the coating has to be taken into account, as the forces applied to orthopedic coatings are significant and the biological fluids highly corrosive. Furthermore, their action is only directed in the space immediately adjacent to the implant, which can be either be an advantage and a disadvantage. The antibiotic cannot fight against the propagation of the infection if avoiding biofilm formation is not enough.

Antimicrobial peptides

Antimicrobial peptides (AMP) are part of the immune system of numerous organisms, including humans. They possess superior antimicrobial characteristics than antibiotics, because they are broad-spectrum agents. They are active against bacterial, fungi, parasites and viruses, by targeting fundamental structures of microorganisms, like the membranes 147 . AMP are produced chemically or biologically using bacteria, yeast or insect cells. Advantage of biological production is the cheaper price of large-scale production.

AMP may be encapsulated in polymeric films 148 , or in calcium phosphate coatings 149,150 . Even if the incorporation is successful, the diffusion of the AMP to the surface is an issue. The outer layer is not the AMP, and bacteria adhering onto the top assembly may block AMP release. Moreover, longterm stability of the coating has to be studied. AMPs adsorbed on calcium phosphate coating were able to kill S. aureus and P. aeruginosa within 30 minutes 149 . Coatings remained active after four cycles of bacteria incubation, even if the activity decreased. No loading nor antibacterial activity was observed when AMPs were adsorbed on Ti surface (see Figure 29). To delay the AMP release from the coating, a thin phospholipid film (palmytoyl-oleoyl phosphatidyl-choline POPC) could be deposited on top of the initial calcium phosphate layer 150 . The presence of the film allowed a continuous drug release up to 72 hours, without initial burst release. These systems were active against S. aureus and P.

aeruginosa after 24 hours of incubation. and XDR-TB), which are a rising threat in the developing world 145 . However, this group is outside of our study as this strain does not concern PJIs.

Antibiotics resistance is mainly due to misuse and overuse of antibiotics in western countries.

80 % of the total antibiotics consumption in the USA is used in animal food production, and almost 30 % of antibiotics prescriptions may have been inappropriate 146 . Resistance may appear only few years after the use of a new drug, and is spread worldwide through contact between people infected by MDR bacteria. Genes for resistance can be transferred among bacteria of different taxonomic and ecological groups. These genes are generally directed against a single family of antibiotics. In case of local delivery of antibiotics, it is important to know that very low concentration of antibiotics induces the resistance, which is correlated with sub-MIC concentration of antibiotic. To fight against antibiotics resistance, it is necessary to reduce antibiotic consumption, together with a better use. Researchers need to develop new antibiotics, as only two new classes of antibiotics have reached the market since the 1970s. New antibiotics may be inspired by natural molecules, which can be used as such or improved by organic synthesis to obtain more efficient drugs 145 . In this part, we will focus on alternative strategies involving natural molecules. First, the use of antimicrobial peptide will be underlined, before a study of natural polyphenols extracted from natural products, known for their numerous biological properties (antibacterial, anti-viral, antioxidant, anti-inflammatory).

In order to face the development of antibacterial resistance, the use of natural products as antibacterial agents has attracted much attention in the last decades 105 . Phytopharmacology is used in traditional medicine over three thousand years mainly as herbal tea, but also in modern medicine since a large proportion of known drugs are molecules that have been identified in plants and used as such or as a derivative. Indeed, it is estimated that at least 25% of all modern medicines are derived, either directly or indirectly, from medicinal plants, primarily through the application of modern technology to traditional knowledge 154 . As a sign of this increasing interest, a list of 442 plants is included in the 2013 version of the European pharmacopoeia for their therapeutic use. A more recent approach is to focus on the most active molecules from plants. Most are secondary metabolites that serve in many cases as plant defense mechanisms against predation by microorganisms, insects and herbivors 155 .

These molecules are responsible for plant odors (terpenoids) and colors (quinones and tannins), and most of them give the plant its flavor. Among them, polyphenols have been demonstrated to possess numerous health benefits such as antioxidation [156][157][158] , anti-inflammation 159,160 and antimicrobial activity 157 . It should be mentioned that these molecules have been investigated mainly for their use in food preservation, in the form of plant extract or essential oils, or extracted molecules [161][162][163] .

The most studied compounds include flavonoids, ubiquitous plant secondary metabolites that are known to be synthesized by plants in response to microbial infection 155,164 . More than 4000 flavonoids have been identified in fruits, vegetables and plant-derived beverages (tea, coffee, and wine). Additionally, a synergistic effect was observed against ciprofloxacin resistant strains 169 . Another class with high biological importance includes carboxylic polyphenols such as chlorogenic acids 158,170,171 , gallic acid 172 , rosmarinic acid 162 , ferulic acid 173 , ellagic acid 174 , that combine polyphenol and carboxylic acid groups. Chlorogenic acids are a family of esters formed between transcinnamic acid and quinic acid. The most common compound is 5-O-caffeoyl-quinic acid (5-CQA), which is the only one commercially available and is therefore commonly called "chlorogenic acid" 170 . It can be found in many foods and beverages, mainly coffee, fruits, vegetables and lamiacae (oregano, basil, marjoram, melissa, peppermint, rosemary, sage, spearming and thyme). The scavenging activity and antibacterial property of methanolic extracts of fennels leafs and seeds have been attributed to chlorogenic acid 157 . It is also one of the major and most active components of the plant Morina persica 175 .

Finally, it is also worth mentioning the terpene family 105 , including carnosic acid. Carnosic acid possess one phenolic group, so that it is often classified as a polyphenol compound, but it is closer to the terpene family 176 . It has been described as the most active compound of rosemary essential oils for antioxidant and antibacterial activity. It is active against Gram-negative and Gram-positive bacteria.

Figure 1.32 Molecular structures of chlorogenic, rosmarinic and carnosic acid

These examples are only a few over the wide range of natural molecules able to kill planktonic bacteria and/or to induce efficient biofilm prevention. Natural products may be used as essential oils or plant extracts, to take advantage of the synergy between compounds, or as pure compounds. An important point is that their MIC are far higher than those of commons antibiotics, meaning that a higher dose is necessary to reach the antibacterial activity. As for their use in the field of body implants, only few studies but promising examples are available in the literature 177-179 .

Conclusion: our strategy

This literature review emphasized the different strategies available to enhance osseointegration of metallic implants and to prevent the development of periprosthetic joint infections. Our goal in this PhD project was to confer both properties to titanium alloy surfaces, Ti6Al4V, the most common alloy used in total hip remplacement.

As studied in this chapter, hydroxyapatite is a material that could enhance osseointegration and support to immobilize antimicrobial agents. The unique FDA approved synthesis of hydroxyapatite is the plasma spraying but coatings is inhomogeneous and the adherence to the substrate is poor.

Moreover, with this method it is not possible to incorporate organics molecules like antibiotics during the process. In order to overcome the issues of plasma spraying, we decided to synthesize hydroxyapatite by a biomimetic deposition in using the Kokubo's simulated body fluid. Hydroxyapatite synthesized with this method is biomimetic in terms of chemistry and morphology, and temperature used in the process is compatible with the incorporation of organic molecules.

Several options are available to add antibacterial properties to a hydroxyapatite process, with inorganics or organics inorganics compounds. The use of metal is controversial because of the toxicity of such agents, and the issue of antibioresistance emerges with the use of antibiotics. Therefore, our choice goes on the use of natural antibacterial molecules, flavonoids and carboxylic acid polyphenols, known to be efficient against resistant-strains and never used in the field of antibacterial coatings for body implant. These four molecules possess carboxylic acid function and/or catechol functions that could significantly interact with hydroxyapatite.

In the next chapter, we will first discuss the antibacterial and antioxidant properties of selected antibiotics, as well as their interaction in solution with calcium ions, to determine which molecule would be the best choice to be associated with the hydroxyapatite coating. Spectrophotometric and spectrometric methods will be used to describe the complexation with calcium, and the behavior of molecules in aqueous solution. In a second step, the proof of concept to develop hybrid materials consisting of natural antibacterial molecules and hydroxyapatite will be presented. Antibacterial and antioxidant activity of mixed baicalein-hydroxyapatite particles will be determined. Finally, biomimetic coating of hydroxyapatite will be deposited on Ti alloy, and the incorporation of baicalein will be performed. In vitro antibacterial and osseointegration properties of the hybrid coating will be explored. Molecular study: interactions between calcium and biologically-active molecules

Introduction

In this first part of my PhD project, we attempted to understand the interaction between calcium and four biologically active molecules (BAMs) in solution at different pHs. The four BAMs studied along this chapter, and the entire thesis, are polyphenols and flavonoids: rosmarinic acid (RA), chlorogenic acid (CA), baicalin (BA) and baicalein (BE). As already discussed in the Literature review (Chapter 1), these molecules are potent antibacterial and/or antioxidant agents to be used in the field of bone implants. In a first instance, the characterization of the antibacterial and antioxidant activities of these molecules will be presented. Secondly, the interaction between rosmarinic and chlorogenic acids with calcium at different pHs will be investigated. As we intent to incorporate BAMs in hydroxyapatite, the interaction with calcium should be considered. This work conducted to the submission of a full paper, presented in the paragraph 2.4. In a third step, the in vitro fate of baicalin and baicalein at different pHs, as well as their interaction with calcium will be studied. In particular, the degradation of both molecules has been mentioned several times in the literature, but without further characterizations. We will try to better understand this phenomenon in the paragraph 2.5. and study how it affects their calcium-binding affinity.

Antibacterial and antioxidant activities of BAMs

The antibacterial activity of RA, CA, BA and BE was characterized by the measure of the minimum inhibitory concentration (MIC), against four bacterial strains: a Gram negative, Pseudomonas aeruginosa ATCC 27853, and three Gram positive, Listeria ivanovii Li4(pvS2), Staphylococcus epidermidis CIP 68.21 and Staphylococcus aureus ATCC 6538. Minimum inhibitory concentration of the molecules was measured by the microdilution method in 96-wells plates. Mueller-Hilton broth (MH) was used for culture of S. epidermidis, Brain Heart Infusion broth (BHI) for L. ivanovii and Lysogeny broth (LB) was used for S. aureus and P. aeruginosa. For each tested strain, a bacterial inoculum of 1 x 10 6 cfu/mL was prepared by diluting an overnight culture in sterile MH. BAM solutions (100 µL of 10 mg.mL -1 stock solution in DMSO) were allotted in a 96-well microplate by two fold serial dilutions.

Growth of bacterial strains was de visu assessed after 24 hours incubation at 37°C. MIC was defined as the minimal concentration of the tested compound that completely inhibited cell growth. BAM were tested at final concentrations ranging from 16 to 0.4 mg.mL -1 for RA and CA, 40.96 to 0.01 mg.mL -1 for BA and 1.35 to 0.033 mg.mL -1 for BE. The highest final DMSO concentration used (10 % v/v) induced no significant bacterial growth inhibition. MIC determination of ciprofloxacin (0.0625 µg.mL -1 for S. epidermidis CIP 68.21 and 0.5 µg.mL -1 for S. aureus ATCC 7574) was performed as a control on each microplate. The accepted variance on MIC value can be estimated to a 2-fold difference due to the microdilution method used. Experiments were performed in duplicate. Results are gathered in Table 2

.1.
First, a difference is noticeable between Gram-positive and Gram-negative bacteria. The MIC of the four molecules against P. aeruginosa is outside the range of tested concentrations, while it was possible to measure MIC for the three other bacteria strains. This suggests that the four molecules are more active against Gram-positive bacteria than Gram-negative. Against the three Gram-positive bacteria, baicalein is the molecule with the lowest MIC, at 34 µg/mL against S. epidermidis and L. ivanovii and 17 µg/mL against S. aureus, meaning that it is the molecule with the best antibacterial activity. Chlorogenic acid is the less active molecule against the three tested bacteria strains, with a MIC of 2043 µg/mL against L. ivanovii, 4087 µg/mL against S. aureus and higher than 4087 against S. epidermidis. Baicalin and rosmarinic acid have intermediary MIC comprised between 512 and 1024 µg/mL depending on the bacteria. Thereafter, the antioxidant activity of the four molecules was also tested. It was assayed by the measure of the scavenging activity on the stable free radical DPPH with a modified Blois' method in which the bleaching percetange of DPPH is monitored at a characteristic wavelength in presence of the sample 1 . The assay was performed as follows: 100 μL of BAM solution (concentration 50 µM) in ammonium carbonate pH 7.7 were mixed with 400 μL of Tris buffer pH 7. To conclude, baicalein is the molecule with the best antibacterial activity against the four tested bacterial strains, while chlorogenic acid is the one with the worst. On the contrary, baicalein has the weakest antioxidant activity, together with baicalin. Rosmarinic acid has an antioxidant activity 6times higher than baicalin and baicalein, and twice that of chlorogenic acid. Rosmarinic is then the best antioxidant molecule in our study. approach is to focus on the most active molecules from plants. Among them, polyphenols have been demonstrated to possess numerous health benefits such as antioxidation [3][4][5] , anti-inflammation 6,7 and antimicrobial activity 4 . The most studied compounds include in particular flavonoids, ubiquitous plant secondary metabolites, whose activity is, at least partly, associated with their chelation of transition metals ions 8 . Such a complexation can not only lead to increased solubility, hydrophilicity and bioavailability, but the metal complexes may also possess different biological properties than the ligand alone 7,9,10 . In particular, iron 7,[11][12][13] and copper 9,11,[14][15][16] playing a key role in living systems, their complexes with flavonoids have been widely studied 17 . Another class with high biological importance includes carboxylic polyphenols such as chlorogenic acids 5,18,19 , gallic acid 20 , rosmarinic acid 21 , ferulic acid 22 and ellagic acid 23 . Complexation with transition metals ions was also largely studied, in particular for caffeic acid derivatives [24][25][26] . In aqueous solutions at neutral pH polyphenolic molecules usually bind to the metal or alkali metal ion by one or more -OH (and-COO -, if present), usually resulting in the formation of weak mono-nuclear complexes 27 . However, their calcium complexes have been poorly characterized, despite the presence of this ion in biological fluids and therefore their possible relevance for pharmaceutical applications as antibacterial or antioxidant compounds. In this context, this study aimed at a detailed in vitro evaluation of calcium complexes of chlorogenic acid and rosmarinic acid (denoted biological active molecules (BAM) in the text) (Figure 2.2). spectroscopy. Effect of calcium complexation on antioxidant activity of both molecules was also assessed. Taken together, these data reveal the large diversity of possible complexation modes of calcium ions with these molecules and their noticeable dependence on pH. However, little if any variation of their biological activity could be evidenced. This suggests that, compared to other inorganic ions, interactions of calcium ions with these molecules should not play a major role in their antioxidant efficiency but may nevertheless be taken into account for their in vivo fate.

MIC (µg

Experimental

Chemicals

Chlorogenic acid (CA) and rosmarinic acid (RA) was purchased from Carbosynth Limited (Compton, Berkshire, UK). Calcium chloride dihydrate was obtained from Jessen Chemica. Phosphate buffer (PB) 0.1 M was prepared by mixing potassium phosphate monobasic and potassium phosphate dibasic at pH 7.2.

Spectroscopic and spectrometric measurements

The formation of complexes between the BAMs and calcium was investigated with several methods. UV-Visible spectroscopy was performed on a Libra Biochrom S60 spectrometer, between 250 and 600 nm. Electrospray ionization mass spectrometry (ESI-MS) studies were conducted on a LTQ orbitrap mass spectrometer, with a capillary voltage of 20 V and tube lens voltage of 70 V, in positive ion mode. Resonant excitation was carried out using collision-induced dissociation (CID), normalized collision energies (NCE) were between 5 and 30 %. All data analysis was performed using Thermo Xcalibur™ software. Samples were introduced at the rate of 20 µL.min -1 with a 500 µL syringe. Solutions were prepared in ammonium carbonate buffer 1 mM at pH 7.8, from stock solutions of BAM in methanol. Complexation of calcium with rosmarinic and chlorogenic acids was studied by 1H and 13C NMR, on a Bruker 300 MHz spectrometer equipped with a BBFO probe. Rosmarinic acid and chlorogenic acid were dissolved in D2O in the presence of calcium chloride with ratio BAM /Ca = 0.1, and pH was adjusted to 7 with NaOD 0.01 M.

Antioxidant activity assays

The scavenging activity on the stable free radical DPPH was assayed by the modified Blois' method in which the bleaching percentage of DPPH is monitored at a characteristic wavelength in presence of the sample Because of its sensitivity to the protonation state of catechol as well as to the influence of the presence of metal ions, UV-vis spectroscopy is a method of choice to study both free polyphenols and their complexes 1,12,15,28,29 .

The absorbance of carboxylic polyphenols recorded in the 250-600 nm range significantly varies with pH, as shown on 255 nm at pH 8. Evolution of absorption behavior of RA is consistent with previous reports by Danaf et al. 30 . The bathochromic shift visible when increasing pH from acidic to basic values was ascribed to the deprotonation of the hydroxyl on the position 3 of the caffeic acid part, whose pKa is reported to be at 8.36, based on potentiometric titration and Density Function Theory calculations. 30 Another clue for a relationship between the absorbance modification and deprotonation of a catechol hydroxyl group is the observed isobestic point at 271 nm and, at lower extent, at 340 nm suggesting that a unique reaction is occurring within this pH range. Noticeably no modification between absorption spectra at pH 3.8 and pH 5.1 are observed for CA, even though the pKa of the carboxylic acid was calculated at 3.90. This is in agreement with previous observations by Cornard et al. 29 and consistent with the position of the carboxylic group that it is carried by the quinic acid moiety and is not conjugated with the catechol ring. Similarly, for RA no difference could be observed on absorption spectra between pH 3 and pH 5, in accordance with the fact that its carboxylate group is not conjugated to the catechol rings.

Chapter 2. Molecular study: interactions between calcium and biologically-active molecules

As we intended to investigate the antioxidant properties of the BAM, the study of complexes formation between BAM and calcium was performed at pH 7.8, near physiological conditions. the two main bands at 300 nm and 323 nm rise in intensity while the shoulder at 380 nm remains unmodified. In the presence of a higher proportion of calcium (CA:Ca 2+ ratio 1:100), the intensity of these two bands does not evolve but the shoulder is less intense. After 24 h, the absolute and relative intensity of the two high-energy bands are modified while that of the low-energy shoulder has increased. Moreover, a new broad band also appears at ca. 600 nm. Quite similar observations prevail for RA, except that the 290 nm band intensity decreases to a limited extent only while the 365 nm shoulder varies more significantly with time.

From these data, one could hypothesize that calcium ions interact with CA and RA. Such interactions affect several bands of the UV-visible spectra, indicating that the chromophore part of the molecules, i.e. the caffeic group, is involved in the reaction. The decrease in the intensity of the bands in the 250-350 nm range and the increased intensity of the 390 nm band in the presence of calcium could indicate a deprotonation event. This, and the appearance of a new band at 600 nm, is therefore in favor of the formation of a BAM-Ca 2+ complex involving the catechol ring. However, because carboxylic acids of CA and RA are not conjugated to the caffeic moiety, their possible involvement in such a complexation cannot be put aside.

Electrospray mass spectrometry

Electrospray ionization mass spectrometry (ESI-MS) has previously shown to provide direct evidence for the formation of the complexes based upon the masses and fragmentation ions of the complex 11,31 .The presence of calcium-BAM complexes was thus investigated by ESI-MS in positive mode. Complementary studies were conducted at pH 5 in ultrapure water allowed the detection of additional peaks -compared to that identified at pH 7-that can be assigned to BAM/Ca 2+ complexes (Figure 2 Moreover, in acidic conditions, a much wider range of complexes are formed. Noticeably, monomolecular complexes have a similar stability for the two BAMs, which would suggest that the catechol ring, that is present on both CA and RA, are mainly involved in their formation. In contrast, the stability of the bimolecular complexes is very different for the two BAMs, which would suggest that carboxylate groups are also involved in Ca 2+ coordination.

NMR spectroscopy

NMR spectroscopy was performed in order to gather more information on potential complexation site between BAM and calcium 13,17,32 . We studied solutions of calcium and BAM with a ratio BAM:Ca = 0.1, as this is the ratio where effects of complexation are visible on the UV-Vis absorption spectra. 1 H and 13 C NMR spectra of CA and RA without or with calcium were recorded in D2O at pH 7, and corresponding chemical shifts were assigned in comparison with literature [33][34][35] .

For CA, H2 was the only proton not affected by the presence of calcium (Figure 2 (1.06 ppm) of C4 is a sign for the complexation around the carboxylate, that we can assume to be between the hydroxyl group of C4 and hydroxyl group of C8 (4-hydroxy-8-hydroxy site), which is consistent with the fact that this site is commonly mentioned as a site of complexation for metals on CA 36 .

It is noteworthy that intensity of both C20 and C19 as well as C8 signals were strongly decreased, which could sign for the complexation on the catechol or on the carboxylate groups.

However, such modifications could also be induced by some relaxation issues, so that they cannot unequivocally be assigned to a complexation process. that could be explained by a mesomeric effect, and to a lower extent to the shift of the vinylic carbons.

Some modifications of the C3-C4 catechol signal was also observed, but to a lesser degree (only 0.06 ppm shift). Finally, a 0.2 ppm shift of C14 signal was observed, which could be an indication the carboxylate group could also be involved in the complexation, although this hypothesis is not confirmed by 1 H NMR results. Altogether, NMR studies suggest that both catechol and carboxylate groups may act as binding sites for calcium for both BAM at pH 7, although the former seems highly favored for RA.

Effect of complexation upon antioxidant activity

The effect of calcium chelation on antioxidant activity was assessed according to the DPPH radical scavenging method (Figure 2.9), for 0.1 molar ratio (BAM/Ca). Rosmarinic acid is the best antioxidant molecule, with a DPPH bleaching percentage of 27 %, followed by chlorogenic acid (13.6%).

The presence of calcium does not affect the antioxidant activity of chlorogenic acid, while the DPPH bleaching percentage is decreased by 29 % for RA. Antioxidant activity is recognized in the literature 37 to arise from the oxidation potential of hydroxyl groups, namely the catechol ones for RA. Therefore, the effect of the presence of calcium on the antioxidant activity of RA designates the catechol as the preferential site for the complexation. Conversely, as there is no effect of the complexation on the antioxidant activity for chlorogenic acid, it could be assumed that the catechol group is less involved in complexation for CA and thus that the carboxylate could be the preferred site of complexation.

Discussion

UV-visible spectroscopy analysis of chlorogenic acid and rosmarinic acid in the presence of calcium ions near neutral pH have evidenced modification of the intensity of several absorption bands and appearance of an additional one, suggesting that complexation does occur and is correlated with partial deprotonation of the catechol ring. However, because the carboxylate groups of both BAMs are not conjugated with the caffeic moiety, this technique did not allow us to conclude on the possible involvement of these groups in the complexation reaction. MS studies confirmed that both molecules can form mononuclear complexes with calcium near neutral pH and evidenced that, at pH 5, multimolecular complexes could also be obtained. The difference in stability of the bimolecular complexes formed with CA and RA suggested that carboxylate groups could also be involved in calcium complexation. Finally, 1 H and 13 C NMR indicated that both catechol and carboxylate groups could interact with calcium at pH 7, although the later appears significantly more efficient in CA than in RA.

In the case of chlorogenic acid, Cornard et al. have shown that carboxylate is first involved in the complexation of Pb 2+ ions, followed by the catechol 29 . For quinic acid, i.e. one of the structural moiety of chlorogenic acid, it is known that two polyol sites : the 1-hydroxy-6-hydroxy and the 4hydroxy-8-hydroxy 36 CO and CO2, i.e. the loss of the carboxylate (Figure 2.12), suggesting that the calcium is not complexed by the carboxylate. Chelation with a stoichiometry 1-1 would therefore occur preferentially on the catechol C19-C20 (on the caffeic part of RA), as it is the one with the lower pKa (Figure 2.13 (c)).

Moreover, this complex is the only one that can be formed at pH 7.8, and NMR studies at this pH show that the complexation is more likely to occur on this site. Indeed, all peaks for protons corresponding to one or the other aromatic cycle are shifted, but those of carbons C19-C20 are more shifted than C3-C5. 13 C NMR study also reveals that the carboxylate is involved at this pH, with the shift of C14 peak.

From UV-Vis absorption studies at pH 7.8 in presence of calcium we know that calcium complexation induces a modification of the spectrum similar to deprotonation, which is less important at high calcium dosages. In conclusion, the complexation at pH 7.

Conclusion

The ability of two natural polyphenols, chlorogenic acid and rosmarinc acid to chelate calcium ions and the impact of such a complexation on their anti-oxidant activity was studied. Complexation for their antibacterial and antioxidant activity. However, several papers 38,39 mentioned their degradation in vivo, supposedly via an oxidation process, by analogy with the well-characterized oxidation of others flavonoids (quercetin, fisetin) [40][41][42] . The final product of degradation of both molecules has not been yet undoubtedly identified. Nevertheless, the degradation was found to be very sensitive to pH, temperature and presence of other flavonoids in solution. In this chapter, we will study the fate of BA and BE at pH 5 and pH 7.8. UV-Vis spectroscopy was used, because both molecules are two colored molecules. We also performed electrospray mass spectrometry (ESI-MS) to determine the molecular mass of the product in solution as well as their potential product of degradation. We were able to propose a scheme for the degradation and oxidation of baicalin and baicalein. With the oxidation of BE and BA in mind, we studied their interaction with calcium at pH 5 and pH 7.8, with the same characterization techniques. The effect of interaction with calcium on the biological activity of baicalein and baicalin was also investigated.

Stability of baicalin and baicalein at different pH

UV-visible spectroscopy

As the literature reports that baicalin and baicalein are likely to undergo degradation in aqueous solution 38 , their stability was assayed over time in phosphate buffer at pH 7.2. Neutral pH was chosen because it is representative of the physiological pH, i.e. conditions where BA and BE biological activity is relevant. In freshly-prepared solutions, both BA and BE UV-visible spectra (Figure 2.15)

exhibit two main bands positioned at 275 and 316 nm, and 271 and 340 nm, respectively, in accordance with literature data. Indeed, flavones are reported to show two major absorption bands in the UVvisible region 1,12,43 , band I and II. Band I, in the range of 300-400 nm, arises from the absorption of Bring conjugated with carbonyl of ring C (cinnamoyl system, see Figure 2.15 (c)). Band II, between 240-300 nm, is related to conjugated system between ring A and carbonyl of ring C (benzoyl system, see The evolution of UV-Vis spectra of BA and BE were recorded more systematically as function of pH. For baicalin (Figure 2.17 Deprotonation of the carboxylic acid of BA is not visible on the UV-Vis absorption spectra, although its pKa was calculated at 3.8. This is consistent with the assumption that the glycosidic part of BA is not conjugated with rings A and C.

We have to be very careful with the interpretation of the evolution of UV-Vis spectra as a function of pH because of the degradation of both molecules at pH ≈ 7. For baicalin, we know that the degradation starts after 4 hours at neutral pH. Consequently, we can assume that spectra recorded within 1 hour at pH 5 and 7.2 can be compared on the basis of pH evolution. In case of baicalein however, the degradation takes place before 4 hours. It is therefore difficult to assign the evolution of the UV-Vis spectrum only to the rise of the pH. We are probably looking at two events at the same time, the evolution due to pH, and the degradation of the molecule.

To conclude, baicalin and baicalein are both unstable in aqueous solution, with different kinetics. Baicalin is stable at acidic pH, and starts to be degraded after 4 hours at pH ≈ 7. Baicalein is degraded at neutral pH within 1 hour, while at pH 5 the degradation has been evidenced only 6 hours after solubilization of the molecule. We have thus to be careful while studying events involving baicalein because of its quick degradation in aqueous medium. Further experiments as a function of pH were consequently performed with solutions prepared for less than one hour.

Mass spectrometry

The degradation of baicalein was studied by electrospray mass spectrometry (ESI-MS) in positive mode, at pH 5 in ultrapure water and pH 7.8 in ammonium bicarbonate buffer (NH4)2(HCO3).

Baicalein was solubilized at 0.01 mol.L -1 in methanol before the dilution in water or buffer at 50 µM.

ESI-MS experimental parameters were the same than presented in paragraph 2.4.2.2. Mass spectra were recorded shortly after preparation of BE solution or 20 hours later. In the case of BA (Figure 2.21 (a)), the presence of Ca 2+ at pH 7.8 does not modify the UVvisible spectrum shortly after the addition of calcium, whatever the BA/Ca ratio tested. After 6 h, spectra are very similar to the one obtained for pure baicalin at pH 10 without calcium, with a drop of the intensity of main absorption band. However, this effect cannot be related to calcium complexation, as the evolution is the same for pure baicalin and baicalin in presence of calcium whatever the BA/Ca ratio. Given the evolution of UV-Vis spectrum of BA at pH 7.8 after 8 hours, we can assume that the degradation is pH-dependent, but also buffer-dependent. Indeed, the degradation at pH 7.2 in phosphate buffer involves only a shift of band II near 270 nm within 8 hours (see Figure 2.15 (a)). In ammonium bicarbonate buffer at pH 7.8, the degradation is quicker, occurring within 6 hours, with a more important modification of the UV-Vis spectrum. The degradation is similar to a deprotonation, which could be related to an event at the catechol group in ring A.

For BE (Figure 2.21 (b)), there is a clear effect of presence of calcium immediately after its addition with a red-shift of the 270 nm peak and the emergence of the 420 nm band that are both enhanced as the BAM:Ca decreases, suggesting the transitory formation of specific complexes. After 6 h, no further band II shift is observed but the overall spectra intensity decreases while a shoulder appears at 293 nm. This modification is not dose-dependent as the spectra are identical whatever the BAM/calcium ratio tested mixtures. Given the degradation of BE at pH 7.2 in phosphate buffer (Figure 2.15 (b)), after 6 hours, we are probably only looking at the degradation of the molecule, with no effect of the presence of calcium. We can also notice that the spectrum of the degraded molecule is similar to the one of BE at pH 10. Degradation has the same effect than deprotonation, suggesting an event at the catechol site.

To conclude, we tried to observe a complexation between calcium and BE or BA at pH 7.8 in ammonium carbonate buffer. As for baicalin, no effect of interaction with calcium was visible, neither at short-term nor at long-term. After 6 hours, the only event is the degradation of the molecule that we assumed to be quicker at pH 7.8 in ammonium carbonate buffer than in phosphate buffer at pH 7.2. For baicalein, a modification of the UV-Vis spectrum on the short-term for molar ratio BE/Ca 1:10 and 1:100 suggests an interaction with calcium. However, after 6 hours, the degradation of the molecule overwhelmed any interaction with calcium. For both molecules, the degradation indices the same modification of the UV-Vis spectrum than the one observed when increasing pH, thzt was interpreted as a deprotonation. For baicalin, in addition to the peak of the protonated molecule at m/z 447.10, two peaks are observed, at m/z 466.07 with z = 2 and at m/z 485.04 with z = 1. The first peak corresponds to a complex

[2BA+Ca] 2+ with a 2 : 1 stoechiometry (BA:Ca) and the second to a complex [2BA-2H+2Ca] + with a 2 :

2 stoechiometry (BA:Ca). This latter complex could be formed by two deprotonated baicalin, or by a fully protonated baicalin and an oxidized form of the molecule, as it was observed for baicalein.

For baicalein, the main peak is the protonated molecule at m/z 271.06, as observed before.

Another peak is observed at m/z 299.04 wih z = 2. This peak corresponds to the adduct [2BE+Ca+H2O] 2+ .

Overall, both baicalin and baicalein are able to form complexes with calcium at pH 5. Stability of complexes was estimated by the calculation of CE50, the necessary energy to dissociate 50 % of the complex. For BA, an intermediate value is obtained for [2BA+Ca] 2+ (9.0 eV) whereas the complex formed by two deprotonated BA (or one BA and one BAox) and two calcium ions [2BA+2Ca-2H] + was found more stable (11.4 eV). Note that such a calculation was not possible for [2BE+Ca+H2O] 2+ as this complex was detected in very low amount and revealed to be highly unstable. To conclude, the complexation with calcium is pH-dependent and varies with the oxidation of BA and BE. At pH 5, BA chelates calcium with a ratio 2:1 and 2:2, with the 2:2 complex being more stable than the 2:1. The 2:2 complex may be constituted by two deprotonated BA or by one fullyprotonated BA and one oxidized BA (see Figure 2.24). For BE, a 2:1 hydrated complex is formed. At pH 7.8, the oxidation process overwhelmed the complexation with calcium. Oxidized BE is not able to form chelate calcium, and only one complex with oxidized BA is formed. However, oxidized BA is unstable and degradation products of BA have been detected by mass-spectroscopy. It is stabilized by adducts with water or calcium. The oxidation of BA was also observed by UV-Vis spectroscopy at pH 7.8, but

we were not able to detect any possible complexation in the presence of calcium with this method.

Complexation site could thus take place on the carboxylate that is not in the conjugated part of the molecule. On the contrary we observed a sign of interaction with calcium in the UV-Vis spectrum of BE while nothing is detected by mass spectrometry. Chapter 2. Molecular study: interactions between calcium and biologically-active molecules 104

Effect of complexation on the biological activity

The effect of the presence of calcium on the antioxidant and antibacterial activities of baicalin and baicalein was finally studied Nevertheless, the MIC of BA is decreased by 8-fold against S. epidermidis. Thus, it appears that the presence of calcium does not affect, or slightly positively affects the antimicrobial activity of the flavonoids. This result is encouraging from the perspective of using the product as an antibacterial agent in a living organism, therefore in the presence of calcium

Conclusions

Baicalin and baicalein are two flavonoids, with two main sites for calcium complexation, the catechol on the A-ring and the 5-hydroxy-4-keto site. In addition, baicalin is a glucuronic acid derivative, so the carboxylate group on the sugar ring is also a potential site for calcium binding.

Interaction between baicalin and calcium is pH-dependent. At pH 5, ESI-MS studies revealed the presence of two complex between calcium and the molecule, [2BA+Ca] 2+ and [2BA-2H+2Ca] + (constituted by two deprotonated BA or one BA and one oxidized BA). At pH 7.8, none of these complexes are formed, but the complex [BAox-H+Ca] + , i.e. a complex between the oxidized molecule and the calcium, is detected, besides the non-oxidized non-complexed molecule. Oxidized BA coexists with non-oxidized BA, but only when stabilized by water or calcium; otherwise it is degraded in BEox.

We can assume that complexation with calcium occurs immediately after oxidation; otherwise a peak corresponding to BAox would be detected. No effect of complexation is visible on UV-Vis absorption spectra, which can be explained by considering that complexation occurs at the carboxylate site, which is not conjugated with the chromophore part of the molecule. Concerning the biological activities of BA, its antioxidant activity is only affected by the oxidation and not the complexation with calcium.

Besides the decrease of MIC of BA against S. epidermidis, complexation of calcium does not affect the antibacterial activity of BA.

Baicalein, which is the aglycone of baicalin, oxidizes faster than baicalin and at a lower pH. At pH 5, the oxidized molecule is detected, as well as a [2BE+Ca+H2O] 2+ complex in a very small quantity compare to the native molecule. As complexation occurs with both baicalin and baicalein at this pH, we can assume that the catechol and 5-hydroxy-4-keto sites are also the preferred complexation sites for baicalin at this pH. At pH 7.8, the entire molecule is oxidized, as shown by ESI-MS and UV-Vis absorption experiments. A small effect of calcium complexation is visible in the first times of UV-Vis studies, but no complex is detected by mass spectrometry. As soon as the complex is formed, baicalein oxidizes, and the calcium leaves the molecule, so no complex can be detected by mass spectrometry.

It therefore seems that the oxidation of the complex is in competition with the oxidation of the noncomplexed molecule, the former being faster. The absence of complex at pH 7.8 with BE supports our previous assumption that, for baicalin at this pH, calcium interacts with the carboxylate moiety. Finally, there is no effect of the presence of calcium on the antioxidant activity, as the molecule spontaneously oxidized in the medium, even without calcium. There is no significant effect of calcium on the antibacterial activity of BE, except a 4-fold decrease of the MIC against S. aureus.

Conclusions

In this section, we aimed at investigate the fate of baicalin and baicalein at different pH, and their interaction with calcium at neutral pH. The degradation of both molecules has already been mentioned, but not deeply investigated. Literature only focused on how to avoid this degradation, and oxidation was only suggested. Here, we show that baicalin is oxidized and hydrolyzed in baicalein at pH 7.8, but remains stable at pH 5. On the contrary, baicalein is more unstable, and is oxidized at pH 5. The oxidation was proven by ESI-MS, with the loss of two protons. Both BE and BA have a weak antioxidant activity at neutral pH, because they already fully oxidized. On the contrary, their antibacterial activity does not vary with oxidation. We can even assumed that the active molecule is the oxidized one in the case of BE, because the oxidation of the molecule is almost immediate at physiological pH. Moreover, the antibacterial assays are performed at 37 °C, which probably increases the kinetics of the reaction. The calcium complexation is pH-dependent. At pH 5, both BE and BA may chelate calcium, but at pH 7.8 complexation is hindered by the oxidation reaction. Oxidized BE cannot chelate calcium, and only a small fraction of oxidized BA form a complex. For BA, oxidation overwhelms the complexation because there is no trace of a complex with non-oxidized BA while there is still a peak of non-oxidized BA alone. Given the information of complexation of both molecules at pH 5 and 7.8, we can assumed that at pH 5, the complexation site is on the catechol or the 5-hydroxy-4-keto site, because both molecules chelate calcium. After oxidation, only BA can form complex, so the site of complexation is more likely the carboxylate, as it is absent on BE that does not form any complex.

Calcium complexation does not have an important effect on the antibacterial activity of baicalin and baicalein. The presence of calcium does not interfere with the biological activity of both molecules, which is encouraging for their utilization in vivo as potent antibacterial agents.

General conclusions

In this chapter, we decided to study four biologically active molecules, chosen for their antibacterial and antioxidant activities. Chlorogenic and rosmarinic acids are two polyphenols that share the same caffeic acid moiety. As for baicalin and baicalein, they are two flavonoids, baicalein being the aglycone form of baicalin. Among the four molecules, baicalein was characterized to be the best antibacterial agent, while rosmarinic acid is the best antioxidant molecule. The interaction of the four molecules with calcium was investigated by UV-Vis spectroscopy and mass spectrometry. For chlorogenic and rosmarinic acid, complexation was found highly pH-dependent, with monomolecular complexes formed at neutral pH and bimolecular complexes at acidic pH. Monomolecular complexes formed with deprotonated molecules possess the best stability. We tried to determine the site of complexation of calcium by NMR, but we were not able to discriminate between catechol and carboxylate groups.

Finally, the effect of calcium complexation on the antioxidant activity was studied, but only a slight decrease of the activity of rosmarinic acid was observed. For baicalin and baicalein, we were first concerned by their degradation at physiological pH, as it was already mentioned in the literature. We highlighted the oxidation of both molecules, with different kinetics of degradation. BE is more unstable than BA, it oxidizes at pH 5, while BA only starts to oxidize at pH 7.2. The kinetics of degradation is pHdependent and medium-dependent. Given the oxidation of the molecules, we can assume that the active form of baicalein is the oxidized one. The complexation with calcium was investigated, knowing that both molecules are degraded at neutral pH. Complexation is possible at pH 5, but in competition with the oxidation at pH 7.8. Indeed, oxidized BE cannot chelate calcium, while the presence of calcium allows the stabilization of oxidized BA that is otherwise degraded in oxidized BE.

To conclude, we studied the complexation of chlorogenic and rosmarinic acid with calcium, as well as its effect on the antioxidant activity of both molecules. The oxidation and degradation of baicalin and baicalein, and their interaction with calcium were investigated depending on pH. Complexation with calcium should play a role for the in vivo fate of the molecules, but the effect on their biological activity was not marked. As we want to associate these molecules in hydroxyapatite, knowing their interaction with calcium could be useful to understand their behavior towards the targeted material. In the next chapter, we will first focus on the preparation of biologically active molecules-hydroxyapatite hybrid nanoparticles.

Introduction

In the previous chapter, we evidenced that the four natural biologically active molecule (BAM), rosmarinic acid, chlorogenic acid, baicalin and baicalein could interact with calcium at different levels.

Rosmarinic and chlorogenic acid formed monomolecular complexes at both pH 5 and 7.8, while the oxidation of baicalin overwhelmed the complexation at neutral pH. Baicalein was the molecule with the lowest affinity for Ca 2+ . Keeping these informations in mind, we here tried to associate these molecules with hydroxyapatite nanoparticles in order to achieve their controlled release in a biological medium. Two strategies were considered, adsorption on pre-formed hydroxyapatite nanoparticles or incorporation of BAMs in the reaction medium during hydroxyapatite synthesis. Considering the size of BAMs compared to the crystal lattice of hydroxyapatite, in both cases BAM would be adsorbed on the surface of HAp particles, and/or in the interparticulate porosity. In the first section, the synthesis and characterization of hydroxyapatite nanoparticles will be described, before reporting the investigation of the two methods for incorporating the molecules in the material. Then, the antibacterial and antioxidant properties of hydroxyapatite nanoparticles incorporating baicalein will be studied.

Hydroxyapatite synthesis

Hydroxyapatite was synthesized by co-precipitation following the neutralization method 1 . Briefly, 0.1 mol of Ca(OH)2 (7.4 g) was dissolved in 100 mL of distilled water during 1h30, and 0.06 mol (6.9 g) of NH4H2PO4 was dissolved in 50 mL of water. Ammonium dihydrogenophosphate solution was added to calcium solution, and the medium was left to react for 48 h at 25 °C under stirring. Temperature was controlled by a thermostat bath. After reaction, white particles were recovered by centrifugation at 5000 rpm for 5 minutes, followed by three washing with distilled water. The powder was dried in a oven at 100 °C for 24 hours. Particles were characterized by X-ray powder diffraction (XRD) and Transmission Electron Microscopy (TEM). N2 physisorption was used to determine the specific area of the powder and its porosity.

The yield of synthesis, as determined by weighting the final powder, was 60 % assuming that only hydroxyapatite was formed. The X-ray diffractogramm (Figure 3.1 (a)) confirmed this hypothesis, by comparison with the PDF card 00-064-0738. TEM image as seen in Figure 3.1 (b) showed plate-like nanoparticles 31±6 nm in length and 9±3 nm in width. Specific surface area calculated with the BET method was 160 m 2 .g -1 . The average pore diameter of the powder, corresponding to interparticle space, was 15 nm (see the isotherm of N2 physisorption on Crystallite size may be calculated using Debye-Scherrer's formula (Equation 3.1). We used the peak at 26 °, corresponding to the plan (002) due to its adequate resolution and loss of interference.

D = K × λ β × cos (θ) Equation 3.1 Scherrer's formula

In the formula, D is the crystallite size, K is a dimensionless shape factor with a typical value of 0.9, λ is the X-ray wavelength, in our case 1.54 Å (Cu K wavelength), β is the full width at half maximum (radians), and θ is the diffraction angle of the corresponding reflection (°). Using this formula, calculated crystallite size for HAp was 21±2 nm.

Incorporation of BAMs

Post synthesis adsorption

As previously mentioned, BAM were selected in particular for their carboxylic acid and catechol functions, presumed to interact with the hydroxyapatite surface. In a first approach, a two-step method was used to synthesize the hybrid particles.

In this approach, 10 mg of ground HAp particles were re-suspended in a 200 mL solution of BAM in distilled water (pH ≈ 5). The BAM concentration was set to 0.05 mM as it is near the limit of solubility of BA and BE, corresponding to an initial molar ratio between Ca and BAM of 1. After 24 hours of contact under stirring at room temperature, particles were recovered by filtration, and dried at 100 °C for 24 hours. The quantity of adsorbed BAM was estimated by TGA and/or UV-Visible spectroscopy. For the UV-Vis method, absorbance of the solution was checked before and after adsorption, and the difference between the two values was used to estimate the adsorbed quantity.

Morphology of particles after adsorption was also observed with TEM. Incorporated quantity measured by TGA and UV-Vis spectroscopy, expressed in mg of incorporated molecule by g of HAp, and particle size as obtained from TEM are gathered in Table 3 which modifies the UV-vis spectrum and therefore induces an error in the estimation.

The variation of pH at the beginning and end of the adsorption process was also monitored (Table 3.2). In all situations, the initial mixture is acidic and the pH is higher after adsorption, close to neutrality. As seen in the TEM images presented in Figure 3.2 adsorption of BAM did not modify the platelet-shape aspect of HAp particles. Determination of particle width and length by image analysis confirmed that adsorption did not significantly modify particles dimension (Table 3.

HAp

1).

As seen in Figure 3.3, X-ray diffractograms after adsorption are similar to pure HAp, confirming TEM observations. The crystallite size, calculated and gathered in Table 3.1 are similar to the one of HAp before adsorption. The background noise is higher for particles after adsorption because the quantity of powder used for the measurement was very small. We can note two remarkable peaks on the diffractogram of HAp + adsorbed RA, at 18 ° and 24 °. The peak at 18 ° deserves our attention. We assigned it to Ca(OH)2, but we cannot give an explanation to the presence of calcium hydroxide in these conditions. This must be checked with another batch of hydroxyapatite after adsorption. The peak at HAp-BA. However, the peak is dissymmetrical, with a more intense part above 170 ppm, which would suggest a type B substitution 4 . In HAp lattice, carbonates can substitute the hydroxyl, which is called an A-type substitution, or the phosphate that is called a B-type substitution. In addition, an intense peak at approximatively 120 ppm is seen on the HAp-BE spectrum, and, to lesser extent, on the one of HAp-BA. This peak corresponds to aromatic carbons of baicalein and baicalin. As NMR is not a very sensitive characterization method, only adsorbed baicalein and baicalin are detected because they are Chapter 3. Formation of hybrid hydroxyapatite -biologically active molecules nanoparticles 123 adsorbed in a larger quantity than CA. These data also confirm the higher affinity of BE for HAp compared to BA and CA. A 2D correlation map between 1 H and 31 P of HAp-BE was recorded. A three units sequence was used, with 1-a saturation on 31 P to remove noise coming from dephasing, 2-an Hahn echo to filter the contribution of amorphous OH in 1 H spectrum and 3-acquisition 31 P with cross-polarization. During a cross-polarization experiment, 1 H is magnetized, before contact with 31 P during the contact time, and then spectrum is acquired on 31 P. We tested four contact times (p15) for cross-polarization, and we decided to focus on the 10 ms condition (Figure 3.7 (a)). The correlation between crystalline 31 P and aromatic 1 H is visible at 2.7 ppm for 31 P and 7.1 ppm for 1H. This correlation is the sign of the adsorption of BE on HAp. The same correlation map was recorded for pure HAp as reference (Figure 3.7 (b)). The only correlation visible is at 2.7 ppm for 31P and 0 ppm for 1H, which corresponds to the correlation between OH in apatitic structure and crystalline phosphorous, that is also visible on HAp-BE spectrum.

In situ synthesis

The second attempt to incorporate the four natural BAM in hydroxyapatite was by incorporation during synthesis. The protocol was the same as for pure hydroxyapatite, except that 100 times smaller concentrations of inorganic precursors were used, molecules were added in the calcium hydroxide solution and the solution left under stirring for 1 h 30 before adding the dihydrogenophosphate solution. Two ratios between calcium and BAMs were studied, 0.0075 and 0.075 (BAM : calcium), except for baicalin for which the highest ratio was 0.04 due to solubility issues.

Colored particles were obtained, with color depending on the incorporated molecule. The crystalline phase of the precipitated powder was first checked by XRD. The morphology of the synthesized From XRD, it is possible to evidence the formation of hydroxyapatite in all conditions (Figure

3.8).

The calculated crystallite size of pure hydroxyapatite is 15 nm and is not significantly modified in the presence of BAMs at this concentration. It is also important to notice that in the low 2 domain, diffractograms of HAp-RA and HAp-BE show an additional peak at ca. 2 = 18°. This peak may correspond to the (001) diffraction peak of Ca(OH)2, which is the most intense for this structure. This peak is not observed for BA and CA.

In parallel, the yield of the hydroxyapatite formation decreases with the incorporation of the BAM, from 90 % for pure HAp, to 50 ± 5 % in the presence of BAMs (Table 3.4). Therefore, the presence of BAMs seems to inhibit the formation of HAp. The amount of incorporated BAM is higher than in the case of adsorption, except for bacailein. However, from the measure of the incorporated quantity, we can calculate the final molar ratio between BAM and Ca in the hybrid material. It is 0.011 for HAp-CA, 0.015 for HAp-RA, 0.02 for HAp-BE and 0.01 for HAp-BA. The highest ratio is again obtained for BE, as observed in the adsorption process. Thus, there is an enrichment in BAM within the material compared to the initial molar ratio in solution (0.0075).

TEM images of HAp-CA and HAp-BA (Figure 3.9 (b) and (d)) show platelet-shaped particles, similar to pure HAp, but apparently more aggregated. HAp-RA and HAp-BE have a less defined shape.

The particles in the four samples are significantly smaller (Table 3.3) than pure hydroxyapatite, which is consistent with crystallite size calculated from XRD data. However, while crystallite size varied only in a non-significant amount, size variation as measured from TEM evidence a noticeable difference between HAp-RA and HAp-CA, and HAp-BA and HAp-BE.

Altogether, BAMs at low concentration decrease the amount of formed hydroxyapatite nanoparticles and the crystallite size, these two parameters being apparently correlated (i.e. lowest yields (RA, BA) are obtained for the smallest particles). The amount of incorporated BAM can also vary, but clear correlation with the other studied factors is not straightforward: for instance BE leads to the highest BAM:Ca ratio but the yield is not very different from other BAMs. Importantly XRD suggests that other phases are present in the final powder, and especially Ca(OH)2. The synthesis with rosmarinic acid was difficult to handle. After washing, almost no powder was collected. Synthesized particles seemed to redissolve in distilled water, so it was impossible to calculate the yield of the reaction and to measure the incorporated quantity of RA by TGA. In order to recover few mg of powder to perform XRD, the washing step was skipped.

Chapter 3. Formation of hybrid hydroxyapatite -biologically active molecules nanoparticles

129 XRD patterns presented in Figure 3.10 showed the presence of hydroxyapatite for HAp-BE, HAp-CA and HAp-BA. Calculated crystallite sizes are similar to the one calculated for HAp particles synthesized in the presence of BAMs with the molar ratio 0.0075, i.e around 15 nm. In presence of RA, the diffractogramm is not the one of hydroxyapatite, therefore we decided to name this compound CaP(for calcium phosphate)-RA. However, after comparison with database, it could be assigned to the calcium carbonate (CaCO3) calcite phase. As a matter of fact, the main diffraction peak of calcite at ca.

29° is also visible on the diffractogramm of HAp-BE, and to a lesser extent in the one of HAp-CA but not with BA. The presence of calcite would explain the redissolution of CaP-RA in water during washing, as calcite is quite soluble in water near neutral pH. Therefore HAp formation seems totally inhibited by the presence of rosmarinic acid. For CA and BE incorporating particles, we have noticed that the apparent yield was lower than with the ratio 0.0075. Thus, a larger quantity of BAM seemed to inhibit HAp formation more. In parallel, incorporated quantity of BAMs was higher, with 73 mg.g -1 of incorporated CA and 135 mg.g -1 of incorporated BE. Yet, whereas the initial solutions were 10 times more concentrated in BAM, the final incorporated quantity is not 10 times higher than the quantity incorporated when the BAM: Ca ratio was 0.0075. The final loadings were at least twice that obtained at the lower ratio but smaller than the initial solution content. Again, BE was the most efficiently incorporated molecule.

Overall, the synthesis of hydroxyapatite in presence of BAM is possible for low molecular ratio between BAM and calcium, even if, when synthesized in the presence of organic molecules, the yield of the reaction decreases and HAp crystals are smaller. At higher ratio, the synthesis is totally inhibited in presence of rosmarinic acid while, despite a stronger effect compared to lower ratio, HAp is still obtained for the other BAMs Variations in yields, as calculated from the weight of recovered powder, must be taken carefully as other phases, Ca(OH)2 at low molar ratio and CaCO3 at high ratio, were also evidenced.

These phases may also be taken into account when calculating the amount of BAM per g of hydroxyapatite. However, it can at least be stated that bacalein is the most efficiently retained BAM in the final product.

Comparison of incorporation methods

We developed two methods to associate BAMs with HAp, either by post-synthesis adsorption or incorporation during synthesis. The first method leads to adsorption of all BAMs without significant modification of the HAp nanoparticles structure, with highest loading being obtained for BE. The second method allows to obtain hybrid nanoparticles for all BAMs and two sets of concentrations, except for RA at the highest BAM:Ca ratio. However, the reaction yield of hydroxyapatite formation is decreased compared to the one of pure HAP synthesis and other phases are recovered in almost all samples.

Adsorption method

Because the underlying mechanisms are expected to be simpler, we can first discuss the second method. Adsorption of biologically active molecules on hydroxyapatite have already been reported in the literature, for delivering antibacterial agents or osteogenic components. For instance, antibiotics such as gentamicin, tobramycin, or gentamicin have been efficiently adsorbed on hydroxyapatite nanoparticles or coatings [5][6][7][8][9] . Others examples are anticancer 10 , anti-inflammatory 11 or hemostatic 12 molecules. Overall, three kind of interactions can be expected between the molecules and the surface: (1) electrostatic, (2) hydrophilic/hydrophobic and (3) complexation with the calcium ions. The first two ones are highly correlated to the surface charge of HAp: at pH below its isoelectric point (IEP), the surface is positively-charged and has good affinity for anionic species, while the mirror situation occurs at pH above IEP. Near the IEP, the HAp surface has a marked hydrophobic character.

The IEP of hydroxyapatite can range between 7 and 8, being therefore positively-charged in the sorption conditions in this work. We measured the pKa of the carboxylic acid of RA, CA and BA by titration with NaOH. For the three molecules, carboxylic acid is deprotonated at pH 5 in distilled water, suggesting that attractive electrostatic interactions can drive their adsorption on the HAp surface.

Surprisingly, baicalein appears to have the strongest affinity for HAp, although being neutral at pH 5.

Moreover, it did not show any interaction with calcium ion in our previous molecular study (see Chapter 2). This result can be interpreted in light of a study by Chirdon et al. 13 that showed that catechol and pyrogallol adsorbed more readily and strongly on HAp surfaces than phenol and carboxylic acids. The proposed explanation is based on cooperative hydrogen-bond interactions thanks to the presence of two or three close phenol groups. The fact that BA is less efficiently adsorbed than BE evidence that the loss of one available OH group on the pyrogallol ring is not compensated by the presence of an additional carboxylate group on the sugar moiety. Accordingly, the catechol ring present on CA and RA is less efficient than the pyrogallol ring of BE to interact with the HAp surface.

Noticeably, it seems that the ability of the BAM to form complexes with Ca 2+ is not relevant to explain the results obtained by the adsorption process. As discussed above, the main reason is that other interactions are also driving the BAM affinity for the HAp surface. Another point to consider is that the coordination sphere of calcium ions within HAp is already occupied by phosphate and hydroxyl groups, limiting its chelation by the BAM ligands.

In situ method

In contrast, the formation of BAM-Ca 2+ could be expected to be relevant for the first in situ method since it involves a preliminary step where Ca(OH)2 is dissolved in the presence of the bioactive molecules. However, two points are to be considered. The first one is related to the pH conditions that are highly alkaline (pH 12). On the one hand, such conditions should favor highly deprotonated forms of the molecules, which should enhance their interaction with calcium ions. On the other hand, experiments described in Chapter 2 made us expect that some degradation of the BAM could occur in these pH conditions.

To check this hypothesis, we investigated the stability of the BAMs at pH 12 by UV-Vis spectroscopy (Figure 3.12). It is clear that the spectrum at pH 12 for the four molecules is different from the one at pH 5 (see Chapter 2), and that spectra evolve with time. For BE and BA, we assume that oxidation is the privileged degradation pathways, as it was described in Chapter 2 for degradation of these molecules at pH 7. As for RA and CA, we assume that the ester function would be hydrolyzed.

Therefore, it is very likely that the structure of BAMs involved in the HAp growth process is different from the one investigated in Chapter 2 during studies in solution, and that the identified complexes are not fully relevant to understand their influence on the in situ process. The second point to consider is that BAM may also influence Ca(OH)2 dissolution and/or favor CaCO3 formation. Indeed, for RA, the Ca(OH)2 diffraction peak observed on particles synthesized with the ration 0.0075 is the highest and CaCO3 formation is the most favored when ratio is 0.075, while for BA, no Ca(OH)2 peak is observed at low ratio and no CaCO3 is found at high ratio.

To understand better this phenomenon, some additional experiments were performed. First, the evolution of the Ca(OH)2 solution in the absence or presence of RA was followed by XRD measurements. In the first case, after 15 minutes, a very low amount of powder could be recovered, suggesting fast dissolution of calcium hydroxide. After 30 minutes, the solid content of the solution was much more significant and XRD indicate that it consists of pure CaCO3. Formation of calcite in these conditions is very favorable and already reported in the literature. A similar evolution was found One important information is that the crystallite sizes are similar for HAp and HAp-CA/BE/BA, while all BAMs induce a decrease in particle size, as estimated on TEM images. If we make the hypothesis that in a first approximation, the crystallite size is the size of the first objects that are formed in solution, if the crystallites have the same size whatever the synthesis condition, this can be interpreted considering that HAp nucleation is not perturbed. If we continue the hypothesis with the fact that the particle size reflects the growth phase, if the particle size is decreased when incorporation of CA, BA and BE, we can suggest that these molecules inhibit the growth of HAp. Such a growth limitation can result either from adsorption of the BAMs on the surface of growing particles of HAp or a depletion in available Ca 2+ . The first possibility is very unlikely, because when we tried to adsorb BAMs on HAp particles, BE was the molecule with the highest affinity for HAp surface while it does not show a particular inhibiting effect here. Accordingly, RA did not exhibit a specifically high affinity for

HAp surface but has the highest inhibiting action on precipitation. Altogether, this would favor the hypothesis that the underlying mechanism is mainly related to BAM interactions with CaCO3 that would limit its dissolution. It has been shown that for selected organics at concentrations lower than 0.01 mol.L -1 the dissolution of CaCO3 is inhibited in alkaline pH 14,15 . Inhibition was attributed to the blocking of dissolution sites by adsorbed carboxylates. RA, with one carboxylate and two catechol functions may act as strong adsorbent on CaCO3, preventing from its dissolution.

Biological activity of hydroxyapatite -baicalein hybrid particles

obtained by adsorption

Introduction

In this part, the biological activity of hydroxyapatite nanoparticles with BAM will be investigated. We decided to focus only on the hybrid hydroxyapatite particles with baicalein. Indeed, as seen in Chapter 2, it is the molecule with the best antibacterial activity. Moreover, it is the molecule with the best incorporation in hydroxyapatite, either by adsorption or during synthesis. As we saw that baicalein is degraded in pH 12 during synthesis of hydroxyapatite, we decided to study only particles after adsorption. First, the release of baicalein from HAp coatings was studied by HPLC, before the study of their antioxidant and antibacterial activities.

Baicalein release in vitro

This work was initiated during my stay in the team of Pr M.F. Desimone at the Universidad de Buenos Aires. Baicalein release from HAp particles (after adsorption) in PBS was studied by HPLC. 10 mg of particles were suspended in 2 mL of PBS at pH 7.4, and 0.5 mL of supernatant was removed at different times for HPLC analysis, and replaced by fresh PBS. Baicalein release was studied over 6 days.

Chapter 3. Formation of hybrid hydroxyapatite -biologically active molecules nanoparticles 135 HPLC analysis was performed with reverse phase on a C18 column. Cumulative release profile, in percentage of the total adsorbed amount, is presented in Figure 3.14.

Figure 3.14 Cumulative release of baicalein

Release profile is characterized by a rapid burst release, with 20 % of the total amount of adsorbed baicalein released in the first 45 minutes. Thereafter, release rate is slower during 24 hours, to reach 36 %. After 6 days, 40 % of baicalein is released from the hydroxyapatite particles. This suggests that a noticeable amount of baicalein strongly binds to hydroxyapatite.

The release profile of baicalein follows a classic profile of drug release from nanoparticles, consisting in initial burst release followed by a slower and more controlled phase 6,7,9,10,16 . The initial release may be longer or shorter, from a few minutes 7 to several hours 9 , depending on the affinity of the molecule for the substrate, and the design of nanoparticles. Our nanoparticles reached a plateau in 24 hours, which is approximatively in the average of systems found in the literature. In some cases, the total quantity is released in few hours 6,7 , while others may achieve a sustained release over several days 10,16 . In the literature, sustained release of baicalein was achieved by encapsulation in organic polymers matrixes, like chitosan nanoparticles 17 or polymers micelles 18 . When encapsulated in such chitosan nanoparticles, baicalein is entirely released after 14 days of experiments.

Antioxidant activity

As seen in Chapter 2, baicalein is a flavonoid with a significant antioxidant activity, thanks to its pyrogallol group. The antioxidant activity relies on the quenching of the reactive oxygen species (ROS) that are iinvolved in the development of diseases such as cancer, and inflammation. This work was also initiated during my stay in the team of Pr M.F. Desimone at the Universidad de Buenos Aires. Antioxidant activity of HAp particles with adsorbed baicalein was tested with the DPPH radical scavenging testing, derived from Blois 19,20 . It is based on the DPPH• scavenging through donation of a hydrogen, which is followed by the reduction of the absorption band at 517 nm, as already seen in Chapter 2. 10 mg of particles were suspended in a 1:1 (v/v) mixture of TRIS buffer at pH 7.4 and DPPH solution in ethanol (100 µM). Samples were let to react for 20 minutes in the dark before centrifugation and recovering of the solution and measure of the absorbance of the solution at 517 nm. In order to see the effect of baicalein release, particles after DPPH testing were incubated in PBS for 24 hours before going to a second DPPH assay. After recovery of the particles they were again placed in fresh PBS. This was done every 24 hours for 5 days. Antioxidant activity of each supernatant was also tested. DPPH bleaching rates of particles are gathered in Table 3.5.

First, it is important to mention that the release medium did not show any antioxidant activity, Incorporation in HAp of flavonoids was already reported with quercetin, which is a hydroxyflavone 21 . After incorporation of the molecule in hydroxyapatite particles during synthesis, radical scavenging activity of the hybrid material was reduced to 50 % compared to pure quercetin.

Authors interpret that results as a consequence of the oxidation of quercetin, which loss its antioxidant activity. On the contrary, materials synthesized by phase transition, meaning first the synthesis of monetite (Ca(HPO4)), another calcium phosphate synthesized at acid pH, in which quercetin is stable, before phase transition to hydroxyapatite in ammonia, have an antioxidant activity close to pure quercetin. Quercetin is not oxidized during the latte synthesis, so it keeps its antioxidant activity. Only a few other examples of hybrid hydroxyapatite nanoparticles with antioxidant activity using flavonoids were reported and it is the first time with baicalein. Besides quercetin mentioned before 16,21 , catechin was also used 22 (Figure 3.15). Catechin covalently linked to nanoscale hydroxyapatite showed an antioxidant activity higher than pure catechin, which is attributed to the accumulation of the molecules on the particles surface.

Antibacterial activity

In order to check if the molecule is still active after oxidation, we tested its antibacterial activity after 24 hours of oxidation in the culture medium. The MIC was measured as presented before in Chapter 2. We observed that the MIC after oxidation of the molecule is the same than the value obtained with a fresh solution of BE, i.e. 34 µg/mL. This is not surprising, because we saw that the oxidation of baicalein in these conditions is very fast. In fact, the molecule oxidized in the first hour in the culture medium, so it was already oxidized in the test using a freshly prepared BE solution. This implies that the weak antibacterial activity of the hydroxyapatite particles with adsorbed baicalein is not due to a loss of activity by baicalein after release.

Antibacterial activity of particles with adsorbed baicalein was first tested with an adapted diskdiffusion method. In the classic disk diffusion method, a paper disk is impregnated with a suspension of particles, and deposited on an agar plate where bacteria are spread 23 . Here, particles were put under the form of pellet of approximatively 10 mg. The pellet was then deposited on a MH-agar plate with spread Staphylococcus epidermidis CIP 68.21. As seen in Figure 3.16, no diffusion disk is visible around the pellet. We assumed that the pellet did not have any antibacterial activity because baicalein could not diffuse from the particles, due to the compact form of the pellet. We decided to move to testing in liquid medium. First and foremost, we can see that the concentration of bacteria after incubation with hydroxyapatite particles with or without adsorbed baicalein is similar to the one of the negative control, whatever the quantity of particles tested. Hybrid particles do not prevent the growth of S. epidermidis in these conditions.

In order to see if our particles possess a bactericidal effect, we tested their antibacterial activity in PBS. In this medium, the bacterial growth is inhibited. The number of bacteria is supposed to be constant during the time of the experiment. The protocol was the same than used for testing in MH.

Results are presented in Figure 3.18.

First, we can observe that the number of bacteria after incubation in PBS is the same for hydroxyapatite particles and hydroxyapatite particles with adsorbed baicalein, whatever the tested amount of particles. This suggests that particles with adsorbed baicalein do not have any bactericidal effect, otherwise a reduction of the counted number of bacteria would have been observed.

Surprisingly, the number of bacteria in the negative control is lower than the number at t0, suggesting that a certain quantity of bacteria died during the experiment. We thought that maybe the initial quantity of bacteria was too important for the activity of baicalein. We decided to perform the same test than the one in MH and PBS, but starting from a lower concentration of S. epidermidis. We chose 10 4 CFU/mL as the initial concentration of bacteria in method, with particles shaped as pellets. This antimicrobial testing method was also used to evaluate the activity of hydroxyapatite nanoparticles loaded with vancomycin, against laboratory strains of S.

aureus and E. coli, and MRSA 27 . Particles were active for 7 days against MRSA, and for more than 20 days against the other strains. In both cases, the antibacterial activity is related to the diffusion of the antibiotics outside the hydroxyapatite pellet. In the first study, hydroxyapatite particles are porous, made from stoichiometric HAp. Porous nanoparticles allow a large loading of antibiotics, and its sustained release. In the second study, vancomycin is loaded in commercial HAp, in a large amount (160 mg/g). The difference of activity between these systems and ours should also be strongly linked to the intrinsic antibacterial activity of the used molecules. These studies described hydroxyapatite loading with classic broad-spectrum antibiotic (vancomycin and tetracyclin), with MIC much below that of baicalein (MIC < 1 µg/mL compared to 34 µg/mL for BE ).

Conclusions

In this part, we investigated the biological activity of hydroxyapatite with adsorbed baicalein. In vitro release, radical scavenging activity and antimicrobial activity against S. epidermidis were tested.

Baicalein release follows a typical profile consisting of initial burst release and slower phase. The maximum quantity released is 40 % of the total amount of baicalein adsorbed, meaning 25 mg of baicalein for 1 g of particles. After adsorption of baicalein on hydroxyapatite, the molecule remains partly active as hybrid particles exhibit antioxidant activities. We assumed that adsorbed baicalein is responsible for both activities, rather than the released baicalein in solution. Released baicalein, oxidized at physiological pH, should lose its antioxidant activity, while particles with adsorbed baicalein exhibit a significant activity for several days. As far as the antibacterial activity is concerned, our system is unfortunately not efficient against S. epidermidis CIP 68.21.These particles are less effective than other antibacterial drug release system with hydroxyapatite found in the literature, but the later use antibiotics such as vancomycin or tetracycline, with MIC below the MIC of baicalein.

Conclusions

In this chapter, we wanted to determine if it is possible to associate rosmarinic acid, chlorogenic acid, baicalin and baicalein with hydroxyapatite nanoparticles. Two methods of incorporation were tested, post-synthesis adsorption and direct synthesis with incorporation of BAM in the reaction medium. Hydroxyapatite nanoparticles were synthesized by precipitation in aqueous medium. We evidenced that the in situ incorporation of molecules is possible, but the presence of organics at high concentration partially or totally inhibited the crystallization of hydroxyapatite.

Moreover, BAM being degraded at alkaline pH, the real form of incorporated molecules is not known.

On the contrary, post-synthesis adsorption did not modify the morphology nor the size of hydroxyapatite crystals. Among the four molecules tested, baicalein was the one with the best affinity for hydroxyapatite, despite the fact that no complex with calcium was identified during the molecular study. As it is the molecule with the lowest MIC towards bacteria of interest, we decided to test the antibacterial and antioxidant activities of baicalein associated with hydroxyapatite nanoparticles.

Particles with adsorbed baicalein showed a significant antioxidant activity, with 80 % of DPPH radical scavenging. The antioxidant activity was maintained over a week, after baicalein release in PBS.

However, the release medium did not show any antioxidant activity, because the molecule oxidized in PBS. Unfortunately, our particles do not possess any antibacterial activity against S. epidermidis CIP 68.21, in the tested conditions. Compared to other systems consisting of hydroxyapatite nanoparticles with incorporated natural molecules, our system is promising in regards to radical scavenging activity, as the activity is sustained over days after incubation in PBS.

To conclude, this chapter aimed at providing a proof of concept for the development of hybrid materials consisting of hydroxyapatite and natural biologically-active molecules. It is the first report of baicalein-integrated hydroxyapatite, with antioxidant properties. These particles may be used in the field of body implant if coated on metallic substrates. However, as the organic molecule is degraded at high temperature, low temperature processes are required.

Chapter 4

Elaboration of hybrid hydroxyapatite -biologically active molecules coating on Ti6Al4V titanium alloys

Introduction

In this chapter, we will focus on the development of a hybrid coating deposited on Ti6Al4V titanium alloy consisting of hydroxyapatite and natural biologically-active molecules. The chosen method has been to form hydroxyapatite from a supersaturated solution called Simulated Body Fluid (SBF) whose composition was directly derived from the one used by Kukubo et al 1 . These authors have shown that the obtained hydroxyapatite was suitable with osteoblast growth, so that the material was qualified as biomimetic. SBF closely resemble the inorganic composition and concentration of blood plasma.

The presence of ions such as Na + and Mg 2+ , and CO3 2-induces substitutions, leading to a carbonated calcium-deficient hydroxyapatite. This method of deposition is widely used in the literature, but with different parameters depending of the authors. Surface pretreatment, temperature of deposition, composition and concentration of the solutions, are particularly discussed in the literature [2][3][4][5] . In this chapter, we will investigate different surface treatments with the composition of SBF derived from the one used by Kokubo et al 1 , in order to determine the best suited for HAP deposition and adhesion.

Secondly, the composition of the solution will be modified, mainly the carbonate content. The biomimetic coating has been deeply characterized. The second part of this chapter deals with the incorporation of BAM (rosmarinic and chlorogenic acids, baicalin and baicalein). In Chapter 3, we proved that the incorporation of BAM is possible either concomitantly with HAp synthesis or by adsorption on pre-synthetized HAp. The same methodology will be used here. First, the incorporation of BAM during synthesis by adding the molecules directly in the SBF will be investigated. Then, the post-synthesis adsorption of baicalin and baicalein on deposited HAp coatings has been performed.

Hybrid coatings synthesized following both methods will be characterized and compared. The last part of this chapter is the characterization of the biological properties of the hybrid materials. Drug release, antioxidant and antibacterial activities will be investigated. A preliminary study of osteoblasts adhesion and proliferation will also be presented.

Deposition of biomimetic hydroxyapatite in SBF 4.2.1 Elaboration of the method

Two parameters are important for the deposition of biomimetic hydroxyapatite in SBF: the formulation of the SBF solution and the pre-treatment of the surface of the alloy. In the literature, it is commonly reported that an alkali-heat treatment, consisting of first hydroxylation of Ti in NaOH as a first step followed by an annealing one at high temperature to crystallize titanium oxide, favors the deposition of HAp 1,2 . In this work, we have studied the effect of the annealing step on the deposition of HAp in a SBF whose composition is similar to the Kokubo's formulation of SBF 1 but 1.5 fold concentrated (1.5-SBF). The latter modification is expected to improve the deposition rate.

Chapter 4. Elaboration of hybrid hydroxyapatite -biologically active molecules coatings on Ti6Al4V titanium alloys

As mentioned in the literature review (Chapter 1), besides the original Kokubo's preparation of SBF, many derived composition have been used 3,6,7 . The main variable is the carbonate concentration.

Consequently, we decided to compare the HAp deposition process using 1.5-SBF or a carbonate more concentrated solution. These two studies allowed us to determine the best parameters to obtain a thick and homogeneous layer of hydroxyapatite on the surface of the Ti alloy Ti6Al4V. The deposition duration and the renewal of the solution was also taken into account.

Surface pre-treatment

Surface preparation

Square plates of Ti6Al4V (1 cm², provided by GoodFellow) were used as substrate for the deposition of HAp. They were polished at three different grid with silicon carbide papers (# 500, # 800 and # 1200).

Plates were then soaked in a 5 M NaOH solution, at 60 °C for 24 hours (30 mL of solution for 8 plates).

After this alkaline treatment, they were washed three times in distilled water and finally left 24 h in water at room temperature, before drying 24 h at 37 °C. For annealing process, alkali-treated samples were heated at 600 °C for 1 h under air, with a ramp of 5 °C/min, before cooling in the furnace.

Deposition of hydroxyapatite

After surface pre-treatment, hydroxyapatite was deposited on the substrates in SBF concentrated 1.5 fold (1.5-SBF). We chose to concentrate the soaking solution in order to speed up the process. Samples were immerged vertically in the solution, with ratio surface/volume ratio S/V = 25 mL.cm -2 . As samples are suspended vertically, the deposition of hydroxyapatite is possible on both faces of the substrate.

The composition of SBF is reminded in Table 4.1. SBF was prepared in ultrapure water, and the pH was checked to be between 7.4 and 7.6. The amount of each constituent required for the preparation of the solution for 1 L is described in Table 4.2, with order of addition of the reagents. Deposition was performed in high density polyethylene bottles (Thermo Scientific™ Nalgene™ Wide-Mouth HDPE Packaging Bottles), because hydroxyapatite may crystallize on the surface of glass bottles. The solution of deposition was renewed every 48 h by freshly prepared SBF. Plates were withdrawn from the medium after either 7 days or 14 days of deposition. They were washed three times in distilled water, and dried 24 h at 37 °C. The morphology of the deposited layer was observed by SEM, and the crystalline phase was controlled by XRD and Raman spectroscopy. growing material densifies at the surface. The morphology is typical for hydroxyapatite synthesized in supersaturated solutions 1,2,6 . As described by Kokubo, hydroxyapatite is formed by the crystallization at the surface of the substrate. Erreur ! Source du renvoi introuvable. and (d)). In addition, there is a broad band between 20 and 35 ° on both diffractograms after 7 and 14

Concentration (mmol.L -1 )

Na + K + Ca 2+ Mg 2+ Cl - HCO3 2-HPO4 2-SO4
days. This commonly refers to an amorphous contribution, probably an amorphous calcium phosphate in that case. From XRD results, it can be concluded that 14 days of soaking on non-annealed substrates seems to be the most effective method to obtain pure hydroxyapatite. (AT= alkali-treated; AHT = alkali-heat-treated)

Raman spectra of surfaces after deposition are presented in Figure 4. 6 The apparatus used is equipped with an optical microscope and an optical fiber, which allows precise control of the surface area to be analyzed. As previously pointed out on the SEM images, we know that annealed substrates lead to the formation of un-homogeneous surfaces after soaking. This was confirmed by optical microscopy analyses. However, thanks to the optical fiber, Raman analyses were performed only areas with deposited material. Spectra after deposition for 7 days on the non-annealed substrates and annealed substrates are similar (see at 430 and 448 cm -1 (ν2), between 579 and 591 cm -1 (ν4), and at 1048 and 1075 cm -1 (ν3) 8 . A peak at 1070 cm -1 , corresponding to the ν1 vibration of CO3 -2 is found, indicating the presence of type B carbonate substitution 9 . The intense ν1 vibration at 960 cm -1 is also visible on spectrum after 7 days of deposition on annealed substrates (see Figure 4.6 (c) and (d)). We can note the presence of the vibrations corresponding to TiO2, anatase and brookite. Peaks of carbonate are absent. After 14 days

Here, after annealing, we obtained a mixture of anatase and brookite. Crystallization of TiO2 was visible on the X-ray diffractogram, and the mixture of phases was also seen on the Raman analysis. In contact with 1.5-SBF, hydroxylated surfaces were fully homogeneously covered with hydroxyapatite while deposition on annealed surface was not efficient. Our results are slightly different from those obtained in the systematic study from Pattanayak et al 2 . The authors varied the pretreatment step pH from acidic to alkaline, and the effect of heat treatment of the titanium plates. For an alkaline pretreatment, they observed the deposition of hydroxyapatite in SBF, which is consistent with our study. The difference with our findings deals with the heat treatment: despite the fact we used the same conditions, they reported that a thicker layer of hydroxyapatite was deposited on heat treated plates.

In our study, heat treatment is unfavorable, because the deposited layer is not homogeneous.

Influence of the composition of the soaking solution

The conventional composition of SBF contains 147.8 mM of Cl -and 4.2 mM of CO3 [2][3][4][5][6][7][8][9][10][11][12] . The latter carbonate concentration is lower than the one in blood plasma (i.e. 27 mM), and was modified by several authors. For instance, Tas and coll. 6 suggested that 27 mM and 103 mM of CO3 2-and Cl - respectively led to increase the deposition rate of a biomimetic hydroxyapatite. Consequently, we decided to compare two different soaking solution: SBF concentrated 1.5 fold (1.5-SBF) and the solution used by Tas and coll. (TSBF) concentrated 1.5 fold (1.5-TSBF). Solutions were renewed every 48 h with freshly prepared 1.5-SBF and 1.5-TSBF. Plates were withdrawn after 7 and 14 days of soaking and characterized by XRD and Raman spectroscopy.

First of all, we observed by optical microscopy that surfaces after deposition in 1.5-TSBF were not homogeneously covered with the deposited material. X-ray diffractograms of surfaces after deposition in 1.5-TSBF are presented in Surfaces coated by deposition in 1.5-TSBF were not observed by SEM as we have already seen that they were not homogeneously covered by optical microscopy. Contrary to what it is reported by Jalota et al. 6 , we did not find that a higher concentration of carbonates was favorable to deposit HAp.

The most plausible explanation is that the 1.5-TSBF solution was highly unstable, and a calcium phosphate material started to crystallize homogeneously in the solution, besides the heterogeneous crystallization on Ti alloy surfaces. On this basis, we decided to keep the conventional composition of SBF to deposit HAp on Ti surfaces for subsequent experiments.

-SBF on Ti6Al4V

After the choice of the surface pretreatment of the substrate and the composition of the supersaturated soaking solution, HAp was synthesized in these conditions, i.e. alkali treatment of the plate before soaking in 1.5-SBF and further characterized.

To determine the thickness of the layer, scratches were made with a scalpel on the edges of the coating, in order to peel it off from the substrate. Samples were then observed by SEM-FEG, with an acceleration voltage of 1 keV, on a tilted sample-holder of 45 °, in order to observe the cross-section of the coating. Images at magnification x1k, x2k and x10k are presented in ), layer thickness is about 12 µm. The layer is thicker than thin films deposited with sol-gel methods, but much thinner that what is deposited with plasma-spray deposition 13 . It is in the average of deposition from supersaturated biomimetic solutions. Energy-dispersive X-ray spectrum of HAp coating is presented in Figure 4.10. The absence of Ti peak confirmed the thickness of the coating is above 1 -2 µm, as it is the depth penetration of this analytical technique. The presence of Na and Mg peaks indicates that the HAp layer may be substituted. It is possible to calculate the ratio Ca/P in the coating, which is 1.4. As HAp might be substituted, we can also calculate the ratio cations/P, i.e. (Ca+Na+Mg)/P, which is 1.5 14 . A stoichiometric hydroxyapatite possesses a Ca/P ratio of 1.67, and in biological HAp, the ratio is closer to 1.5. This suggests that HAp is calcium-deficient, with a biomimetic composition. We tried to measure the incorporated quantity of BAM in the coating by thermogravimetric analysis, with the use of a TGA sorption/desorption apparatus. The loss of weight between room temperature and 600 °C was investigated. However, the weight difference is measured on the whole sample, meaning the substrate, the coating and the incorporated BAM. Considering the very low quantity of molecule that can be expected to be incorporated, the loss of weight is almost negligible.

Moreover, unfortunately, the signal was very noisy, as seen in Figure 4.17 for HAp-BE and HAp-BA.

Moreover, the measured loss of weight was higher in the case of pure HAp coating than for coatings containing BE or BA. From several measurements, we could estimate an incorporated quantity of baicalein of 0.2 mg for one coating but did not analyze further the other coatings. not prevent the crystallization of hydroxyapatite, but some differences exist between pure HAp coating and coatings deposited with BAM. First of all, the morphology of coating is different, with a more fragile and thinner layer that does not cover the entire surface in the case of rosmarinic acid. XRD and Raman spectroscopy suggested the presence of hydroxyapatite as the only calcium phosphate phase, even if the Ca/P ratio calculated by XPS for the top surface of the coating is lower than for pure HAp.

Unfortunately, the quantification of BAM incorporated in the coating was not possible, as detection techniques are missing. The traditional technique consisting in measuring the UV-Vis spectrum before and after synthesis is not very feasible, as the synthesis occurs in 14 day with renewal of the solution every 48 hours. Moreover, as BAM are not very stable at physiological pH, especially baicalin and baicalein, UV signatures of the molecules are not reliable. In addition, the homogeneous precipitation of BAM is visible in the deposition vessel, meaning that a measure of BAM concentration in solution after synthesis would misrepresent the real incorporated quantity. Another possibility would be to dissolve the HAp to release the molecule, before measuring the concentration by analytical technique such as HPLC.

Post-synthesis adsorption of BAM

The second method to add biological function to hydroxyapatite coatings is the adsorption of biologically active agents on the hydroxyapatite. This technique is widely used to incorporate antibiotics to obtain antibacterial coatings 4,16,18 . Adsorption force depends on the interaction between the organic molecules and hydroxyapatite. Antibiotics presenting carboxylic acid or catechol functions are supposed to have a strong affinity for hydroxyapatite 18 . We already shown in Chapter 3 than the adsorption of the four BAM used, chlorogenic and rosmarinic acids, baicalin and baicalein is possible on hydroxyapatite nanoparticles. In this part, we propose to investigate the adsorption of BAM on hydroxyapatite coatings synthesized in 1.5-SBF on Ti6Al4V alloys. With the purpose of using these hybrid coatings for their antibacterial properties, we decided to only study the adsorption of baicalein and baicalin on HAp coatings, as these molecules have the highest antibacterial properties.

Hydroxyapatite coatings were synthesized in 1.5-SBF on alkali-treated Ti6Al4V substrates, as presented in 3.2.2. For the adsorption, Ti alloy plates coated with hydroxyapatite are immerged in an 2 mL/plate aqueous solution of either BA or BE at 1.10 -4 mol.L -1 for 24 hours. At this concentration, and for this volume, the quantity of BA or BE in solution is 2.10 -7 mol for one sample. Ti alloy plates dimensions are 1 cm² for each face. As plates are soaked vertically in the 1.5-SBF, both faces are coated with HAp, so the total surface covered with HAp available for adsorption is 2 cm², if we neglect the specific surface area of the hydroxyapatite layer, as it appears very dense. To sum up, the theoretical quantity of BA or BE that can be incorporated is 1.10 -7 mol.cm -2 , i.e. 6.10 2 molecules.nm -2 .

Several attempts were made to measure the adsorbed quantity, with the same issues encountered than for the quantification when BAM are incorporated during the synthesis. UV-Visible spectroscopy was first used to measure the concentration of BE or BA in solution before and after adsorption. However, as expected, the UV-Vis spectrum after 24 hours is different from "fresh" solutions of the molecules. Moreover, the spectrum of BE after adsorption does not exhibit the same maxima than the one of BE after 24 hours in water, as seen in Figure 4.18. After 24 h in water, the peak at 274 nm is shifted at 264 nm, and the peak at 323 nm is shifted at 350 nm, with a broadening. This is consistent with the observations made in Chapter 2 about the degradation of baicalein in solution.

After adsorption, the peak at 323 nm is shifted at 360 nm, which is similar with baicalein at pH 10 or after total degradation. For both spectra, the shoulder at 420 nm is visible. Two events happen at the time, the degradation of the molecule in water, and the release of ions from hydroxyapatite, notably OH -, contributing to increase the pH of the solution, and thus modifying the state of the molecule. Due

For baicalein, we decided to take advantage of its fluorescence properties and measure the fluorescence response of coatings to detect its presence on the coating surface. First, the fluorescence signal of baicalein in solution in DMSO was measured, with an excitation wavelength at 320 nm. The fluorescence spectrum of baicalein exhibit several peaks (Figure 4.20 (a)) at 366, 373, 386 and 540 nm.

In the spectrum of the hydroxyapatite with adsorbed baicalein (HAp-BE, the enlargement is made on the small particles. This charging effect suggests that these smaller particles are composed by a material more insulating than hydroxyapatite, which would be the Chapter 4. Elaboration of hybrid hydroxyapatite -biologically active molecules coatings on Ti6Al4V titanium alloys 176 expected case of organic molecules. We can therefore assume that small particles are composed by baicalein, which precipitated on the hydroxyapatite surface during the adsorption step, or by a calcium phosphate material, with a morphology and a composition different from hydroxyapatite. To answer this question, EDX spectroscopy may be useful, in order detect the presence or absence of calcium and phosphate in this area. As a comparison, images of hydroxyapatite coating after adsorption when the plate is vertically placed are presented in Figure 4.22. At the lowest magnification, the morphology is similar than for horizontal adsorption. However, at higher magnification, the second type of particles are not present. This strengthens the hypothesis that they are composed of precipitated baicalein, that settle on the surface when the latter is placed horizontally, whereas when it is placed vertically the deposition is not possible. In order to verify that the "dissolution" of the coating is actually due to the adsorption process, a control of adsorption was performed and sample were observed with SEM. Briefly, Ti alloy plates with hydroxyapatite coatings synthesized in SBF were placed either horizontally or vertically in a vessel, a immerged in water for 24 hours. Their morphology was then observed, and is presented in The morphology is similar for both horizontal and vertical controls. It consists of the dense and homogeneous layer of spheroids typical for hydroxyapatite. This suggests that the immersion of coatings in water is not responsible for the dissolution of the coating during adsorption. Therefore, baicalein adsorption seems to induce a remodeling of the surface, characterized by a dissolution of the hydroxyapatite coating, and the precipitation of baicalein if adsorption is performed horizontally.

To conclude, baicalin and baicalein were adsorbed on hydroxyapatite coatings on Ti6Al4V surfaces. SEM showed that the adsorption is responsible for a remodeling of the surface, and that the molecule precipitates again on the surface if the adsorption is performed in a horizontal configuration.

Vertical adsorption would thus be privileged. XPS was useful to determine that baicalein interacts with both calcium and phosphorous on the surface, which was not seen in the case of incorporation during synthesis. However, despite the number of characterization techniques used, we cannot quantify the adsorption. We are still searching for an efficient quantification method.

Conclusions

In this part, we investigated the incorporation of BAM on the hydroxyapatite coatings by two methods, incorporation during synthesis of HAp or post-synthesis adsorption on pre-formed HAp layers. For the first method, BAM were incorporated in the 1.5-SBF prepared for the deposition of hydroxyapatite, with a ratio in solution between calcium and BAM BAM: Ca = 0.0075. Hydroxyapatite is efficiently synthesized in these conditions, even if the presence of BAM induces some changes in the morphology of the material. With all BAMs, the layer is thinner and more fragile for all molecules, and does not cover the entire surface with rosmarinic acid. The ratio Ca/P calculated from XPS data is lower than the ratio of pure hydroxyapatite coating, suggesting the presence of BAM at the hydroxyapatite surface and/or precipitation of a P-rich amorphous calcium phosphate phase. Unfortunately, it was not possible to quantify the incorporated amount of molecules in the coatings.

The second method used is the post-synthesis adsorption. After hydroxyapatite deposition in 1.5-SBF, samples were immerged in an aqueous solution of baicalein (or baicalin) for 24 hours. We observed by SEM that the adsorption induces a remodeling of hydroxyapatite surface. Fluorescence spectroscopy confirmed the incorporation of baicalein in the coating. However, we were not able to quantify the adsorbed amount of the molecule on the hydroxyapatite surface, due to a lack of quantification technique. HPLC would be an effective technique to measure the remaining concentration of baicalein in solution, but we were not able to perform this experiment, notably due to a lack of sensitivity of our apparatus.

Quartz crystal microbalance experiments were undertaken. For the first, we have established the feasibility of measuring the adsorption of baicalein by this method by adsorbing baicalein on The number of bacteria after 3 hours of incubation is the same, within the experimental error in absence of presence of baicalein. The adhesion on hydroxyapatite coating is slightly higher than on bare Ti surfaces. The presence of hydroxyapatite may enhance bacterial adhesion, as its surface is rougher than polished bare Ti. From this assay, we can conclude that hydroxyapatite coatings do not have any positive effect on the adhesion of S. epidermidis CIP 105.777 and that the presence of baicalein does not influence bacterial adhesion.

The growth ability of adhered bacteria was also assessed. The beginning of the experiment was as stated above (3 h of incubation at 37 °c in 2 mL of S. epidermidis suspension in PBS at 9.7.10 5 CFU/mL), samples were carefully rinsed to remove any poorly adhered bacteria, before being The number of proliferated bacteria is the same for HAp and HAp-BE coatings. A slight effect on the proliferation on (HAp + adsorbed BE) is significantly visible (p value = 0.004 compared to HAp), but with less than a log variation. We can conclude that the coating (HAp + adsorbed BE) can slightly prevent the proliferation of adhered S. epidermidis after 24 hours in rich medium. In contrast the HAp-BE coating with baicalein incorporated during the synthesis, does not show any anti-proliferation effect. From these two first assays, we can conclude that hydroxyapatite coatings incorporating baicalein do not have a very significant antibacterial effect. Whereas we could expect a reduction on initial adhesion, we showed that the presence of hydroxyapatite in fact enhances the bacterial adhesion compared to bare Ti. The HAp coating with incorporation of baicalein during synthesis did not have any effect on bacteria proliferation, but a reduction of the number of bacteria was visible on the coating with adsorbed baicalein. These results suggested that baicalein is not active in three hours, but can reduce the bacteria growth on a longer timescale. On this basis, we hypothesized that the kinetics of baicalein activity was slow, comprised between 3 and 24 hours. as bacteria cannot grow in a poor medium. In presence of baicalein, the bacteria concentration was the same than the control until 5 hours, but started to decrease between 10 and 24 hours. After 24 hours, the bacteria concentration in presence of baicalein was lower by one order of magnitude compared to the control. In culture broth (MH) without baicalein, the bacteria concentration increased from 3 hours to 10 hours and remained stable at 9 log. In presence of baicalein, it remained stable during 5 hours, when it started to increase, but to a lower extent than in the control. From these results, we can assume that the baicalein is an antibacterial molecule with a slow kinetics of action. In PBS, the effect is visible after 24 hours, while in MH the presence of baicalein only delayed the bacteria growth. The activity of baicalein in MH is visible after 8 hours. To sum up, we observed an inhibition of bacterial growth by the coatings (HAp + adsorbed BE) by one order of magnitude compared to the controls. Unfortunately, the coating HAp-BE did not exhibit any antibacterial activity. We can conclude that adsorption of baicalein is the most efficient method to incorporate baicalein in HAp coating in order to add an antibacterial property.

Conclusions

We highlighted the antibacterial activity of hydroxyapatite coating with adsorbed baicalein.

However, its activity is lower than the activity of other calcium phosphate coatings incorporating antibiotics from the literature. Hydroxyapatite coatings with adsorbed antibiotics such as tobramycin or cephalotin are efficient against S. aureus in 16 h 18 . Tobramycin was also adsorbed on phosphate calcium coating on fixation pin, with a sustained antibacterial activity for 7 days, despite an initial burst release in 15 min 17 . However, the comparison of antibacterial activity is not obvious, because the method of testing is different from the one we used. Authors used an adaptation of disc diffusion method by placing the plate directly on a seeded agar plate. Yet, the major difference between these coatings and ours is the antibacterial activity of the antibiotics alone. Synthetic antibiotics are at least 100 times more active (i.e. have MIC two orders of magnitude smaller) than natural antibacterial molecules on susceptible strains. This issue is all the more problematic here as the concentration of the solution that can be used for baicalein adsorption is limited by the poor solubility of BE in water (1.10-4 mol.L -1 ).

Preliminary testing of the cytotoxicity of hybrid coatings

Cytotoxicity studies were performed on Human Osteoblasts, cultured in cell culture medium consisting of DMEM supplemented with glucose, antibiotics, serum and dexametasol. Cells were cultured for three weeks before the experiments. For our studies, samples were seeded with 50 000 cells. We tested the toxicity of our materials towards the osteoblasts at 24 hours, and the proliferation of cells on the plates at 7 days. After 24 hours of cellular culture at 37 °C in an incubator with 5 % of Indeed, the manipulation of samples at 24 hours to perform the AlamarBlue test may have cause a mortality. This would be consistent with the large experimental error observed for the control, with percentage of viability going from 50 to 100 %. Further experiments will be undertaken, taking more experimental precautions. We investigated the deposition of hydroxyapatite on titanium alloy, and the incorporation of biologically active molecules (BAM) in the coating. The deposition was biomimetic in a 1.5-fold concentrated simulated body fluid, taking place at physiological pH and temperature. The deposition protocol was optimized, both for the substrate pretreatment and the composition of the deposition solution. We found that an alkali pretreatment was suitable to allow hydroxyapatite deposition, without the requirement of an annealing step. For the composition of SBF, we decided to keep the Kokubo's solution with low carbonate content, but concentrated 1.5 fold (1.5-SBF), as the synthesized layer was more homogeneous. In these conditions, after 14 days of deposition with renewal of the solution every 48 h, a 12 µm thick carbonated and substituted hydroxyapatite coating was obtained.

The deposition of biomimetic hydroxyapatite was thus successfully achieved.

Two methods were tested for the incorporation of BAM : 1) co-solubilisation of BAM in the SBF, 2) adsorption of BAM on pre-formed HAp. The presence of BAM in the SBF did not prevent the hydroxyapatite crystallization, but coatings presented a morphology different from pure hydroxyapatite. In the case of rosmarinic acid, the coating did not cover the entire surface of the substrate. XRD and vibrational spectroscopies evidenced the deposition of hydroxyapatite as the only calcium phosphate phase. The effect of BAM incorporation was visible on XPS, with lowering of Ca/P ratio. In case of the post-synthesis adsorption, plates after hydroxyapatite deposition were immerged in an aqueous solution of BAM. Adsorption induced a reshaping of the hydroxyapatite surface.

Unfortunately, we were not able to quantify the amount of BAM in the coating, either after incorporation during synthesis or adsorption.

The biological activity of coatings was assayed with baicalein, as it is the molecule with the best antibacterial activity. Coatings with baicalein incorporated by the two methods did not possess any antioxidant activity. It can be related to a weak quantity incorporated (which should be checked by finding an appropriate technique to characterize the incorporation), and for the molecule incorporated during synthesis to the oxidation of the molecule in SBF. The antibacterial activity of both coatings was investigated against S. epidermidis CIP 105.777. For both coatings, no effect on bacterial adhesion and growth was observed after 6 hours of incubation, but their proliferation was partly inhibited by the coating (HAp + adsorbed baicalein) after 24 hours. The latter coating inhibited the bacterial growth in 16 h in rich medium. Coatings with baicalein incorporated during synthesis did not exhibit any antibacterial activity. Preliminary studies on human osteoblasts were inconclusive. A high cell mortality was observed at 7 days. Further experiments need to be done to better characterize the osseointegration ability of hydroxyapatite coatings in the presence of adsorbed baicalein.

Chlorogenic acid

Chlorogenic acid is an ester formed by a caffeic acid and a quinic acid moiety. We characterized its weak antibacterial activity (MIC = 4 mg/mL against S. epidermidis CIP 68.21), but significant antioxidant properties. The molecule is stable at acidic and neutral pH. We first demonstrated that the complexation of CA with calcium is pH-dependent. At acidic pH, monomolecular [CA-H+Ca] 2+ and bimolecular [2CA+Ca] 2+ complexes are formed, with the monomolecular complex more stable than the biomolecular complex. At neutral pH, only the [CA-H+Ca] 2+ complex is detected. NMR studies suggested that the preferential complexation site would be the catechol ring rather than the carboxylate. The complexation does not modify the antioxidant activity of CA. The ability of CA to form a complex in solution with calcium is not correlated with an affinity for hydroxyapatite surface. Indeed, CA is one of the molecule with the lowest affinity for the surface of HAp nanoparticles in our study.

Regarding the incorporation of the molecule during synthesis of Hap nanoparticles, it was found that CA is poorly incorporated. At the lowest CA:Ca ratio (0.0075), the presence of RA decreases the yield of the reaction. HAp particles with CA are smaller than pure HAp particles, but the crystallite size is equal. At the highest CA:Ca ratio (0.075), besides HAp, a small fraction of CaCO3 is observed by XRD.

HAp synthesis is hindered, that may be explained by an affinity of CA for calcite that is not fully dissolved. Among the studied molecules, CA belongs to the one with the lowest effect on HAp synthesis, either under the form of nanoparticles or coatings. Despite a difference in the morphology of the coating, the presence of CA in the deposition solution does not prevent the HAp synthesis.

Overall, despite a strong complexation between calcium and chlorogenic acid in solution, CA does not show a specific affinity for the hydroxyapatite surface.

Rosmarinic acid

Rosmarinic acid is an ester formed by a caffeic acid and a 3, 4-dihydroxyphenyllactic acid moiety. Its antibacterial activity is higher than the one of chlorogenic acid (MIC = 1.0 mg/mL against S. The monomolecular complex is the only detected one at neutral pH. NMR studies did not allow the discrimination between sites of complexation. On the contrary of CA, the complexation has a small effect on the antioxidant activity of rosmarinic acid. We can assumed that if the catechol is the function responsible for the antioxidant activity, the loss of antioxidant property may be due to the complexation of calcium at the catechol site. From antioxidant assays, we can assume a higher probability of complexation on the catechol site rather than on the carboxylate. Despite a significant complexation between RA and calcium, RA does not have a significant affinity for the HAp surface. Looking back at the chemical structure of the four molecules, we have already pointed out that the higher affinity of BA/BE compared to CA/RA for the HAp surface can be attributed to the presence of a pyrogallol ring in the former compared to a catechol ring for the latter. This assumption is strengthened by the fact that in BE this pyrogallol ring is unsubstituted while it bears a sugar ring in BA, such a difference accounting for the highest affinity of the former for the HAp surface compared to the latter. Such a difference between catechol and pyrogallol was attributed to the fact that the presence of three close aromatic hydroxyl groups allows for cooperative interactions strengthening adsorption to the surface. On the opposite, the catechol group seems more favorable than the pyrogallol one for interaction with calcium ions. Again, as RA bears two catechol groups while CA has only one, it would explain while the former has a more marked inhibitory effect than the latter. the oxidation of the pyrogallol group of BA/BE should be consider to explain the lower complexation affinity of those molecules with calcium ions compared to RA/CA.

Chapter 4. Elaboration of hybrid hydroxyapatite -biologically active molecules coatings on Ti6Al4V titanium alloys 194

To summarize the results, we can conclude that among the four tested BAM, baicalein is the most relevant molecule to develop an antibacterial coating with hydroxyapatite. The molecule has the best antibacterial activity among the four molecules tested, and interacts the most with hydroxyapatite surface. Baicalin may also be considered of interest because it has a non-negligible affinity for hydroxyapatite surface, but its antibacterial activity is 10 times lower than the one of BE.

Both molecules have a slight inhibition effect on the hydroxyapatite synthesis while added in the solution medium, but HAp is still the major phase synthetized. Rosmarinic and chlorogenic acids do not have any affinity for hydroxyapatite surface. Furthermore, rosmarinic acid is a strong inhibitor of hydroxyapatite crystallization, probably by preventing the dissolution of calcite formed in the first steps of HAp synthesis because of their high affinity for calcium. The weak antibacterial activity of RA and CA, in addition of their poor affinity for hydroxyapatite, do not make these molecules choice alternatives to develop antibacterial coatings with hydroxyapatite.

General conclusions and perspectives General conclusions and perspectives

The goal of my PhD work was the development of a hybrid coating consisting of biomimetic hydroxyapatite and natural antibacterial molecules, with the goal to add antibacterial properties and enhance the osseointegration of titanium implant (see Figure 5.1). This work falls within the fight against periprosthetic joint infections that are one of the concerns for hip arthroplasty. The other issue it aims at addressing is the lack of interaction between implant and bone that can be improved by the presence of a layer of hydroxyapatite on the implant surface. We focused on four biologically active molecules (BAMs) extracted from natural products, divided in two classes: polyphenols for rosmarinic and chlorogenic acids and flavonoids for baicalin and baicalein. One of the main objectives was to understand the interaction between those molecules and hydroxyapatite, in order to choose the best candidate for the development of the antibacterial coating.

First, we characterized the biological activities of the four molecules, and we investigated their interaction in solution with calcium. Secondly, we attempted to incorporate the biologically active molecules within hydroxyapatite nanoparticles. Finally, we elaborated the hybrid coating with hydroxyapatite and biologically active molecules on titanium alloy. At each step, biological activity of synthesized materials was tested.

By characterization of the DPPH radical scavenging activity, we showed that rosmarinic acid is the molecule with the best antioxidant activity. We measured the minimum inhibitory concentration of the four molecules against Gram-negative and Gram-positive bacteria. Baicalein is the molecule with the best antibacterial activity against S. aureus, S. epidermidis, and L. ivanovii (Gram-positive strains), but none of the molecule was active against P. aeruginosa (Gram-negative strain).

Complexation of rosmarinic acid and chlorogenic acid with calcium was studied with spectroscopic and spectrometric methods. Electrospray ionization mass spectrometry was useful to determine the presence of complexes and their stoichiometry. The complexation was found highly pHdependent. We used NMR spectroscopy to determine the site of complexation, but we were not able to discriminate between the carboxylate and the catechol functions. The complexation does not have a high impact on the antioxidant properties of both molecules. As for baicalin and baicalein, we carefully characterized their stability in aqueous solution at different pH by UV-Vis spectroscopy and ESI-MS. The kinetics of degradation of both molecules is faster when pH rises, and it is related to an oxidation. The oxidation does not affect the antimicrobial activity of baicalin and baicalein, but, as expected, decreases their antioxidant ability. While complexes with calcium are detected for both molecules at pH 5, at pH 7.8 the oxidation overwhelmed the complexation.

Biologically active molecules were incorporated on/within hydroxyapatite nanoparticles by two methods, by adsorption or during the synthesis of hydroxyapatite. Baicalein is the molecule adsorbed with the highest amount on hydroxyapatite, as it was characterized by thermogravimetric analysis and solid-state NMR. Hydroxyapatite synthesis by precipitation in aqueous medium was partially or totally inhibited by the presence of BAMs if incorporated during synthesis. Crystallite size is not modified by the presence of organics, but particle size is decreased. At high concentration in presence of rosmarinic acid, only calcite is recovered at the end of the synthesis. Altogether, we assume that BAMs prevent the dissolution of calcite, which is a transitory phase in HAp synthesis.

Finally, considering the degradation of BAM at pH 12 during the synthesis, and the fact that baicalein is the molecule with the best adsorption rate on hydroxyapatite nanoparticle, we decided to study the biological properties of nanoparticles with adsorbed baicalein. 40 % of the total adsorbed amount of baicalein was released from hydroxyapatite nanoparticles, as measured by HPLC. We showed a sustained antioxidant activity of particles with adsorbed baicalein over 5 days, with an initial DPPH radical scavenging of 80 %. However, particles did not exhibit any antibacterial activity against S. epidermidis CIP 68.21.

Finally, we deposited hydroxyapatite on titanium alloy by a biomimetic process at physiological pH and temperature in the Simulated Body Fluid (SBF). Parameters of deposition (composition of soaking solution, pretreatment of the substrate) were optimized before the incorporation of BAMs.

Without BAMs, we synthesized a calcium-deficient and substituted hydroxyapatite layer, with 12 µm of thickness. The presence of BAMs in SBF partially inhibits the deposition of hydroxyapatite, especially in presence of rosmarinic and chlorogenic acid. Layer is thinner, and the Ca/P ratio is decreased.

Interaction between BAM and calcium in the HAp coating was evidenced by XPS. BAMs were also incorporated in hydroxyapatite coating by post-synthesis adsorption, inducing a remodeling of the surface. Unfortunately, we did not find a pertinent technique of characterization to measure the incorporated quantity of BAMs in the coatings. We studied the antibacterial and antioxidant activities of coatings with baicalein, incorporated during synthesis or after adsorption. Coatings synthesized by both methods did not exhibit any antioxidant activity. On the contrary, we were able to evidence some antibacterial effect of the coating hydroxyapatite with adsorbed baicalein on S. epidermidis CIP 105.777.

A first issue we met during our project is that the behavior of the four molecules is highly pHdependent. Therefore, it is very difficult to compare the three parts of this work, because adsorption, nanoparticles synthesis and biomimetic synthesis do not occur at the same pH. Despite a molecular study conducted at pH 5 and pH 7.8, it is not easy to correlate the results of calcium complexation with what we observed during incorporation of BAMs in hydroxyapatite, whatever the method.

The second limitation of our work is the weak antibacterial activity observed for the hydroxyapatite coatings, which is absent for hydroxyapatite nanoparticles. Nevertheless, whereas it

was not expected at the beginning of this work, hydroxyapatite nanoparticles with adsorbed baicalein possess sustained antioxidant activity. It could be interesting to try with other BAMs tested in this study, in particular rosmarinic acid as it is the molecule with the best antioxidant activity.

The weak antibacterial activity of hydroxyapatite coatings and nanoparticles with baicalein is explained by the fact that the molecule is not incorporated in a sufficient quantity, and by its weak intrinsic antibacterial activity. The novelty of this PhD work was the use of natural antibacterial molecules, to take advantage to their biological activity and to avoid any phenomenon of antiobioresistance because they have never been used in the field. However, those molecules have antibacterial activity far lower than the one of traditional antibiotics (for example, the MIC of ciprofloxacin is 0.5 µg/mL against S. aureus ATCC 6538 while it is 34 µg/mL for baicalein). We used rosmarinic acid, chlorogenic acid, baicalin and baicalein because they bear carboxylic acid and/or catechol functions that we assumed to have affinity for hydroxyapatite. However, some of these molecules are not stable at physiological pH, and their poor solubility in water prevents their incorporation in high quantity in hydroxyapatite.

If we think of other natural biologically active molecules, flavonoids are reported in the In terms of materials, we had expected that the in situ approach led to hybrid "supra"particles where the active molecules would be have been buried within hydroxyapatite nanoparticles, which could have limited their release rate. This is clearly not the case here, at least for the nanoparticles. If we focus on the adsorption strategy, then a parameter of major importance will be the porosity of the particles/coatings. While mesoporous hydroxyapatite nanoparticles are not easy to obtain, one can think of porous bioglass particles as interesting alternatives. Considering the coating, a better control of the deposition process may allow to tune the interparticle porosity.

Introduction

Le corps humain est constitué de 206 os connectés par plus de 290 articulations. La hanche est un exemple d'articulation qui relie la cuisse au bassin, grâce à la tête fémorale et la cavité cotyloïde (aussi appelée acetabulum) qui est formée à la jonction de trois os du bassin (l'ilium, le pubis et l'ischium).

Le rôle de l'articulation de la hanche est de supporter le poids du corps, lors de la marche ou en position statique. Cependant, avec l'âge, une douleur au niveau de la hanche, causée par le développement

Résumé en français

Résumé en français

Formation de nanoparticules hybrides

Abstract

This thesis aims to develop a hybrid coating based on biomimetic hydroxyapatite and natural antibacterial molecules, with the goal to add antibacterial properties and enhance the osseointegration of titanium (Ti6Al4V) implants. Four natural biologically active molecules (BAMs) were tested, rosmarinic acid, chlorogenic acid, baicalin and baicalein. To understand the interaction between those molecules and hydroxyapatite, we present here three complementary studies: in solution with calcium ion, with hydroxyapatite nanoparticles and with biomimetic hydroxyapatite coatings on titanium alloy. The complexation between BAMs and calcium ion in solution is highly pHdependent. When incorporated within hydroxyapatite nanoparticles or coatings during synthesis, BAMs inhibit the formation of the mineral phase. On the contrary, they can be efficiently adsorbed on pre-synthesized particles. Finally, we present the biological properties of hydroxyapatite nanoparticles and coatings with adsorbed baicalein. Hybrid nanoparticles are potent antioxidant agents, but they do not exhibit any antibacterial activity, whereas hybrid coatings have a significant effect on the bacterial growth of Staphylococcus epidermidis CIP 105.777. This work originality stands on the use of natural antibacterial molecules, but we highlight the difficulty to handle them, due to their poor solubility and stability in physiological pH, which should be considered for further use in biomedical applications.

Key-words: hydroxyapatite, biomimetic coating, natural molecules, antibacterial, antioxidant, titanium implant

Résumé

Cette thèse porte sur l'élaboration de revêtements de surface hybrides composés d'hydroxyapatite biomimétique et de molécules antibactériennes naturelles, dans le but d'ajouter des propriétés antibactériennes et d'améliorer l'osséointégration d'implants en titane (Ti6Al4V). Quatre molécules naturelles biologiquement actives (MABs) ont été étudiées, l'acide rosmarinique, l'acide chlorogénique, la baicaline et la baicaléine. Pour comprendre l'interaction entre ces molécules et l'hydroxyapatite, nous présentons ici trois études complémentaires : en solution avec l'ion calcium, avec des nanoparticules d'hydroxyapatite et avec un revêtement de surface d'hydroxyapatite biomimétique sur alliage de titane. En solution, le pH influence la complexation entre les MABs et le calcium. Les MABs inhibent formation de l'hydroxyapatite lorsqu'elles sont incorporées pendant sa synthèse, sous forme de nanoparticules ou de revêtements. Elles peuvent cependant être efficacement adsorbées à la surface de particules ou de revêtements pré-synthétisés. Enfin, nous présentons les propriétés biologiques des nanoparticules et de revêtements hybrides avec de la baicaléine adsorbée. Les nanoparticules hybrides sont de potentiels agents antioxydants, mais n'ont pas d'activité antibactérienne. Les revêtements de surface hybrides sont quant à eux efficaces pour limiter la croissance bactérienne de Staphylococcus epidermidis CIP 105.777. L'originalité de ce travail repose sur l'utilisation de molécules antibactériennes naturelles, mais nous soulignons ici la difficulté de les utiliser, de par leur faible solubilité et stabilité à pH physiologique, ce qui devra être considéré lors de leur future utilisation dans des applications biomédicales.

Mots-clés : hydroxyapatite, revêtement de surface biomimétique, molécules naturelles, antibactérien, antioxidant, implant en titane

Figure 0 . 1

 01 Figure 0.1 Hip joint anatomy, position of the implant at the place of the joint and description of a classical implant

Figure 1 . 1

 11 Figure 1.1 SEM images of human osteoblasts on TiAlV samples after 14 days of cell cultures (a) on mechanically polished surface with P4000 SiC paper and (b) on sandblasted surface with 500 µm alumina particles 14

Figure 1 . 4

 14 Figure 1.4 FE-SEM image of (a) the friction-hardened sample heated at 600 °C for 1 h after immersion in NaOH 10 M at 30 °C for 12 h and (b) adipose-derived stem cells cultured on this surface 22

Figure 1 . 5

 15 Figure 1.5 SEM images of (a) anodized titanium; (b) anodized titanium coated with collagen; Human mesenchymal cells after 2 h of culture on (c) anodized titanium and (d) anodized titanium coated with collagen 28

Figure 1 . 6

 16 Figure 1.6 Schematic of peptide-functionalized poly-L-Lysine grafted polyethylene glycol (PLL-g-PEG)

Figure 1 . 7 F

 17 Figure 1.7 F-actin staining images of human osteosarcoma MG-63 cells grown on (a) Ti, (b) Ti after BMP-2 adsorption (BMP-2/Ti), and (c) heparin-conjugated Ti surface after BMP-2 adsorption (BMP-2/Hep-Ti) after 24 h incubation; (d) release kinetics of BMP-2 from BMP2/Ti and BMP-2/Hep-Ti 36

Figure 1 . 8 (

 18 a)). Because of a crystal lattice allowing substitutions (see Figure 1.8 (b)), the calcium ions can be substituted by several ions found in the biological fluids like Mg 2+ , Na + , K + … whose charge is compensated by oxygen lacunas. Moreover, OH -and PO4

  in which hydroxyl is substituted by fluoride that increases mechanical and physical properties of the compound. Due to its very low solubility, and toxicity of fluorides at high doses, fluoroapatite is rarely used for bone regeneration. On the opposite of fluoroapatite, amorphous calcium phosphate (ACP, Ca/P = 1.15-1.67) is poorly if even crystallized, and very unstable in biological fluids. In aqueous solution, it forms hydroxyapatite by a dissolution/reprecipitation process.

Figure 1 . 8

 18 Figure 1.8 (a) XRD pattern of a synthetic ("artificial") HAp and three bone specimens 45 and (b) representation of the unit cell of hexagonal HAp. (Ca in green, O in red, PO4 tetrahedra in brown)46

Figure 1 . 9

 19 Figure 1.9 TEM micrographs and electrodiffraction patterns (inset) of HAp samples obtained at (a) 25 °C, (b) 90 °C and (c) 90 °C with 24 h of reaction 51

Figure 1 .

 1 Figure 1.10 SEM image of the ultralong HAp micro/nanoribbons synthesized by hydrothermal method with pH controlled by urea decomposition 57

2 ) 1 . 2 ( 1 )

 2121 H 2 O →M-OH +R-OH (1) M-OH+YO-M→M-O-M+Y-OH (Equation Hydrolysis and (2) condensation of metal alcoxide by sol-gel route Before thermal treatment, the gel is composed of amorphous calcium phosphate. Calcination is necessary to obtain hydroxyapatite, which is one of the disadvantage of sol-gel method. Calcination temperature depends on the previous reaction parameters. Temperature used are comprised between 350 °C and 1200 °C64,65 . Very high crystalline hydroxyapatite is obtained after high sintering

  and hybrid material 68 for bone regeneration. Raucci et al. synthesized a scaffold composed of gelatin and hydroxyapatite by sol-gel method and freeze drying. Needle-like nanoparticles were homogeneously distributed in the porous polymeric matrix, with intimate interaction between both components (Figure 1.11).

Figure 1 . 11

 111 Figure 1.11 Gelatin hydroxyapatite scaffold obtained by freeze drying process and crosslinked (a), SEM image (b) and TEM image (c) of the scaffold 68

Figure 1 .

 1 Figure 1.12).Temperature of calcination is also to consider, as at temperature under 1000 °C, mixtures of HAp, DCP, DCPD, TCP and CaO are formed69 . This method necessitates a lot of energy to obtain pure hydroxyapatite, but may be interesting to obtain biphasic compositions like biphasic calcium phosphate (BCP, mixture of HAp and β-TCP), used in bone regeneration. In order to reduce consumption of time and energy, microwave heating may be an interesting process for solid-state synthesis73 . An original approach of Wu et al. allows the formation of carbonated hydroxyapatite from DCPD and eggshell powders, in order to reuse waste. Eggshells are mainly composed by calcium carbonate. 10 hours of reaction at 1000 °C are necessary to form pure hydroxyapatite 70 .

Figure 1 .

 1 Figure 1.12 SEM image of HAp obtained after 8 h milling in acetone (a) and water (b) and heat treatment at 1100 °C for 1 h 72

  Figure 1.13.

Figure 1 .

 1 Figure 1.13 SEM image of hydroxyapatite deposited by (a) plasma-spray technique 77 , (b) ion-beam assisted deposition 78 and (c) dip coating 79

Figure 1 .

 1 Figure 1.14 SEM image of surface (left) and cross section (right) of apatite layer formed in SBF on an alkali heat-treated titanium surface 90

Figure 1 .

 1 Figure 1.15 Before (a) and after (b) heat treatment at 600 °C. FE-SEM images of surfaces of Ti soaked in SBF for 3 days after exposure to solutions with different pHs92

Figure 1 . 16 .

 116 Along time, thickening of the coating is visible on the EDX spectrum, as the Ti contribution decreases while Ca and P increase. The substitutions are visible by the presence of Na and Mg contributions.

Figure 1 .

 1 Figure 1.16 SEM images and EDX spectra of HAp coatings on titanium alloy after different times of immersion in SBF: 12 h (a), 4 days (b), 8 days (c) and 14 days (d)97

Figure 1 .

 1 Figure 1.17 SEM images of (a) Ti samples treated by alkali heat treatment and subsequently soaked at 57 °C in 3 x SBF for (b) 1 days, (c) 2 days and (d) 4 days. (e) -(h) are high magnification images98

Figure 1 .

 1 Figure 1.18 SEM images of (a) SBF-A coating and (b) SBF-B coating after 24 hours; (c) XRD diffractogramm of (a) bone, (b) SBF-A coating, and (c) SBF-B coating after 24 hours 99

  haemolyticus, S. capitis, and S. hominis. The principal mechanism in the development of infections is the biofilm formation 104 on a surface. Bacteria live in two states, the planktonic state when they are in solution, and an adhered biofilm state on a surface. The stages of biofilm development are 1-Initial attachment and adherence of planktonic cells to an abiotic or biotic surface; 2-recruitment of adhered cells and production of extracellular polymeric substance (EPS); 3-maturation and development of full biofilm architecture; and 4-dispersion of the biofilm to yield back planktonic cells 105 (Figure1.20).Being in a biofilm state increases the resistance of bacteria to biocides. Dispersion is the deleterious stage, as bacteria are released in biological fluids in the case of biofilm formed on implant surface, spreading the infection in the entire body105 . Combating biofilm formation is very important to avoid the development of PJIs. Two main strategies are currently implemented, alone or in combination: avoid bacterial adhesion to the surface of the implant, and/or kill the bacteria under contact or in solution by the release of a biocide agent.

Figure 1 . 20

 120 Figure 1.20 Bacterial life cycle 105

Figure 1 .

 1 21 (a).These surfaces were tested against Gram + and Gram -bacteria. Bacterial adhesion and biofilm formation were reduced after treatment, with an enhanced effect for the longest treatment. Gramnegative bacteria were more impacted than gram-positive (Figure1.21 (b)), probably because of the difference between their outer membranes.

Figure 1 .

 1 Figure 1.21 (a) Contact angle of water on non-treated Ti surface (NP), after 2 min (P2) and 10 (P10) minutes of plasma treatment; (b) Fluorescent images showing the live (green) and dead (red, arrow) stained bacteria Staphylococcus aureus and Klebsiella oxytoca adherent to titanium disc 107

Figure 1 .

 1 Figure 1.22 (a) Water contact angle on different substrates before and after laser treatment; Fluorescence images for the untreated (b, d) and laser-treated (c, e) samples after 24 h of S. aureus ATCC 65.38 culture: Ti (b, c) and TiAlV (d, e) 106

Figure 1 .

 1 23 (a)), but exhibited cytotoxicity, as shown in Figure 1.23 (b) with a reduced of number of primary rats osteoblasts compared to controls. The toxic burst release of silver ions may be delayed by coating loaded TNT by a polyelectrolyte films 116 . This protective coating did not affect the antibacterial activity of Ag loaded TNT, but prevented the toxicity towards osteoblasts and played a key role in adhesion and spreading.

Figure 1 . 23

 123 Figure 1.23 Fluorescence images of bacteria on samples after 7 days of incubation (a), and primary rat osteoblasts cell numbers on samples after 1 and 4 days of incubation (b) 114

Figure 1 .

 1 Figure 1.24 SEM images of pure Ti (PT), titania coating (MT), Zn-incorporated titania coating (MT-Zn) and Zn/Sr-incorporated titania coating (a); antibacterial rate of surfaces against S. aureus (b) 115

Figure 1 . 25

 125 Figure 1.25 Molecular structures of some antibiotics immobilized on Ti surfaces

1. 26 (

 26 a)). Nevertheless, this coating showed a diminution of S. epidermidis adhesion and prevention of biofilm formation (Figure 1.26 (b)).

Figure 1 . 26

 126 Figure 1.26 Cumulative amounts of gentamicin released from surfaces with adsorbed gentamicin directly on Ti (Gent-Ti), or on carbonated hydroxyapatite (Gent-CHACs) (a); Relative number of viable bacteria (S. epidermidis) on the different samples after culturing for 24 h (b) 142

Chapter 1 .

 1 Literature review monolayers 134,135,133 . As a first step, titanium surface was etched with a mixture of H2SO4 and H2O2, before covalent immobilization of silanes like aminopropyltriethoxysilane, to obtain an amine-rich surface. The antibiotics were covalently coupled in a second stage using a linker, to take the drug at a certain distance from the surface. Vancomycin-Ti surfaces significantly prevented S. aureus biofilm formation over 30 hours incubation, and after seven re-incubations, but did not show any activity against E. coli biofilm 135 (see Figure1.27). According to the authors, this confirmed that the activity is due to the presence of the antibiotics, and not to a modification in surface roughness or chemistry, because vancomycin is only active against Gram + bacteria. Studies on osteoblasts showed the noncytotoxicity of these coating 133 .

Figure 1 . 27

 127 Figure 1.27 Bacterial colonization Control and Vanc-Ti rods after 30 h of incubation with S. epidermidis, a gram positive organism that falls within the spectrum of activity of vancomycin, or E.

Figure 1 . 28

 128 Figure 1.28 Antimicrobial activity of Ti-AMP and CaP-AMP against (a) P. aeruginosa and (b) S.aureus.149

Figure 1 .

 1 Figure 1.29 (a) Representation of peptide immobilized copolymer brush on surface; Antibacterial activity of peptide immobilized titanium wire against (b) P. aeruginosa and (c) S. aureus. 151

  Flavonoids can be subdivided into many subclasses: flavonols, flavones, flavanones, anthocyanidins, flavanols, and isoflavones (Figure1.30)164 . Catechins and epigallocatechin gallate, mainly found in tea, are parts of the flavan-3-ol class. They are the most studied natural bioactive molecules, for their antibacterial and antifungi activities. Flavonols, such as myricetin, morin and quercetin, have received much attention thanks to their remarkable activity against both Grampositive and Gram-negative bacteria. Flavones share a common skeleton with flavonols, without the hydroxyl on the central aromatic cycle. Baicalin and its aglycone baicalein are parts of this class (Figure1.31). They are extracted from the root of the plant Scutellaria baicalensis, known in the Chinese pharmacopeia for centuries for its anti-inflammatory 165 , antioxidant, antiviral, anticancer 166 and antibacterial161,167 properties. These two molecules have been characterized as ones of the most active molecules in Scutellaria baicalensis extracts161,167 . Moreover, antibacterial activity of baicalein was tested against S. aureus and MRSA168,169 . Baicalein alone was able to eradicate 7-days biofilm in a dosedependent manner, for concentrations above MIC168 . Use in combination with ciprofloxacin, baicalein at concentration below MIC was able to restore the antibacterial activity of ciprofloxacin against MRSA.

Figure 1 . 30 Figure 1 . 31

 130131 Figure 1.30 Chemical structures of the polyphenol classes and their biological properties 164

Figure 1 . 33

 133 Figure 1.33 Our strategy to develop a natural antibacterial molecule -biomimetic hydroxyapatite coating

  4 and 500 μL of fresh solution of DPPH at 100 μM in ethanol. Absorbance was measured at 517 nm after 20 minutes of reaction in the dark. DPPH inhibition was calculated by the equation: % inhibition = [1 -(Asample/ ADPPH solution)] × 100, with A the measured absorbance.

Figure 2 . 1

 21 Figure 2.1 Antioxidant acitivity of the four biologically active moleculesFigure 2.1 presents the DPPH bleaching percentage of the four BAMs. RA is the molecule with the best antioxidant activity, with a radical scavenging activity of 29 %. BE and BA have the same antioxidant activity, with a DPPH bleaching rate of ca. 5 %. CA have an intermediary behavior with 13.6 % of bleaching rate.

Figure 2 . 1

 21 Figure 2.1 Antioxidant acitivity of the four biologically active molecules

4

 4 Interactions of calcium with natural polyphenols exhibiting antioxidant properties 2.4.1 IntroductionPhytopharmacology has attracted much attention in the research field in the last decades not only because of its use as in traditional medicine over three thousand years mainly as herbal tea, but also in modern medicine since a large proportion of known drugs are molecules which have been identified in plants and used as such or as a derivative. Indeed, it is estimated that at least 25% of all modern medicines are derived, either directly or indirectly, from medicinal plants, primarily through the application of modern technology to traditional knowledge 2 . As a sign of this increasing interest, a list of 442 plants is included in the 2013 version of the European pharmacopoeia for their therapeutic use. However, control quality to ensure safety and efficacy in production of traditional medicines is difficult, thus precluding from ensuring the efficiency of a drug based on a plant extract. A more recent

Figure 2 . 2

 22 Figure 2.2 Molecular structures of (a) chlorogenic acid and (b) rosmarinic acid

Figure 2 . 3 .

 23 The position of the bands as well as their shift with pH are very similar for RA and CA (Figure 2.3 (a), (b)), which share the same caffeic acid moiety (Figure 2.3 (c)). In acidic pH, a maximum is observed at 325 nm for RA together with a shoulder at 288 nm (see Figure 2.3 (b)). At basic pH, the absorption maximum showed a bathochromic shift up to 365 nm. In addition, a new shoulder was observed around 307 nm. In parallel, an additional absorption band is visible at c.a.

Figure 2 . 3

 23 Figure 2.3 UV-Visible spectra of (a) chlorogenic acid and (b) rosmarinic acid at different pHs, BAM concentration 50 µmol.L -1 ; (c) structure of caffeic acid

Figure 2 . 4

 24 displays the absorbance of mixtures of BAM and calcium chloride at three different BAM:Ca molar ratios 1, 0.1 and 0.01 recorded in ammonium carbonate buffer, immediately after mixture and 24 hours later.

Figure 2 . 4

 24 Figure 2.4 UV-Visible spectra of (a) chlorogenic acid and (b) rosmarinic acid with calcium at pH 7.8 in (NH4)2(CO3) 1mM, BAM concentration 50 µM, molar ratios BAM: Ca 2+ . (c) and (d) inset between 550 and 650 nm

Figure 2 . 5

 25 shows mass spectra of equimolar solutions of BAMs with calcium at pH 7.8 in ammonium carbonate buffer. Rosmarinic acid and chlorogenic acid spectra exhibit the peak corresponding to the protonated molecule, at m/z 361.09 ([RA+H] + ) and 355.10 ([CA+H] + ), respectively, as the main peak. In addition, [BAM-H+Ca] + species were detected for CA and RA at m/z 394.10 and 393.05, respectively, with a significant intensity (≈5 % of that of the main peak). Additional peaks on

Figure 2 . 5 (

 25 Figure 2.5 (a) and (b) can be attributed to RA or CA species associated with ammonium.

Figure 2 . 5 8 Figure 2 . 6

 25826 Figure 2.5 MS spectra of chlorogenic (a) acid and rosmarinic (b) acids with calcium (AM/Ca = 1) at pH 7.8

13 C

 13 .7). Aromatic and vinylic protons were shifted up-field, while protons from the quinic part of the molecule were shifted low-field. A complexation of calcium at the carboxylic acid site leading to an electronwithdrawing effect on the adjacent carbons of the quinic moiety of CA, can explain the up-filed shift of the corresponding protons. In parallel, a deprotonation/complexation at the catechol site of the molecule could explain the low-field shift of aromatic and vinylic protons. Indeed, a facilitated deprotonation of one hydroxyl group complexed with calcium would induce a positive mesomeric effect along the conjugated moiety of the molecule. NMR studies confirmed the presence of the two complexation sites. Mesomeric effect to the conjugative part of the molecule lead to the shifting low-field of carbons in ortho and para positions compared to the deprotonated hydroxyl, and the shifting up-field of meta position. High low-field shift

Figure 2 . 1 H

 21 Figure 2.7 1 H (a), and 13 C (b) NMR spectra of chlorogenic acid pure (red line) and in presence of calcium, CA/Ca = 0.01 (blue line)

Figure 2 .

 2 Figure 2.8 1 H (a), and 13 C (b) NMR spectra of rosmarinic acid pure (red line) and in presence of calcium, RA/Ca = 0.01 (blue line)

Figure 2 . 9

 29 Figure 2.9 Antioxidant activity of BAM alone or in presence of calcium (molar ratio BAM/Ca 0.1)

Chapter 2 .

 2 2+ and [CA-H+Ca] + are the more intense ones. Additional fragmentation experiments performed for [2CA+Ca] 2+ showed the formation of two ions following the hydrolysis of the ester function: (i) a complex constituted by one CA, the calcium ion and the quinic moiety of the second CA, and (ii) the Molecular study: interactions between calcium and biologically-active molecules 84 caffeic acid part of the second CA (Figure 2.10). Calcium would therefore be complexed on the quinic acid part of the molecule, meaning on the carboxylate or on the 4-hydroxy-8-hydroxy site, as shown on Figure 2.11 (a). It is also possible that one molecule chelates the calcium by one of these sites, and the other by the catechol (Figure 2.11 (b)).

Figure 2 .

 2 Figure 2.10 MS/MS spectrum of chlorogenic acid with calcium (CA: Ca = 1), m/z = 374.08

Figure 2 . 11

 211 Figure 2.11 Calcium complexation by chlorogenic acid at pH 5 (a) and (b), and pH 7.8 (c)

  8 is more likely to take place on the catechol C19-C20, and the carboxylate is involved only if the concentration of calcium rises. Effects on the antioxidant activity, which is lowered in presence of calcium, strengthens this hypothesis. Unlike for chlorogenic acid, only two catechols of RA are responsible for the antioxidant activity so the complexation of one of them should have a strong effect on the antioxidant activity of the molecule.

Figure 2 . 04 Figure 2 . 13

 204213 Figure 2.12 MS/MS spectrum of rosmarinic acid with calcium (RA: Ca = 1), (a) m/z = 380.07, (b) m/z = 399.04

2 . 5

 25 was found highly pH-dependent, with monomolecular complexes formed at neutral pH and bimolecular complexes at acidic pH. Monomolecular complexes formed with deprotonated molecules possess the best stability. Both catechol and carboxylate groups are able to act as binding sites for calcium. Only the antioxidant activity of rosmarinic acid was decreased in the presence of calcium, due to the inactivation of the catechol moiety. Considering that calcium level in blood is ca. 1 mM, such interactions may play a role in bio-availability and in vivo efficiency of both rosmarinic and chlorogenic acid Stability of baicalin and baicalein at different pH and effect on calcium complexation 2.5.1 Introduction Baicalin (BA) and baicalein (BE) (Figure 2.14) are two flavonoids extracted from the root of the plant Scutellaria baicaleinsis. As mentioned in the literature review (Chapter 1), they are interesting

Figure 2 . 14

 214 Figure 2.14 Molecular structures with pKa (calculated with ADC iLab software) of baicalin (a) and baicalein (b)

Figure 2 .

 2 Figure 2.15 (c)). Z.S. Markovic et al 1 published an experimental UV spectrum of BE at pH 7 with two main peaks at 264 and 359 nm, which is consistent with our observations.

Figure 2 . 15

 215 Figure 2.15 Stability of baicalin (a) and baicalein (b) as a function of time in 0.1 M PBS pH 7.2, at a concentration of 50 µmol.L -1 , and structure of flavonoids (c)

Figure 2 . 16

 216 Figure 2.16 Stability of baicalin (a) and baicalein (b) as a function of time in ultrapure water, at a concentration of 50 µmol.L -1

  (a)), an increase of the pH clearly led to the collapse of band I (317 nm) above pH 7 while band slightly decreases II (276 nm) in intensity. In addition, a new band with a maximum at 432 nm appears, which could sign for the deprotonation of the catechol with the lowest pKa that is 9.2 (Figure2.14 Molecular structures with pKa (calculated with ADC iLab software) of baicalin (a) and baicalein (b). For baicalein (Figure2.17 (b)), band II (274 nm) undergoes a progressive slight red-shift while band I (323 nm) shifts towards longer wavelengths (≈50 nm), according to the supposed facilitated deprotonation of catechol12 . A new shoulder near 450 nm is observed at pH 10.

Figure 2 .

 2 Figure 2.17 UV-Visible spectra of (a) baicalin and (b) baicalein at different pHs, BAM concentration 50 µmol.L -1

Figure 2 .

 2 Figure 2.18 presents mass spectra at pH 5. Shortly after preparation (Figure 2.18 (a)), the main peak is detected at m/z 271.06, corresponding to a protonated adduct of baicalein [BE+H] + . Besides this peak, a peak with low relative abundance is present at m/z 269.05, ascribed to a molecule of baicalein that would have lost two protons. The hydrated adduct of this molecule is also found at m/z 287.22, with a higher relative abundance, suggesting that the modified BE is more stable when hydrated. Spectrum after 20 hours (Figure 2.18 (b)) is very similar. The only difference is the relative abundance of the peak at m/z 269.05 that is slightly higher than in Figure 2.18 (a). Spectra at pH 7.8 are presented in Figure 2.19. The same peaks than in pH 5 are present, with different relative abundances. The main peak is at m/z 285.05 that corresponds to adduct of BE that would have lost two protons and H2O. No difference between the two spectra are visible, meaning that the degradation of the molecule in ammonium carbonate buffer at pH 7.8 occurs within the time of preparation of the solution and injection in mass spectrometer.

Figure 2 .Figure 2 .Figure 2 . 20

 22220 Figure 2.18 ESI-MS spectra of baicalein in ultrapure water at pH 5 shortly after preparation of the solution (a) and 20 hours after (b). BE concentration = 50 µM

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

 225312 Figure 2.21 UV-Visible spectra of baicalin (a) and baicalein (b) with calcium at pH 7.8 in 1mM of buffer (NH4)2(CO3) immediately after mixture and after 6 hours. BAM concentration 50 µM; molar ratios are expressed regarding BAM: Ca concentration

At pH 7 . 8 ,

 78 baicalin exhibits only one peak corresponding to a complex with calcium, with a very low relative abundance, at m/z 483.03 (Figure2.23 (a)). It corresponds to a complex formed with calcium and BA that lost two protons, in other words an oxidized BA. The complex would have the formula [BAox-H +Ca] + . Other peaks in the spectrum are assigned to the protonated molecule at m/z 447.10 ([BA+H] + ), an hydrated adduct of the oxidized molecule at m/z 463.09 ([BAox+H+H2O] + ), and two peaks corresponding to oxidized BE at m/z 269.05 and 287.05 ([BEox+H] + and ([BEox+H+H2O] + , respectively). This is in agreement with the fact that baicalin is unstable at pH 7.8, and that the sugar moiety is hydrolyzed during degradation, as suggested by Xing et al.40 On the ESI-MS spectrum of baicalein at pH 7.8 (Figure2.23), no peak corresponding to a complex between BE or its oxidized form with calcium is detected. The same three peaks that were observed on the ESI-MS spectrum of BE at pH 7.8 without calcium are detected: [BE+H] + , [BEox+H] + and [BEox+H+H2O] + , at m/z 271.06, 269.06 and 287.22 respectively.Overall at pH 7.8, the oxidation process strongly decreases the calcium-binding efficiency of BA and BE. Oxidized BA seems to be unstable, as its final products of degradation (BEox+H] + and Chapter 2. Molecular study: interactions between calcium and biologically-active molecules 101 ([BEox+H+H2O] + ) are observed in large quantity. The oxidized molecule is only stabilized by adduct with water or calcium.

Figure 2 .Figure 2 .

 22 Figure 2.22 ESI-MS spectra of baicalin (a) and baicalein (b) in ultrapure water at pH 5 in presence of calcium at ratio BAM/Ca = 1:1. BAM concentration = 50 µM

Figure 2 . 24

 224 Figure 2.24 Complexes formed by baicalein and calcium at pH 5

3 .

 3 Formation of hybrid hydroxyapatite -biologically active molecule nanoparticles ........................ 3.1 Introduction ............................................................................................................................... 3.2 Hydroxyapatite synthesis .......................................................................................................... 3.3 Incorporation of BAMs .............................................................................................................. 3.3.1 Post synthesis adsorption ................................................................................................... 3.3.2 In situ synthesis .................................................................................................................. 3.3.3 Comparison of incorporation methods .............................................................................. 3.4 Biological activity of hydroxyapatite -baicalein hybrid particles obtained by adsorption ...... 3.4.1 Introduction ........................................................................................................................ 3.4.1 Baicalein release in vitro ..................................................................................................... 3.4.2 Antioxidant activity............................................................................................................. 3.4.4 Antibacterial activity ........................................................................................................... 3.4.5 Conclusions ......................................................................................................................... 3.5 Conclusions ................................................................................................................................ References .......................................................................................................................................

Figure 3 .

 3 1 (c)).

Figure 3 . 1 X

 31 Figure 3.1 X-ray diffractogramm (a), TEM image (b), and N2 physisorption isotherm of synthesized hydroxyapatite particles

Figure 3 . 2 Figure 3 . 3 X 1 H, 13 C 3 . 4 .

 323311334 Figure 3.2 TEM image of hydroxyapatite after adsorption of (a) rosmarinic acid, (b) chlorogenic acid, (c) baicalein and (d) baicalin

Figure 3 .

 3 Figure 3.4 1 H solid-state NMR spectra of HAp, HAp-CA, HAp-BE and HAp-BA (initial concentration for adsorption = 0.05 mM) (* indicates residual peaks of acetone and ethanol)

Figure 3 . 5

 35 Figure 3.5 13 C cp-mas solid-state NMR spectra of HAp, HAp-CA, HAp-BE and HAp-BA (initial concentration for adsorption = 0.05 mmol.L -1 ). NS = 47752, d1 = 1s, p15 = 2 ms.

Figure 3 . 6

 36 Figure 3.6 31 P solid-state NMR spectra of HAp, HAp-CA, HAp-BE and HAp-BA (initial concentration for adsorption = 0.05 mmol.L -1 ).

Figure 3 . 7

 37 Figure 3.7 2D hetero correlation map 1H-31P of HAp-BE (a) and HAp (b). NS = 256, d1 = 1 s, p15 = 10 ms, TD = 180

Figure 3 . 9

 39 Figure 3.9 TEM images of (a) HAp-RA, (b) HAp-CA, (c) HAp-BE and (d) HAp-BA

Figure 3 . 3 . 11 .

 3311 Figure 3.10 XRD pattern of hydroxyapatite particles synthesized in the presence of rosmarinic acid (HAp-RA), chlorogenic acid (HAp-CA), and baicalein (HAp-BE) (Ca:BAM = 0.075), and comparison with patterns of hydroxyapatite (JCPDS 64-0738) and calcite (JCPDS 05-0586)

Figure 3 .

 3 Figure 3.11 TEM images of (a) HAp-BE and (b) CaP-RA (initial BAM:Ca = 0.075)

Figure 3 . 12

 312 Figure 3.12 Stability of (a) RA, (b) CA, (c) BE and (d) BA at pH 12.

  in the presence of RA, except for a trace of remaining Ca(OH)2 after 30 min. This would indicate that RA has only a minor influence on Ca(OH)2 dissolution. It is also important to keep in mind that upon ammonium phosphate addition, without BAM, only HAp is formed while in the presence of RA at high concentration only CaCO3 is identified with intermediate situations, i.e. both CaCO3 and HAp being present, for other BAMs. Therefore RA may either inhibit CaCO3 dissolution or inhibit HAp formation.

Figure 3 .

 3 Figure 3.13 XRD of powder recovered after 30 min in solution of Ca(OH)2 and Ca(OH)2 + RA., and comparison with XRD of calcite (PDF card 05-0586)

Table 3 . 5

 35 despite the fact that baicalein is released by 40 %. Two explanations are possible: either the released quantity is too low for detection of antioxidant activity, or baicalein released in PBS is degraded and not active any more. Indeed, as said in Chapter 2, oxidized baicalein does not have antioxidant properties. On the contrary, particles have a radical scavenging activity of about 80 % after the first test. After 24 hours in PBS, particles are still active, with an antioxidant activity of about 50 %. Particles loose a part of their activity in PBS, as after 4 days in PBS activity is only 18 %. Interestingly, at day 5, one day after the previous testing, activity reaches again 50 %. To conclude, immobilized baicalein is still active, and a sufficient quantity remains after release to maintain the antioxidant activity of particles. It is worth to note that the antibacterial activity of hydroxyapatite particles without baicalein should be tested shortly. Antioxidant activity of HAp -adsorbed baicalein particles before and after immersion in PBS (m = 10 mg).

Figure 3 . 15

 315 Figure 3.15 Structure of (a) quercetin and (b) catechin

Figure 3 . 16

 316 Figure 3.16 Disk diffusion assay on a MH-agar plate seeded with S. epidermidis CIP 68.21

Figure 3 . 17

 317 Figure 3.17 Antibacterial activity of HAp particles with adsorbed baicalein, test in MH

Figure 3 . 18

 318 Figure 3.18 Antibacterial activity of HAp particles with adsorbed baicalein, test in PBS

Figure 3 . 19 .

 319 Figure 3.19. The concentration of bacteria for particles with adsorbed baicalein and adsorbed baicalein after incubation in PBS are similar to the one of hydroxyapatite particles. The concentration in lower than initial concentration, but unfortunately we do not have the data concerning the negative control, meaning the concentration of bacteria after 24 hours of incubation in PBS. Nevertheless, this result suggests than even with a lower initial concentration of bacteria, particles with baicalein do not exhibit any antibacterial activity.

Figure 3 . 19

 319 Figure 3.19 Antibacterial activity of HAp particles with adsorbed baicalein, before and after incubation in PBS, test in PBS

Figure 4 . 1

 41 Figure 4.1 shows the X-ray diffraction pattern of plates before and after annealing. Diffraction peaks corresponding to α-titanium and β-titanium are visible for the plate after alkali treatment. After annealing, the diffraction pattern of sample signs for a more crystalline materials, with narrower peaks and a better signal to noise ratio. Peak at 25 ° is characteristics of TiO2 in an anatase and brookite mixture, both being "kinetics" phases of titanium oxide. Peaks are weak because titanium oxide is only present at the surface of the substrate. No peak of TiO2 rutile are detected, probably because the duration of annealing treatment was not enough to transform anatase to rutile.

Figure 4 . 1 X

 41 Figure 4.1 X-ray diffraction patter of alkali-treated Ti6Al4V samples (a) before and (b) after annealing

Figure 4 . 2

 42 Figure 4.2 SEM images of hydroxylated Ti6Al4V surfaces (a,b) before and (c,d) after annealing. (H = hydroxylated; A = annealed)

Figure 4 . 3 and

 43 Figure 4.3 and Figure 4.4 show the deposited layer of material after immersion in 1.5-SBF of the hydroxylated surfaces and the hydroxylated/annealed surfaces, respectively. After 7 days of deposition on the non-heat-treated surface (Figure 4.3 (a)), we observe a bottom layer covered by a cauliflowershaped material, more visible on the image at higher magnification (Figure 4.3 (b)). After 14 days (Figure 4.3 (c)), the material is denser, consisting of plates separated by cracks. The cauliflower shape is still visible, but the surface is fairly flat, compare to the surface after 7 days of deposition. The image at higher magnification (Figure 4.3 (d)) show the morphology of the head of the cauliflower. Some cracks are formed after 14 days, probably due to drying of a very dense and thick layer. The material grows from the bottom to the top, which is visible on Figure 4.3 (a). Between 7 days and 14 days, this

Figure 4 . 4 (

 44 a) shows the alkalitreated and annealed surface after 7 days of deposition. It is visible that the surface is not homogeneously covered, as the deposited material is only located at the center of the image, looking like a small island. The thickness of the material increases from the edges of the spot to the center. We Chapter 4. Elaboration of hybrid hydroxyapatite -biologically active molecules coatings on Ti6Al4V titanium alloys 155 can recognize the cauliflower-shaped material at the center, with plates of dense material in the middle-height of the small island. At the edges, on the bottom, small round particles are visible. On this image, we can observe all the steps of the formation of the synthesized material after deposition in SBF. The image at higher magnification (Figure 4.4 (b)) shows a non-covered part. The morphology is the same than the Ti6Al4V surface after hydroxylation and annealing, before deposition, showing that no material has been deposited.

Figure 4 . 4 (

 44 c), showing the annealed surface after 14 days of deposition, is split in two parts. On the left side, we can observe a layer of deposited material, with a crack between two dense plates. The right side of the image consists of the non-covered surface of Ti6Al4V. A difference in height between the two parts of the image is clearly visible, suggesting that the deposited layer is thick.

Figure 4 . 4 (

 44 d) shows a plate with very thin layer of material, and some small spots of deposited material, that charge under the electronic flow of the microscope.From the observation of the images presented inFigure 4.4, we can conclude that the annealing step does not favor the uniform deposition of hydroxyapatite. Surfaces are not fully covered by the material but a very dense and thick layer can be formed locally. On the contrary, surfaces that were only hydroxylated are fully covered with a homogeneous layer of material (Figure 4.3).

Figure 4 . 3 Figure 4 . 4 4 . 5 .

 434445 Figure 4.3 SEM images of hydroxylated Ti6Al4V plates after (a,b) 7 days and (c,d) 14 days of soaking in 1.5-SBF

Figure 4 . 5 X

 45 Figure 4.5 X-ray diffractograms of substrates after 7 days (a,c) and 14 days (b,d) of deposition in 1.5-SBF: (a) and (b) alkali-treated Ti6Al4V; (c) and (d) alkali-treated and annealed Ti6Al4V.

Figure 4 . 6 (

 46 a) and (b)), characterized by the intense phosphate vibration ν1 at 960 cm -1 , typical of hydroxyapatite. Other phosphate vibrations in HAp are also visible,

Figure 4 . 7 ,

 47 and compared with surfaces after deposition in 1.5-SBF (Figure 4.7 (a) and (b) already shown in 4.2.1.1.2). After 7 days of deposition, diffraction peak of HAp at 26° and 31° are absent from the diffractogram (see Figure 4.7 (c)), that may be explained by several hypotheses: either the deposited layer is too thin or amorphous. More, as evidenced by optical microscopy analysis, the layer was not continuous. The amount of calcium phosphate deposited on the surface was maybe too small to be detected by diffraction. On the contrary, after 14 days of soaking, HAp peaks at 26° and 31° are clearly visible (see Figure 4.7 (d)), suggesting a significant deposition of HAp.

Figure 4 . 7 X 4 . 8 .

 4748 Figure 4.7 X-ray diffractograms of substrates after 7 days (a), (c) or 14 days (b), (d) of deposition in 1.5-SBF (a), (b) or 1.5-TSBF (c), (d)

Figure 4 . 8 (

 48 Figure 4.8 (c) and (d) respectively, the noise is very important. After 7 days, the ν1 vibration of phosphate is distinguishable, while other phosphate vibrations are absent, or merged in the noise.After 14 days, besides the ν1 vibration at 960 cm -1 , ν2 vibration at 430 cm -1 and ν3 vibration at 1075 cm -

Figure 4 . 8

 48 Figure 4.8 Raman spectra of substrates after 7 days (a), (c) or 14 days (b), (d) of deposition 1.5-SBF (a), (b) or 1.5-TSBF (c), (d). Green lines: phosphate vibration of hydroxyapate; pink lines: carbonates vibration in carbonated hydroxyapatite

Chapter 4 . 4 . 2 . 2

 4422 Elaboration of hybrid hydroxyapatite -biologically active molecules coatings on Ti6Al4V titanium alloys162 Characterization of SBF deposited in 1.5

Figure 4 . 9 (

 49 a), (b) and (c) respectively. Taking into account the tilt angle (45°) of the measure (thickness= measured distance sin 45

Figure 4 . 9

 49 Figure 4.9 SEM images of HAp layer after 14 days of deposition of Ti6Al4V, tilt = 45 °. Magnification = x1k (a), x2k (b) and x10k (c)

Figure 4 .

 4 Figure 4.10 EDS spectrum of HAp layer

Figure 4 .

 4 Figure 4.12 SEM images of HAp coatings with BAM incorporation: rosmarinic acid (a), (b); chlorogenic acid (c), (d); baicalein (d), (e); and baicalin (g), (h).

Chapter 4 .

 4 Elaboration of hybrid hydroxyapatite -biologically active molecules coatings on Ti6Al4V titanium alloys 168 X-ray diffraction was performed to verify the crystalline phase of the deposited coating with chlorogenic acid and baicalein.

Figure 4 .

 4 13 presents the X-ray diffractogramms between 20 and 37 ° of HAp-CA and HAp-BE compared with pure HAp. For both coatings with BAM, the peak at 26 ° and the range at 32 ° are present. No difference between the three coatings are visible. The deposition of hydroxyapatite in presence of BAM is therefore confirmed.

Figure 4 .

 4 Figure 4.13 X-ray diffractograms of hydroxyapatite deposited on Ti alloy in presence of chlorogenic acid and baicalein, and comparison with pure hydroxyapatite. 2θ = 20 -37 °

Figure 4 . 14

 414 Figure 4.14 Raman spectra of HAp coatings deposited (a) in presence of chlorogenic acid (HAp-CA) and (b) baicalin (HAp-BA)

Figure 4 .

 4 15 presents the overlay of spectra between 800 and 1300 cm -1 for coatings deposited with the four molecules, and their comparison with pure HAp coating. The presence of the most intense phosphate vibration of HAp at 1031 cm -1 is present on all spectra, as well as shoulder at 1062 cm -1 8 . This confirms the deposition of hydroxyapatite in presence of BAM.

Figure 4 .Figure 4 .

 44 Figure 4.15 ATR-IR spectra of BAM-incorporating HAp coatings, and comparison with pure HAp coating

Figure 4 . 17

 417 Figure 4.17 Thermogravimetric analysis of coatings on TiAlV (a) pure Hap, (b) HAp-BE, and (c) HAp-BA

Figure 4 .

 4 20 (b)), the peak at 518 nm was assigned to BE. The shift from 540 to 518 nm probably results from the modification of the environment of the molecule on the hydroxyapatite surface, compared to in a DMSO solution. HAp has an intrinsic fluorescence at 395 nm, as seen on Figure 4.20 (b), which is also visible on the spectrum of HAp with adsorbed baicalein. Thus, fluorescence spectroscopy confirms the adsorption of baicalein on the hydroxyapatite surface. However, even if the incorporation of baicalein is visible, fluorescence does not allow the measure of the exact adsorbed quantity.

Figure 4 . 20 Figure 4 .

 4204 Figure 4.20 Fluorescence spectra of (a) baicalein 50 µM in DMSO, λex = 320 nm and (b) hydroxyapatite coatings before and after adsorption of baicalein, λex = 300 nm

Figure 4 .Figure 4 .

 44 Figure 4.21 SEM images of hydroxyapatite coating on Ti6Al4V after horizontal adsorption of baicalein. Magnification: 500 (a), 1.00 k (b) and 3.00 k (c)

Figure 4 .

 4 23 (a), (b) for the horizontal adsorption, and (c), (d) for vertical adsorption.

Figure 4 .

 4 Figure 4.23 SEM images of hydroxyapatite coatings on Ti6Al4V after immersion in water for 24 hours, horizontally (a), (b), or vertically (c) and (d). Magnification: 500 (a), (c) and 2.5 k (b) and (d)

Chapter 4 .

 4 Elaboration of hybrid hydroxyapatite -biologically active molecules coatings on Ti6Al4V titanium alloys hydroxyapatite coated quartz used as received from the supplier.

Figure 4 .(Equation 4 . 1 )Equation 4 . 1

 44141 24 (a) represents the monitoring of frequency and dissipation in function on time during baicalein adsorption at a concentration of 5.10 -5 M in ultrapure water, with a flux of 25 µL.min -1 . As the dissipation is lower than one, we can consider that the hydroxyapatite layer is rigid, and we can use the Sauerbrey's relation to obtain the adsorbed weight (Δw, ng) from the frequency variation (ΔF, Hz): the harmonic order and Cf = 17.7 ng.Hz -1 .cm -2 (constant) Sauerbrey's relation During this experiment, after air and water stabilization, we started an adsorption step for 2 hours (1 st injection). A release phase, corresponding to the passage of ultrapure water, followed the adsorption step and lasted for 75 minutes (1 st release). Another adsorption step was conducted for 260 min (2 nd adsorption), before a second release step (2 nd release) for 66 minutes.

Figure 4 .

 4 24 (b) represents the adsorbed weight quantity in function on time, obtained with the Sauerbrey's relation. During the 1 st adsorption step, 136 ng of baicalein were adsorbed, corresponding to 0.5 nmol. After the 1 st release step, 70 ng were released, corresponding to a remaining quantity of baicalein of 66 ng (0.28 nmol). 44 % of adsorbed baicalein were released in 75 min. It is interesting to note that after the 1 st release it was still possible to adsorb baicalein, because 89 ng were adsorbed on top of the 66 ng remaining. The total amount adsorbed after the 2 nd adsorption step is 155 ng, corresponding to 0.57 nmol. During the 2 nd release step, 47 % of the total adsorbed baicalein were released. At the end of the experiment, 101 ng of baicalein are adsorbed on the hydroxyapatite functionalized quartz, corresponding to 0.37 nmol.

Figure 4 . 24

 424 Figure 4.24 Baicalein adsorption monitoring by QCM on a hydroxyapatite functionalized quartz (a), and adsorbed weight obtained from adsorption monitoring (b)

Figure 4 . 25

 425 Figure 4.25 Antiadhesion effect of hydroxyapatite coatings incorporating baicalein on S. epidermidis (NS: non significant, *: p ≤ 0.05)

  broth (MH) for 24 hours at 37 °C. After incubation, samples were processed as presented before to recover the bacteria present on the surface and plate counting. Results expressed as the log of the number of proliferated CFU are presented in Figure4.26. First, bacteria proliferated on the four different plates, as the number of bacteria grows from ca. 5 log to ca. 8 log for each surface.

Figure 4 . 26

 426 Figure 4.26 Effect of hydroxyapatite coatings incorporating baicalein on S. epidermidis proliferation in rich medium after 3 h of initial adhesion (NS: non significant, *: p ≤ 0.05, **: p ≤ 0.01)

Figure 4 . 27 4 . 28 .

 427428 Figure 4.27 Antibacterial activity kinetics of baicalein against S. epidermidis CIP 105.777 in rich and poor medium, at a concentration twice the MIC

Figure 4 .

 4 Figure 4.28 S. epidermidis growth inhibition in rich medium by HAp coatings incorporating baicalein (NS: non significant, **: p ≤ 0.01)

2 :Figure 4 . 29

 2429 Figure 4.29 Percentage of viability of human osteoblasts cultured on titanium alloy, hydroxyapatite coating and hydroxyapatite with adsorbed baicalein, compared with plastic culture plate as control

Figure 4 . 30

 430 Figure 4.30 Fluorescence microscope images of human osteoblasts cultured on plastic culture plate (a), (b), titanium alloy (c), (d), hydroxyapatite coatings (e), (f), and hydroxyapatite coatings with adsorbed baicalein (g), (h), after 24 hours (a), (c), (e), (g) and 7 days (b), (d), (f) and (h). Magnification x10, blue: DAPI, green: phalloidin

Chapter 4 .4. 6 Summary

 46 Elaboration of hybrid hydroxyapatite -biologically active molecules coatings on Ti6Al4V titanium alloys 191In the next paragraphs, we will try to sum up the conclusions about the behavior of the four biologically active molecules from the three previous chapters of the manuscript, i.e. the molecular study (Chapter 2), the formation of hybrid hydroxyapatite -biologically active molecules nanoparticles (Chapter 3), and the development of hybrid biomimetic hydroxyapatite -baicalein coatings on Ti6Al4V alloy (Chapter 4).

Figure 5 . 1

 51 Figure 5.1 Research strategy of this PhD work

literature 1 -

 1 4 to oxidize at different kinetics rate at physiological pH. Any new study with other natural molecule should start with the determination of the stability of the molecule at different pHs. Moreover, in traditional medicine, plant extracts containing several antibacterial molecules acting synergistically are used, and not single components. Hydroxyapatite coatings or nanoparticles incorporating several antibacterial molecules from the same plant could be a solution to improve the General conclusions and perspectives 204 antibacterial activity of the material. For example, in Scutellaria baicalensis, baicalin and baicalein are in presence of wogonoside, daidzein and maackiain (see Figure 5.2). The immobilization of one or several molecules in addition to baicalein could help to improve the antibacterial activity of the material. Moreover, it has been shown that in presence of other flavonoids, stability of baicalein is improved 5 .

Figure 5 . 2

 52 Figure 5.2 Molecules found in Scutellaria baicaleinsis extracts

  d'arthrose, est commune. L'arthrose est une maladie qui caractérisée par la disparition du cartilage favorisant le glissement de la tête fémorale dans la cavité cotyloïde. Pour réduire la douleur et retrouver la fonction de l'articulation dans le but d'améliorer la qualité de vie des patients, l'articulation défectueuse est remplacée par un implant pendant une arthroplastie totale de hanche. C'est l'une des chirurgies orthopédiques les plus communes, avec plus de 100 000 personnes opérés chaque année en France 1 .Depuis toujours les Hommes ont essayé de remplacer des parties défectueuses du corps humain par des implants en matériaux naturels ou synthétiques. Pour la prothèse de hanche les plus anciens essais enregistrés datent de 1891 en Allemagne, avec utilisation de l'ivoire (matériau naturel, composant des dents et des défenses d'éléphants) pour remplacer la tête fémorale 2 . Les implants utilisés de nos jours sont proches de ceux développés dans les années 1960 par Sir John Charnley. Ces implants sont constitués de trois parties, une tête fémorale métallique, une cavité cotyloïde en polyéthylène et un ciment en acrylique. Le ciment permet d'améliorer la fixation de la prothèse, en agissant comme une colle. Aujourd'hui, trois types de prothèses sont disponibles, métal-sur-polyéthylène, métal-sur-métal, et céramique-sur-céramique, chacun avec des avantages et des inconvénients. Des méthodes n'utilisant pas de ciment sont également développées, car celui-ci peut être dégradé pendant la marche, ce qui cause une réponse inflammatoire et peut entrainer le retrait de l'implant.

Figure 1 .

 1 Figure 1. Anatomie de la hanche, position de l'implant à la place de l'articulation et description d'un implant classique

Figure 2 .

 2 Figure 2. Stratégie développée dans cette thèse

  Les quatre MABs ont par la suite été incorporées dans des nanoparticules d'hydroxyapatite par deux méthodes. D'un côté elles ont été incorporées concomitamment à la synthèse des nanoparticules, directement dans le milieu réactionnel, et de l'autre côté elles ont été adsorbées après la synthèse sur des nanoparticules préformées. L'adsorption de molécules biologiquement actives sur l'hydroxyapatite a déjà été rapportée dans la littérature, pour la délivrance d'agents antibactériens ou ostéogéniques. L'adsorption des quatre molécules ne modifie pas significativement la structure des nanoparticules, comme cela a été montré par microscopie électronique à transmission. La molécule incorporée en plus grande quantité, mesurée par analyse thermogravimétrique, est la baïcaléine. Ceci peut être expliquée par la présence de la fonction pyrogallol sur la baïcaléine, qui a été montré comme étant adsorbée plus vite et avec une interaction plus forte sur l'hydroxyapatite, comparée aux fonctions acide carboxylique et catéchol. Dans le cas de l'incorporation des quatre MABs pendant la synthèse des nanoparticules, deux concentration de MABs ont été testées, avec des ratios MAB:Ca = 0.075 et 0.0075. La présence des molécules organiques inhibe partiellement ou totalement la synthèse des nanoparticules. A bas ratio, l'hydroxyapatite est bien synthétisée, mais avec un plus faible rendement et une taille des cristallites plus petite qu'en absence de MAB. A plus grand ratio, dans le cas de l'acide rosmarinique ce n'est pas de l'hydroxyapatite mais le carbonate de calcium calcite qui est synthétisé. Cette phase est également trouvée dans les particules synthétisées en présence de baïcaléine et d'acide chlorogénique. La calcite est un intermédiaire lors de la synthèse de l'hydroxyapatite selon notre méthode. Il se forme après dissolution de l'hydroxyde de calcium. Les molécules organiques peuvent s'adsorber à la surface de la calcite, ce qui pourrait limiter voire inhiber sa dissolution et donc la formation de l'hydroxyapatite. Par ailleurs, nous avons souligné la dégradation des MABs à pH basique, qui est le pH employé lors de la synthèse des nanoparticules. En conclusion, la voie de synthèse par incorporation concomitante à la synthèse des nanoparticules n'est pas la plus adaptée pour obtenir des nanoparticules hybrides. C'est la raison pour laquelle nous avons décidé de Résumé en français XII tester uniquement l'activité biologique des nanoparticules obtenues après adsorption de molécule active. Notre choix s'est porté sur les particules après adsorption de baïcaléine, car c'est la molécule adsorbée en plus grande quantité, et qui présente la meilleure activité antibactérienne. La libération in vitro de la molécule dans un tampon phosphate salin (PBS) a été étudié par chromatographie liquide haute performance (HPLC). Il suit un profile typique avec une forte quantité libérée initialement, suivie d'une phase de libération plus lente. La quantité libérée en 5 jours correspond à 40 % de la quantité totale incorporée, ce qui signifie 25 mg de baïcaléine libérée pour 1 g de nanoparticules. Après adsorption sur l'hydroxyapatite, la baïcaléine reste active. En effet, les nanoparticules montrent une activité antioxydante prolongée sur plusieurs jours. Nous suggérons que c'est la molécule adsorbée qui est responsable de l'activité antioxydante, plutôt que la molécule relarguée dans la solution. En effet, comme la baïcaléine est oxydée à pH physiologique, elle perd son activité de piégeage des radicaux libres. Concernant l'activité antibactérienne, notre système n'a pas montré d'activité significative contre S. epidermidis. Nos nanoparticules hybrides sont moins efficaces que d'autres systèmes de relargage d'antibiotiques trouvés dans la littérature, mais ceci peut s'expliquer par l'utilisation de molécules naturelles, qui possèdent des CMI bien plus faibles que les antibiotiques classiques utilisés comme la tétracycline ou la vancomycine.

Figure 3 . 3 .

 33 Figure 3. Incorporation des molécules actives biologiquement dans/sur des nanoparticules d'hydroxyapatite par incorporation dans la solution de dépôt ou par adsorption. Images de microscopie électronique à transmission et comparaison des diffractogramme des rayons X.

Figure 4 .

 4 Figure 4. Incorporation de la baicaléïne dans/sur des revêtements d'hydroxyapatite sur des alliages de titane par incorporation dans la solution de dépôt ou par adsorption. Images de microscopie électronique à balayage, diffractogramme des rayons X et spectroscopie de photoélectron X de l'oxygen O 1s. Comparaison de l'effet antibactérien sur Staphylococcus epidermidis CIP 105.777.

  3-can easily be substituted by carbonate ions.

	Ca/P	Name	Formula
	0.5	Monocalcium phosphate monohydrate (MCPM)	Ca(H2PO4)2 .H2O
	0.5	Monocalcium phosphate anhydrous (MCPA)	Ca(H2PO4)2
	1.0	Dicalcium phosphate dihydrate (DCPD)	Ca(HPO4). 2 H2O
	1.0	Dicalcium phosphate anhydrous (DCPA)	CaHPO4
	1.33	Octacalcium phosphate (OCP)	Ca8(HPO4)2(PO4)4. 5 H2O
	1.5	α -Tricalcium phosphate (α -TCP)	α -Ca3(PO4)2
	1.5	β -Tricalcium phosphate (β -TCP)	β -Ca3(PO4)2
	1.2 -2.2	Amorphous calcium phosphate (ACP)	CaxHy(PO4)z . nH2O n = 3-4,5; 15-20% H2O
	1.5 -1.67	Calcium-deficient hydroxyapatite (CDHA)	Ca10-x(HPO4)x(PO4)6 -x(OH)2-x (0 < x < 1)
	1.67	Hydroxyapatite (HAp)	Ca10(PO4)6(OH)2
	1.67	Fluoroapatite (FAp)	Ca10(PO4)6F2
	2.0	Tetracalcium phosphate (TTCP)	Ca4(PO4)2O

Table 1 .

 1

1 (Ortho)calcium phosphate family. Minerals with their names and formula are sorted by their Ca/P ratio.

Hydroxyapatite is the more stable calcium phosphate phase, to the exception of fluoroapatite,

Table 1 .

 1

	Essential	Requirement
	Thickness	Not specific
	Ca/P ratio	1.67 -1.76
	Phase purity	95 % minimum
	Crystallinity	62 % minimum
	Tensile strength	> 50.8 MPa
	Shear strength	> 22 MPa
	Density	2.98 g/cm 3
	Heavy metals	< 50 ppm
	Abrasion	Not specific

Two main strategies are available to form hydroxyapatite coatings. On one hand, several deposition techniques exist to coat a substrate with presynthesized hydroxyapatite particles; on the other hand, it is possible to get the mineral to crystallize directly at the substrate surface. Current requirements for hydroxyapatite coating in bone regeneration are listed in Table

1

.2, as expected by the American Food and Drug Administration (FDA) for commercialization of the implant, following ISO standards. Coatings should be pure hydroxyapatite with good crystallinity. It is interesting to note that the Ca/P ratio corresponds to stoichiometric or even sur-stoichiometric hydroxyapatite, meaning that FDA requirements are distant from the characteristics of biomimetic hydroxyapatite coatings. This chapter will focus first on different methods to synthesize hydroxyapatite powders, followed by the presentation on the available coating techniques. 2 HAp coating requirements by FDA, following ISO standards

49

  large number of deposition methods are available to coat a surface, metallic or polymeric with hydroxyapatite, and in a larger extent calcium phosphate mineral. A number of books or reviews deals with methods to deposit calcium phosphates and more precisely hydroxyapatite on metallic substrates[START_REF] León | Thin Calcium Phosphate Coatings for Medical Implants[END_REF][START_REF] Mohseni | [END_REF][76] . Table1.3 gathers some of coating process used to deposit hydroxyapatite on metallic substrates. Plasma-spray technique is the only one that is approved by the American

	1.3.2 Coatings process
	1.3.2.1 From preformed particles

A

Table 1 .

 1

	Hamdi et	al.

81 Choi et al. 78 Asri et al. 82 Bosco et al. 83 Asri et al. 82 Hsieh et al. 3 Deposition of HAp via different coating techniques 1.3.2.2 Wet process 1.3.2.2.1 Electrochemical deposition and micro-arc oxidation coatings

Table 1 .4 Ionic compositions of blood plasma and different SBF formula

 1

see Table 1.4), but

  both are at physiological pH. In this study, the titanium surface underwent an acidic treatment in H3PO4 and an alkali heat treatment in NaOH solutions for 5 hours at 140 C° under 3 bars of pressure. Deposition from FCS and HBSS solutions lasted 14 days at 37 °C with replenishments of the solution every day. By SEM, they showed a homogeneous deposition of HAp but the surface was not fully covered (

	Chapter 1. Literature review
	HBBS contains more diverse ions than FCS (
	The Dulbecco's phosphate
	buffered saline (DPBS) (see composition in Table 1.4) allowed to deposit hydroxyapatite with 7 days at
	60°C on commercially pure titanium, pretreated by physical vapor deposition to obtain a TiO2 anatase
	surface 101 . Hypercalcified solution like Fast calcification solution (FCS) and Hank's balanced salt solution
	(HBSS) are also available to deposit hydroxyapatite on titanium surface after surface pretreatment 102 .

Figure 1.19). Figure 1.19 SEM

image of titanium surface after 2 weeks immersion in HBSS (a) and enlargement (b) Cell culture medium containing the appropriate concentrations of calcium and phosphate ions is also a potential choice to deposit hydroxyapatite on titanium substrates. With this idea, Faure et al. 103 used Dulbecco's modified Eagles medium (DMEM), which is a non-buffered acellular culture medium containing inorganic ions but also amino acids, vitamins and glucose (see composition in

Table 1 .5). Titanium

 1

		was pretreated in NaOH and soaked in DMEM at 37 °C for 4 or 15 days. After 15 days,
	the surface was fully covered with hydroxyapatite, with 10-15 µm coating thickness. It was suggested
	that the deposition mechanism is similar to SBF, with first the nucleation of amorphous calcium
	phosphate on the surface, followed by phase transformation in hydroxyapatite and crystal growth. A
	major advantage of this method is the commercial availability of DMEM that avoid the fastidious
	preparation of SBF solutions.
		Na +	K +	Mg 2+	Ca 2+	Cl -	HPO4 2-	SO4 2-	HCO3 2-
	DPBS	448	10	4.1	2.5	242	14	-	-
	FCS	137	3.71	-	3.10	145	1.86	-	-
	HBSS	142	5.81	0.898	1.26	146	0.779	0.406	4.17
	DMEM	154.56	5.37	0.8	1.82	120.5	1.0	0.8	44
	SBF	141.8	5.0	1.5	2.5	148.0	1.0	0.5	4.2
	Table 1.5 Ionic compositions of solution to deposit HAp on titanium and comparison with SBF

Table 2 .

 2

	/mL)	S. epidermidis CIP 68.21	S. aureus ATCC 6538	L. ivanovii Li4(pvS2)	P. aeruginosa ATCC 27853
	Rosmarinic acid	512	4087	512	> 4087
	Chlorogenic acid	> 4087	4087	2043	> 4087
	Baicalin	1024	256	512	> 2048
	Baicalein	34	34	17	> 68

1 Antibacterial activity of the four biologically active molecules against four bacterial strains

  1 . The assay was performed as follows: 100 μL of BAM solution (concentration 50 µM) in ammonium carbonate pH 7.7 were mixed with 400 μL of Tris buffer pH 7.4 and 500 μL of fresh solution of DPPH at 100 μM in ethanol. Absorbance was measured at 517 nm after 20 minutes of reaction in the dark. DPPH inhibition was calculated by the equation:

%inhibition = [1 -(Asample/ ADPPH solution)] × 100, with A the measured absorbance. For calcium effect testing, a ratio of 0.1 (BAM/Ca) was used, and solution of BAM with calcium was prepared 5 hours before the assay.

2.4.3 Results and discussion

2.4.3.1 Spectroscopic studies of calcium-polyphenol interaction

2.4.3.1.1 UV-visible spectroscopy

  are likely to chelate metals. In our study, we noticed that the behavior of the molecule regarding calcium ion is different at pH 5 and pH 7.8 in ammonium carbonate buffer. At pH 5, when carboxylate is deprotonated several complexes are detected with ESI-MS, but[2CA+Ca]

Table 2 . 2

 22 The antioxidant activity was tested as presented in the paragraph 2.3, to the exception that for the solution with calcium, mixture were preapred 6 hours before the DPPH assay. Results are presented in Figure2.25. The antioxidant activity of baicalein is similar with or without calcium. The low activity is related to the fact that in this solution, BE is fully oxidized even without calcium. Antioxidant activity of BA drastically decreases after 6 hours in presence of calcium. Given the fact that baicalin is oxidized at p 7.8 in 6 hours, we can assume that the reduction of it radical scavenging activity is more likely due to its oxidation rather than an effect of calcium complexation.The antibacterial activity of BA and BE in presence of calcium was studied, against S. epidermidis CIP 68.21, S. aureus ATCC 6538, L. ivanovii Li4(pvS2) and P. aeruginisa ATCC 27853. Calcium was added in equimolar concentration compared to BA or BE. From Table2.2, we observe that MIC in presence of calcium are identical to MIC of pure BA or BE, within the experimental error, except in two cases. MIC of BA was decreased by 8 fold (from 1024 to 128 µg.mL -1 ) against S. epidermidis in presence of calcium. Minimum inhibitory concentration of baicalin and baicalein against four bacterial strains, alone and in presence of calcium (molar ratio 1:1)2.5.3.2.3 ConclusionsBiological activities of baicalin and baicalein appear more dependent on their oxidation state than on the presence of calcium. Indeed, calcium does not have any effect on the antioxidant activity of BA and BE. The presence of calcium does not affect MIC of BA and BE against L. ivanovii and P. aeruginosa.

	2.5.3.2.1 Antioxidant activity

Although significant because above the experimental error, such a decrease remains modest. Similarly, MIC of BE was reduced from 34 to 8.5 µg.mL -1 against S. aureus. It is important to note that BE and BA are oxidized in culture medium.

Table 3 .

 3 .1.

	Sample	Incorporated quantity (mg.g -1 ) (UV)	Incorporated quantity (mg.g -1 ) (TGA)	Incorporated quantity (molecules.nm -2 ) (TGA)	Particle length/width (nm)	Crystallite size (nm) (XRD)
	HAp-RA	20	31	0.3	31±10/8±2	21±5
	HAp-CA	10	27	0.3	43±11/12±1	23±5
	HAp-BE	89	63	0.9	33±10/15±3	24±5
	HAp-BA	60	37	0.3	27±4/8±3	24±5
	HAp	-	-	-	31±6/9±3	21±2

1 Estimation of adsorbed quantity of BAM on HAp measured by TGA or UV-Vis spectroscopy and particle dimension as obtained from TEM From UV-vis and TGA, baicalein is the molecule with the highest adsorption rate. Nonetheless, it is important to note the difference in the adsorbed quantity if measured by UV or by TGA. For baicalin and baicalein, the reason is probably the beginning of degradation of the molecule after 24 hours,

Table 3 . 2

 32 Initial and final pH of HAp suspensions alone or in the presence of BAMs. Calculated pH with the weak acid dissociation equation under brackets for RA, CA and BA. Weak acid dissociation In more details, all initial values are close or lower to the control sample, in agreement with the acidic nature of BAMs. We calculated the theoretical pH for a solution of RA, CA or BA, thanks to

			HAp-RA		HAp-CA	HAp-BA	HAp-BE
	BAM (mM)	0	0.05		0.05	0.05	0.05
	Initial pH	4.7	3.7 (3.5)		4.6 (4.1)	4 (4.05)	5
	Final pH	6.6	7.2		7.1	6.6	7
			pH =	1 2	(pka-log C)
			Equation 3.2

the equation of weak acid dissociation (see Equation 3.2). Considering final values, they are larger than for pure HAp, except for BA. The increase of pH observed when the dried HAp powder is placed in an acidic aqueous environment was previously reported 2 and attributed to the protonation of phosphate groups on the HAp surface and possible release of OH -and carbonate species. This previous work also evidenced that the final pH could be modified if adsorbing molecules were present. Here, a final higher pH compared to HAp alone may indicate that the adsorption process involves a deprotonation of the BAM.

Table 3 .4.

 3

	Sample	Yield (%)	Crystallite size (nm)	Incorporated quantity (mg.g -1 )	Final molar ratio BAM : Ca
	HAp	90	15±3	-	-
	HAp-CA	43	14±2	73	0.02
	HAp-BE	42	14±3	135	0.05
	HAp-BA	50	18±2	111	0.025
	Table 3.4. Apparent yield of the synthesis, crystallite size and incorporated quantity of BAM and of
	HAp-BAM (BAM: Ca = 0.075).

Table 4 . 1

 41 Ionic composition of SBF and comparison with blood plasma

	2-

Table 4 .

 4

2 1.5-SBF preparation for 1 L of solution

Table 4 . 3 .

 43 Calculated ratio Ca/P and Ca/O are compared with the one in stoichiometric HAp (Ca10(PO4)6(OH)2). The position of peaks of Ca and P are consistent with the position of peaks reported in the literature for stoichiometric HAp15 . Ca/P ratio in HAp coating is comprised between 1.3 (Table 4.3) and 1.6 depending on the sample. It is lower than the one of stoichiometric Chapter 4. Elaboration of hybrid hydroxyapatite -biologically active molecules coatings on Ti6Al4V titanium alloys 164 hydroxyapatite. The presence of carbon, which is normally absent in hydroxyapatite, may be due from the presence of adventitious organic contamination, because samples were stored in air after synthesis, or from the presence of carbonates. The peak of C 1s is composed by four contributions: C-C and C-H at 284.8 eV, C-O at 286.4 eV, C=O at 288.5 eV and metal carbonates at 290.3 eV. The contribution of metal carbonates represents 9.1 % of the total peak of carbon in the hydroxyapatite layer. It means that the adventitious carbon contamination represents 90.9 % of the total of carbon. Considering the atomic percentage of carbon in the hydroxyapatite layer, the percentage of adventitious carbon contamination is 14.2 % of the total amount of atoms in hydroxyapatite layer. Moreover, the presence of carbonates confirms the carbonatation of the hydroxyapatite coating synthesized in 1.5-SBF. Ca2p has spin-orbit components with Ca 2p1/2 at 352 eV and Ca 2p3/2 at 347 eV. P 2p has closely spaced spin-orbit components, responsible for the asymmetric peak envelope, and visible on the decomposition, with 2p1/2 at 134.2 eV and 2p3/2 at 133 eV.

	Element	Sample	C 1s	O 1s	Ca 2p3/2	P 2p	Mg 1s Na 1s
		HAp	286.0 532.0 347.6 134.0 1304.2 1072.0	Ca/P Ca/O
	BE (eV)	Stoechiometric HAp 15	285.4* (± 0.2)	531.8 (± 0.2)	347.8 (± 0.2)	133.8 (± 0.2)	-	-
		HAp	15.6	50.4	19.3	14.5		0.2	1.3	0.4
	% At	Stoechiometric HAp	-	59.1	22.7	13.6	-	-	1.67	0.4

hydroxyapatite

(1.67)

. The ratio calculated by XPS reflects the upper surface of the material, which explains why the Ca/P ratio is lower than the one calculated from EDS. However, it confirms that the layer is calcium-deficient. On the contrary, the ratio Ca/O is equal to the ratio Ca/O in stoichiometric

Table 4 .3 Position

 4

and atomic percentages of carbon, oxygen, calcium, phosphorous, magnesium and sodium in hydroxyapatite coatings and comparison with stoichiometric hydroxyapatite

15

(*carbon from adventitious organic contamination)

  To confirm this hypothesis, we decided to study growth of S. epidermidis CIP 105.777 in presence of BE in solution at a concentration C = 2*MIC, in rich medium (MH), and poor medium (PBS), for 24 hours, and to compare with bacterial growth in both culture media without BE. Results are presented in Figure4.27. In PBS without baicalein, the density of bacteria in solution remained stable,

		9,00		*	**
				NS
		8,00	8,01	8,02	8,08	7,69
	NUMBER)	7,00
	LOG (CFU	6,00
		5,00
		4,00
			Ti6Al4V	HAp	HAp-BE	HAp + adsorbed BE

  Chapter 4. Elaboration of hybrid hydroxyapatite -biologically active molecules coatings onTi6Al4V titanium alloys 192 monomolecular complex has a stability similar to [CA-H+Ca] + , but the bimolecular complex is unstable.

epidermidids

CIP 68.21)

, and it possesses a significant antioxidant activity. As CA, the molecule is stable at acidic and neutral pH. Its affinity for calcium in solution is very similar to the one of CA. At acidic pH, monomolecular [RA-H+Ca] 2+ and bimolecular [RA+Ca] 2+ complexes are formed. The deprotonated

  The molecule is not significantly adsorbed on HAp particles. While incorporated during the synthesis, RA has a strong effect on the HAp synthesis, but only a low amount is incorporated (50 mg.g-1 ). Besides a lowering of the synthesis yield at low RA:Ca ratio, particles are smaller than pure HAp (14±2 nm in length compared to 30±7 nm), but with a similar crystallite size. At high ratio, RA totally prevents the HAp synthesis. It is connected with the inhibition of the dissolution of calcite (formed at the beginning of HAp synthesis). The presence of RA in SBF also affects the deposition of HAp on Ti, despite a mechanism of HAp crystallization different from the homogenous precipitation. The deposited layer is thinner than pure HAp coating. Overall, RA has a strong effect on the hydroxyapatite synthesis, either under the form of nanoparticles or coatings, despite a low affinity for HAp surface, as hypothesized from XPS analysis. The inhibition of the synthesis is not related to the complexation between RA and calcium, even if it is significant at neutral pH. RA is not a molecule of choice for the development of hybrid coating, due to its low affinity for HAp surface and its low antibacterial activity. At neutral pH, we evidenced the oxidation of the molecule that we assumed to occur before the complexation with calcium. Consequently, at physiological pH, if any, only complexes involving the oxidized form of baicalin are likely to form. We suggested that the site of complexation is the carboxylate, by comparison with baicalein. As far as interactions of BA with hydroxyapatite are concerned, the molecule has an affinity for the HAp surface. It is adsorbed with higher quantity than RA and CA on HAp nanoparticles. While incorporated during synthesis, BA does not have a significant effect on the HAp crystallization. We can only notice a lower yield and smaller particles. Same comment can be done for the incorporation on BA in the 1.5-SBF solution for the deposition of HAp.BaicaleinBaicalein is the aglycone of baicalin, with interesting antioxidant activity and high antibacterial activity (MIC = 34 µg/mL against S. epidermidids CIP 68.21). The molecule is highly unstable at physiological pH, as we described the oxidation of the molecule at neutral pH at short-term and at acidic pH at long-term. The oxidation of the molecule hampers complexation with calcium, at both acidic and neutral pH. However, BE was found to be the molecule with the highest affinity for hydroxyapatite surface, as it is the molecule adsorbed in the highest amount on HAp nanoparticles.Despite interesting antibacterial activity of BE in solution, HAp nanoparticles with adsorbed baicalein do not have a significant antibacterial activity, but they exhibit an antioxidant activity over 5 days.Among the four tested molecules, BE is the one with the highest amount incorporated while adding during HAp nanoparticles synthesis. Furthermore, it slightly prevents the HAp crystallization, with

	Baicalin Baicalin is a flavonoid, with interesting antioxidant and antibacterial activity (MIC = 1.2 mg/mL against S. epidermidids CIP 68.21). At acidic pH, monomolecular [BA-H+Ca] + and bimolecular [2BA+Ca] 2+ complexes with calcium are formed, the monomolecular complex being more stable than the Ti6Al4V titanium alloys molecule does not prevent HAp synthesis if incorporated during synthesis of either nanoparticles or bimolecular one. Chapter 4. Elaboration of hybrid hydroxyapatite -biologically active molecules coatings on coatings.
	193

The morphology of the coating is different from pure HAp, but BA does not inhibit HAp synthesis.

Overall, BA has a significant affinity for both calcium ion in solution and hydroxyapatite surface. The lower yield, smaller particles size. At high BE:Ca ratio (0.075), it inhibits the dissolution of calcite, but not in the same rate than RA. Due to BE oxidation and degradation in alkaline medium, HAp nanoparticles with BE incorporated during synthesis do not exhibit any antibacterial effect. The molecule may also be incorporated in HAp coatings on Ti substrate. If added in the SBF solution, BE is incorporated in the coating, in interaction with the calcium. Its presence modifies the morphology of HAp coating, but not in the same extent than RA. Baicalein adsorption on HAp coating remodels the surface. HAp coatings with adsorbed BE have some antibacterial activity while HAp with baicalein incorporated during synthesis are inactive. In both cases coatings do not have any antioxidant effect.

Table 1 .

 1 Récapitulatif du comportement des quatre molécules actives biologiquement en fonction du pH et de la présence de calcium.

		Complexation	Oxydation
		pH 5	pH 7,8	pH 5	pH 7,8
	Acide rosmarinique			X	X
	Acide chlorogénique			X	X
	Baïcaline			X	
	Baïcaléine		X		

E. Tranquillo, F. Barrino, G. Dal Poggetto and I. Blanco, Materials, 2019, 12, 155.

are visible. The absence of clear peaks at 1103 cm -1 and 1070 cm -1 after 7 days indicates that carbonates are not incorporated into the hydroxyapatite in significant amounts. This is surprising, as carbonate concentration is higher in 1.5-TSBF than in 1.5-SBF:

27.0 mM and 4.2 mM, respectively. However, carbonate peaks may be evidenced after 14 days of soaking in SBF.

González-Martín, M. Monjo and J. M. Ramis, Advanced Healthcare Materials, 2015, 4, 540-549. M. I. Alvarez Echazú, C. E. Olivetti, C. Anesini, C. J. Perez, G. S. Alvarez and M. F. Desimone, Materials Science and Engineering: C, 2017, 81, 588-596. Chapter 4. Elaboration of hybrid hydroxyapatite -biologically active molecules coatings on Ti6Al4V titanium alloys Chapter 4. Elaboration of hybrid hydroxyapatite -biologically active molecules coatings on Ti6Al4V titanium alloys

particles was studied by TEM. Quantity of incorporated molecule was measured by thermogravimetric analysis (TGA).

We will first describe the synthesis with the molar ratio BAM:Ca = 0.0075. Table 3.3 gathers the yield of the reaction, calculated from recovered powder weight assuming that hydroxyapatite was the only formed phase, the particle dimension as obtained from TEM, the crystallite size obtained from XRD, the incorporated quantity of BAM measured by TGA and the molar ratio BAM: Ca in the materials, calculated from the incorporated quantity of BAM measured by TGA. 3.3 Yield of the synthesis, particle and crystallite size, incorporated quantity of BAM and final molar ration BAM:Ca of HAp-BAM (BAM: Ca = 0.0075). Altogether, Raman analysis confirms that the deposition of HAp is more efficient on non-annealed surfaces. Green lines: phosphate vibration of hydroxyapatite 8 ; pink lines: carbonates vibration in carbonated hydroxyapatite 10 ; blue and red lines: vibrations of titanium oxide (brookite and anatase respectively 11 ).

Sample

(AT= alkali-treated; AHT = alkali-heat-treated)

Conclusion: choice of the surface pretreatment

In the latter part of the work we have investigated the effect of alloy surface pretreatment on the deposition of hydroxyapatite from SBF. In the literature, it is commonly assumed that the hydroxylation of Ti in NaOH allows a good crystallization of hydroxyapatite on the surface, thanks to the exchange of Na + ions with Ca 2+ Overall, we managed to synthesize a hydroxyapatite layer with biomimetic characteristics. The spheroids morphology observed with SEM is classical for hydroxyapatite deposited in supersaturated biomimetic solutions 5 . XRD and Raman spectroscopy confirmed the deposition of hydroxyapatite, with a certain extent of carbonatation, as it was visible on the Raman spectrum and XPS decomposition of carbone. EDX spectroscopy showed that the presence of Na and Mg in the deposited hydroxyapatite.

That was expected, as we used the SBF solution containing Na + and Mg 2+ ions. From EDX, we calculated a ratio (Ca+Mg+Na)/P of 1.5, which is also a biomimetic characteristic. The HAp in the coating is calcium-deficient, and substituted. The ratio was also calculated from XPS for the upper surface, and it is comprised between 1.3 and 1.5. XPS confirmed the carbonatation of hydroxyapatite, and revealed an important adventitious contamination.

Incorporation of BAM during the synthesis

Rosmarinic and chlorogenic acids, baicalin and baicalein were incorporated in the hydroxyapatite coatings in a one-pot synthesis, by addition of the molecule in the SBF solution. Due to solubility issues, only one ratio between BAM and calcium have been tested, BAM:CA = 0.0075 that is comparable with the lowest ratio used for the incorporation of BAM on the HAp nanoparticles (see Chapter 3). This ratio corresponds to a BAM concentration in SBF of 0.019 mM. During 1.5-SBF preparation, BAM is the last component to be incorporated. It was checked that the presence of BAM did not modify the pH of the solution (pH ≈ 7.7). After 14 days of soaking of the plates in the solution which was renewed every 48 hours, morphology of the coatings were observed with SEM, and the crystallinity of the deposited mineral was checked by XRD.

On the SEM images at low magnification (Figure 4.12 (a), (c), (e) and (g)), it is visible than the quantity of deposited material is more important when baicalein and baicalin are present, compared to rosmarinic and chlorogenic acids. In presence of RA, the surface is not fully covered with material, as seen on We tried to measure the thickness of the coating with BE by peeling, but it was not possible to perform the scratches, because the layer is more friable than pure HAp coatings.

The ratio Ca/P of hydroxyapatite deposited with BAM was measured by XPS for the top surface (analyzed depth < 11 nm) of the coatings. Ratio are equivalent for the 4 coatings, comprised between 1.1 and 1.2 (see Table 4.4). The ratio is decreased compared to the biomimetic HAp coating (ca. 1.5), meaning that either the calcium is enriched with phosphorous or depleted with calcium. The presence of Ti in the spectrum of HAp-RA confirmed SEM observations. Depth of analysis with of XPS being approximatively 10 nm, the HAp-RA coating is either ultra-thin, or inhomogeneous with the presence of non-coated areas. In accordance with SEM, the more probable hypothesis is the inhomogeneity of the coating. Overall, XPS indicate that, in presence of BAMS, the Ca/P ratio is significantly decreased compared to the pure HAp coating. One possibility to explain the decrease of Ca/P ratio is that, because of the lowest availability of calcium ions forming complexes, a P-rich calcium phosphate is formed.

Sample

However it should be amorphous and in low amounts as it could not be evidenced by XRD nor by Raman.

to all of these considerations, the quantification of the quantity of the adsorbed molecule was not possible by UV-Vis method. For both coatings, we can estimate an adsorbed quantity of 0.1 mg adsorbed. For baicalein, this quantity is overvalued, as the maximal quantity that can be adsorbed is 2.10 -7 mol, corresponding to 0.05 mg. It is also overvalued in the case of baicalin, as 2.10 -7 mol corresponds to 0.09 mg, but the error is less important. Once again, this method is not adapted to measure the adsorbed quantity of molecule on hydroxyapatite coatings, because the loss of weight due to the molecule is very low.

Chapter 4. Elaboration of hybrid hydroxyapatite -biologically active molecules coatings on Ti6Al4V titanium alloys

In a second step, we have successfully synthesized hydroxyapatite on titanium quartz in SBF.

However, results of adsorption studies are not concluding, because the coating seems to be degraded by the flux during the experiment. Nevertheless, this method is worth considering and further experiments need to be performed.

The incorporation of BAM in the hydroxyapatite coating therefore appears effective by both methods, although the precise amount of immobilized BAM could not be achieved. In the next part we will investigate the effect of these coatings as antioxidant and antibacterial agents, as well as promoter of osseointegration.

Biological activity of hydroxyapatite-baicalein coatings 4.4.1 Introduction

In this part, the biological activity of hydroxyapatite with BAM will be investigated. We decided to focus only on the incorporation of baicalein since it is the molecule with the highest antibacterial activity, as evidenced in Chapter 2, and as a matter of comparison with hydroxyapatite nanoparticles incorporating baicalein studied in Chapter 3. Antioxidant and antibacterial activities of coatings were studied. Preliminary results on the osseointegration properties of the coating with adsorbed baicalein are presented in the last section.

We intented to quantify baicalein release from hydroxyapatite coatings with HPLC as an analytical method using an equipment available at LRS. However, it appeared that the diode array detection device was not sensitive enough to detect baicalein at relevant concentrations. To date, no suitable device was available, so that these studies are for now pending. Studies with UV-Vis spectroscopy were also attempted, but because of the modification of UV-Vis spectrum of baicalein in function of time due to its oxidation, it was not possible to analyze the results.

Antioxidant activity

Antioxidant activity of HAp coatings with baicalein (incorporated during synthesis or adsorbed)

was tested with the DPPH radical scavenging testing, derived from Blois 22,23 (see 3.4.2 for more details).

Each sample was immerged in 2 mL of solution corresponding to a 1:1 (v/v) mixture of TRIS buffer at The radical scavenging activity of coatings are very low, 1.4 % for the coating with baicalein incorporated during synthesis (HAp-BE), and 2.0 % for hydroxyapatite with adsorbed baicalein (HAp + adsorbed BE). The absence of activity is not surprising when baicalein is incorporated during the synthesis, as we know from the molecular study that baicalein oxidized at neutral pH. The molecule is likely oxidized in the SBF solution, and incorporated in hydroxyapatite as the oxidized form. After oxidation, the molecule is no longer active against radicals. The low activity for coatings with adsorbed baicalein is probably due to their low amount.

Overall, hydroxyapatite coatings incorporating baicalein, whatever the incorporation method, do not have any significant antioxidant activity.

Antibacterial activity

Antibacterial assays

The -εox,600 = molar extinction coefficient of rezaruzin at 600 nm = 117.216 L.mol -1 .cm -1 -εox,570 = molar extinction coefficient of rezaruzin at 570 nm = 14.652 L.mol -1 .cm -1 -εred,600 = molar extinction coefficient of resarufin at 600 nm = 80.586 L.mol -1 .cm -1 -εred,570 = molar extinction coefficient of resarufin at 570 nm = 117.216 L.mol -1 .cm -1 -A°570 = absorbance of control (AlamarBlue solution) at 570 nm -A°600 = absorbance of control (AlamarBlue solution) at 600 nm -A570 = absorbance of sample at 570 nm -A600 = absorbance of samples at 600 nm Equation 4.2 Percentage difference compared to control of reduction of rezaruzin in resarufin, corresponding to the percentage of viability

The preliminary results at 24 hours tend to show that viability of cells on HAp coatings with or without baicalein is lower than on titanium surfaces and plastic control. This is confirmed by the visualization with fluorescence microscope Figure 4.30 (a), (c), (e) and (g). On plastic control, cells are well spread. On titanium, the number of cell is higher than on the plastic control plate, with the totality of the surface covered by spread osteoblasts. On the contrary, it is more difficult to find cells on sample with hydroxyapatite, and even worst with hydroxyapatite with adsorbed baicalein. However, cells on these materials are well spread.

Characterization techniques

Spectroscopic and spectrometric studies in solution UV-Visible spectroscopy was performed on a Libra Biochrom S60 spectrometer, between 250 and 600 nm, with stepsize 0.5 nm, bandwith 2 nm and slow scanspeed.

Electrospray ionization mass spectrometry (ESI-MS) studies were conducted on a LTQ orbitrap mass spectrometer, with a capillary voltage of 20 V and tube lens voltage of 70 V, in positive ion mode.

Resonant excitation was carried out using collision-induced dissociation (CID), normalized collision energies (NCE) were between 5 and 30 %. All data analysis was performed using Thermo Xcalibur™ software. Samples were introduced at the rate of 20 µL.min-1 with a 500 µL syringe. These experiments were performed with the help of Cedric Przylbylski at the Institut Parisien de Chimie Moléculaire.

NMR liquid spectroscopy was performed in the NMR platform of the Institut Parisien de Chimie Moléculaire. 1 H and 13 C spectra were recorded on a Bruker 300 MHz spectrometer equipped with a BBFO probe. Number of scan was 32 for 1 H and 3072 for 13 C.

Experimental section II Characterizations of hydroxyapatite nanoparticles XRD X-ray diffractogramm were recorded on a D8 diffractometer (Bruker), equipped with a source CuKα (1.54 Å) and a Bragg-Brentano Θ -Θ geometry, between 7 ° and 80 ° with a variable slit V12. For powder analysis, step was 0.03 ° and scanspeed 1 s/point. For coating analysis, step was 0.05 ° and scanspeed 0.1 s/point, and the record was repeated 5 times to decrease the signal over noise ratio.

TGA

Thermogravimetric analysis was performed with a SDT Q600 apparatus from TA Instruments, between 25 °C and 700 °C, with a ramp of 10 °C/min, under air. Sample weights were typically comprised between 10 and 20 mg.

Specific surface area

Surface specific area was measured with a physisorption apparatus (Belsorpmax), by measuring the adsorbed quantity of N2 in function of pressure, at T = 77 K. Before measurement, vacuum degree was 3.202.10 -6 Pa. Sample weight was 0.9340 g.

Solid state NMR

Transmission electronic microscopy

Samples were observed with Tecnai spirit G2 microscope with a Gatan Orius camera.

Characterizations of hydroxyapatite coatings

Vibrational spectroscopies

Raman spectra were collected with a Kaiser Optical system, Raman Analyzer RXN1 microprobe equipped with a laser diode at 785 nm (P = 10 mW, accumulation time: 10s, 30 scans per spectrum), and a Leica microscope with x50 long working distance lens.

ATR-IR spectra were recorded on a Nicolet spectrometer (256 scans/spectrum), with a MCT detector.

Scanning electron microscopy

Morphology of coatings was observed by scanning electron microscopy on a Hitachi S-3400N microscope (acceleration voltage 10 kV, probe current 70). Samples are covered with carbon before imaging. Energy dispersive X-ray spectroscopy was performed thanks to an Oxford instrument -X max.

Images of HAp coatings tilted with an angle of 45 ° were recorded on a SEM-FEG Hitachi SU-70 with an acceleration voltage at 1 kV.

XPS

XP spectra were collected on an Omicron Argus X-ray photoelectron spectrometer, using a monochromated Al Kα (hn = 1486.6 eV) radiation source having a 300 W electron beam power. The emission of photoelectrons from the sample was analyzed at a takeoff angle of 45° under ultra high vacuum conditions (1.10 -8 Pa). XP spectra were collected at pass energy of 20 eV for C1s, O1s, Ti2p, Ca2p, P2p, and Mg2s core XPS levels. After data collection, the binding energies were calibrated with respect to the binding energy of the C1s peak at 284.7 eV. The peak areas were determined after subtraction of a linear background. The atomic ratio calculations were performed after normalization using Scofield factors. Spectrum processing was carried out using the Casa XPS software package.

Photoluminescence

Fluorescence spectra of coatings were obtained with a Fluorolog 3 apparatus from HORIBA Jobin Yvon.

Excitation was set at λext = 300 nm with 5 nm of bandpass and emission spectrum was recorded between 320 and 650 nm with 1 nm of bandpas. Increment was set at 1.0 nm and integration time at

Experimental section IV 0.5 s. The surface was tilted from the incident beam (source Xenon, 450 W), and spectrum was collected with an angle of 22 °.

QCM

QCM apparatus is a QCM-E4 from Q-Sense® (Götenborg, Sweden). Solutions are injected with a perilstatic pump at 25 µL.min -1 . Temperature was set and regulated at 20 °C. Commercial quartz were provided by LOT-Quantum design