Silver Doping Mechanism in Bioceramics-From Ag+: Doped HAp to Ag°/BCP Nanocomposite

: The results presented in this paper, based on the powder X-ray diffraction technique 8 followed by Rietveld analyses, are devoted to the mechanism of silver incorporation in Biphasic 9 Calcium Phosphates. Results were confirmed by SEM observation. Samples were synthesized via 10 the sol-gel route, followed by heat treatments. Two incorporation sites were highlighted: Ca 2+ 11 replacement by Ag + into the calcium phosphates (HAp: hydroxyapatite and β-TCP: TriCalcium 12 Phosphate), and the other as metallic silver Ag° nanoparticles (formed by autogenous reduction). 13 The samples obtained were thus nanocomposites, written Ag°/BCP, composed of closely-mixed Ag° 14 particles of about 100 nm at 400 °C (which became micrometric upon heating) and calcium 15 phosphates, themselves substituted by Ag + cations. Between 400 °C and 700 °C the cationic silver 16 part was mainly located in the HAp phase of the composition Ca 10- x Ag x (PO 4 ) 6 (OH) 2- x (written 17 Ag + :HAp). From 600 °C silver cations migrated to β-TCP to form the definite compound 18 Ca 10 Ag(PO 4 ) 7 (written Ag + :TCP). Due to the melting point of Ag°, the doping element completely 19 left our sample at temperatures above 1000 °C. In order to correctly understand the biological 20 behaviour of such material, which is potentially interesting for biomaterial applications, its complex doping mechanism should be taken into consideration for subsequent cytotoxic and bacteriologic studies. both metallic Ag° cationic Ag + Synthesized composites closely-mixed Ag° nanoparticles and Ag + calcium phosphate phases. HAp silver-to-calcium doped Ca 10- Ag 4 ) (OH) 2- 600 Ag cations β-TCP definite Ca 10 Ag(PO ) 3- 2.857 Ag 0.286 (PO ) composite aspect of the prepared silver the results, study of biological properties the biological conditions of metallic silver nanoparticles from of substituted cationic + in terms of bactericidal power and release rate/kinetics in the human


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The utilization of synthetic materials for bone reconstructive surgery is generally necessary, 28 because autograft and allograft practice is limited by the quantity of available material, and in the 29 first case entails a second surgical procedure [1]. Among the numerous synthetic materials 30 investigated for bone replacement and/or prosthesis coating, hydroxyapatite (HAp, Ca10(PO4)6(OH)2) 31 is the most often-used material due to its chemical and structural similarities with the bone mineral

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Hydroxyapatite is therefore an interesting biocompatible and osteoconductive material, but it has 37 limited antibacterial properties [8], even though bacterial infections are the main cause of 38 postoperative problems [9]. Bacterial overgrowth on the surface of orthopaedic implants -that is, 39 biofilm formation -potentially leads to serious complications during reconstructive surgery, with 40 severe physiological damage, significant patient discomfort and additional costly surgical 41 procedures [10][11][12]. About 1% of total joint hip arthroplasties, and about 3% in the case of knees, require a second (or multiple) surgical intervention(s) because of bone healing process complications, 43 which makes it a real societal problem on a global scale [13][14][15][16][17]. Nowadays, an antibacterial effect at 44 the surgical site is ensured by systematic antibiotic administration via the blood, which potentially 45 generates toxicity, poor penetration into the surgical site, and also the problem of bacterial resistance.

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The delivery, or the presence, of a bactericidal agent directly at the surgical site would ensure a really 47 promising alternative [18,19]. Among the various possibilities, the use of silver -well known in 48 medicine since ancient times in the treatment of bacterial infection -seems very promising by 49 combining broad spectrum and long term antimicrobial activity with the absence of microorganisms 50 developing resistance [20][21][22][23][24][25]. A particularity of the antiseptic properties of silver is the possibility of 51 using it in metallic or ionic form, without a loss of efficiency [26][27][28]41]. For these reasons, research 52 on silver incorporation into hydroxyapatite has developed in recent years, and has shown the high 53 potential of the synthesized materials [17,22,[29][30][31][32][33][34][35]

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The purpose of this paper is to fully characterize the silver-HAp doping mechanism in the case 66 of sol-gel synthesis followed by gradual thermal treatment leading to silver auto-reduction. The 67 study is a continuation of our previous work on the BCP     Rietveld plot is shown in Figure SEI1. of acquisition on three wide areas (magnification x100) before averaging. Some specific isolated 120 measurements were performed using x5000 magnification. A 4QBSD detector was used to acquire 121 chemical contrast images to highlight silver particles.

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Ag° particles leave the sample, but the calcium phosphate phases were also completely free of Ag +   Table 2 for the 25Ag-T series. These results confirm that nanoparticles were exclusively composed of 264 silver: metallic Ag° as shown in area 3 (and also area 2) in the 25Ag-400 sample. Indeed, the results 265 from the local zone '3' are in favor of particle containing only the silver element (the minor Ca and P 266 contents come from the not really punctual electron beam), and therefore of a metallic nature (i.e. Ag°

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Dotted lines indicate the nominal silver amounts, and error bars correspond to cumulative standard 302 deviation considering the tree phases.

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Nanoparticles of metallic silver therefore formed very quickly in our samples by the autogenous 304 reduction of silver nitrate. The set of samples can thus be considered as a Ag°/BCP composite.

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However, at low temperatures, we also observe the formation of a silver-doped Ag + :HAp phase 306 whose composition is close to the nominal composition (i.e. Ca9.75Ag0.25(PO4)6(OH)1.75 for the 25Ag-T

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The underestimation of silver content from 800 °C upwards suggests that the Ag + amount in the TCP 317 phase continues to increase; however, no experimental proof has been brought to support this.

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The characterization of the doped Ag + :HAp phase with increased lattice parameters, by 319 comparison with an undoped HAp lattice, has already been reported in the literature. This is notably 320 the case in the study of Geng et al. [32], where the Ca10-xAg2x(PO4)6(OH)2 doped composition was 321 considered. This nominal composition suggests two kinds of doping mechanism: only half of the 322 silver cations (x value) can substitute calcium cations into the Ca1 or Ca2 crystallographic sites of the 323 HAp crystal structure. The second half of silver cations (to reach the 2x doping level) must be located 324 at interstitial sites. Our crystallographic study, based on Rietveld refinement, did not enable us to 325 highlight the presence of silver cations at interstitial crystallographic sites, not to mention that this 326 situation is not preponderant for large cations like Ag + . For these reasons we preferred to note the 327 chemical composition of the doped Ag + :HAp phase as follows: Ca10-xAgx(PO4)6(OH)2-x, in which only 328 the substitution mechanism was considered in agreement with the paper by Badrour et al. [61].

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However, we must admit that our Rietveld refinements did not make it possible to clearly quantify 330 the silver substitution rates at both the Ca1 and Ca2 crystallographic sites in the HAp structure.

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During the past decade, several studies have been devoted to silver incorporation in HAp (or 333 BCP) samples due to their high potential for biomaterial applications, namely because of the well-334 known bactericide properties of silver. Unanimously, the results have shown a very interesting 335 bactericidal effect following the doping of calcium phosphates with silver. However, the material aspect of the samples studied in the literature showed some disparities, in particular due to a lack of 337 understanding of the doping mechanism. Some works mention metallic silver nanoparticles; others 338 focus on cationic substitution in phosphate phases. The results presented in the present paper are 339 especially devoted to the description of the mechanism by which silver is incorporated into BCP 340 samples. This work follows the experience previously acquired on the BCP doping mechanism of the 341 first-row transition elements. It appears that both electronic states are simultaneously present in the