Design and properties of novel gallium-doped injectable apatitic cements.

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Design and properties of novel gallium-doped injectable apatitic cements Charlotte Mellier a, Franck Fayon b, Florian Boukhechba a, Elise Verron c, Myriam LeFerrec a, Gilles Montavon e, Julie Lesoeur c, Verena Schnitzler a, Dominique Massiot b, Pascal Janvier d, Olivier Gauthier c, Jean-Michel Bouler d,⇑, Bruno Bujoli d,⇑ a

Graftys SA, 13854 Aix en Provence Cedex 3, France CNRS, UPR3079, CEMHTI, 45071 Orléans, France Université de Nantes, INSERM, UMR 791, LIOAD, Faculté de Chirurgie Dentaire, 44042 Nantes Cedex 1, France d Université de Nantes, CNRS, UMR 6230, CEISAM, UFR Sciences et Techniques, 44322 NANTES Cedex 3, France e Université de Nantes, CNRS-IN2P3/Ecole des Mines de Nantes, UMR 6457, SUBATECH, 44307 Nantes Cedex 3, France b c

a r t i c l e

i n f o

Article history: Received 20 February 2015 Received in revised form 6 May 2015 Accepted 22 May 2015 Available online xxxx Keywords: Calcium phosphate cements Gallium Design Handling properties Biocompatibility

a b s t r a c t Different possible options were investigated to combine an apatitic calcium phosphate cement with gallium ions, known as bone resorption inhibitors. Gallium can be either chemisorbed onto calcium-deficient apatite or inserted in the structure of b-tricalcium phosphate, and addition of these gallium-doped components into the cement formulation did not significantly affect the main properties of the biomaterial, in terms of injectability and setting time. Under in vitro conditions, the amount of gallium released from the resulting cement pellets was found to be low, but increased in the presence of osteoclastic cells. When implanted in rabbit bone critical defects, a remodeling process of the gallium-doped implant started and an excellent bone interface was observed.

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Statement of Significance

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The integration of drugs and materials is a growing force in the medical industry. The incorporation of pharmaceutical products not only promises to expand the therapeutic scope of biomaterials technology but to design a new generation of true combination products whose therapeutic value stem equally from both the structural attributes of the material and the intrinsic therapy of the drug. In this context, for the first time an injectable calcium phosphate cement containing gallium was designed with properties suitable for practical application as a local delivery system, implantable by minimally invasive surgery. This important and original paper reports the design and in-depth chemical and physical characterization of this groundbreaking technology. Ó 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

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1. Introduction The use of synthetic calcium phosphate-based bone substitutes in human bone surgery is continuously expanding [1–6]. These bioactive implants have the unique capacity to be resorbed in vivo according to bone remodeling kinetics and replaced by natural bone. While first generation products were dominated by porous ceramics, injectable calcium phosphate cements (CPCs) are currently under intense investigation [7–13] since they offer additional advantages, including primary mechanical properties

⇑ Corresponding author. E-mail address: [email protected] (J.-M. Bouler).

similar to those of cancellous bone, along with an injectability suitable for implantation of the composite under minimally invasive surgery and adapted to any shape of defects. In addition, the development of calcium phosphate cements opened up new applications in the field of drug delivery systems. Indeed, when implanted, CPCs combined with drugs may not only act, as their main primary function, as mechanically resistant sacrificial calcium phosphate source for bone reconstruction, but are also well-suited to address bone-related diseases or infections [14]. Therefore, CPCs have been considered as carriers for local and controlled supply of antibiotics, anti-inflammatory or anti-cancer agents [15–24], thus potentially providing a reliable strategy for producing efficient pharmacological effects only to specifically intended target sites.

http://dx.doi.org/10.1016/j.actbio.2015.05.027 1742-7061/Ó 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Please cite this article in press as: C. Mellier et al., Design and properties of novel gallium-doped injectable apatitic cements, Acta Biomater. (2015), http:// dx.doi.org/10.1016/j.actbio.2015.05.027

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In this context, bisphosphonate antiresorptive drugs (BP), one of the most conventional treatments of osteoporosis worldwide, have been successfully combined to deficient calcium apatite. Pre-clinical experiments using a large animal osteoporosis model (femoroplasty surgical approach) have thus shown both resorbability of the implanted calcium phosphate material and replacement by newly formed bone, with a therapeutic value stemming from both the attributes of the calcium phosphate matrix and the intrinsic biological activity of the bisphosphonate [25]. Then, a novel injectable BP-loaded calcium phosphate cement was designed and shown to be suitable in terms of (i) rheology, in order to be worth considering for implantation using minimally invasive surgery, and (ii) setting time and strength for reinforcing fragile bone sites [26]. In addition, this CPC-BP combined device provided cement-driven primary mechanical properties (bone augmentation) and offered a better bioavailability of the drug locally [7,27]. More recently we have explored the potential of gallium-doped calcium phosphate materials. Indeed, many studies have shown that gallium(III) ions inhibit bone resorption [28–34], and for example gallium nitrate has been approved by the FDA for the treatment of cancer-related hypercalcemia and Paget’s disease [35–38]. Therefore, we have investigated the in vitro effects of gallium nitrate, using well-established osteoclastic and osteoblastic models [39,40]. It was shown that gallium reduced the resorption activity, differentiation and formation of osteoclasts by non-cytotoxic mechanisms, in a dose-dependent (0–100 lM) manner. In addition, gallium did not induce any adverse effect on osteoblastic bone forming cells. These results suggested that gallium may offer a promising option for regulating the excessive osteoclastic activity taking place in osteoporosis or in some osteolytic bone tumours [41]. Interestingly, other groups in the literature have also reported that Ga3+ ions show antimicrobial properties as well as a capacity for reducing arthritis-related pain [42–46] and inflammation [47]. Finally, it is interesting to note that 67 Ga citrate is used in nuclear medicine for tumor imaging by scintigraphy, especially for lymphoma [48–51]. However, the bioavailability of gallium nitrate is very low and thus requires a long and continuous IV administration. For this reason, we have investigated the possibility to combine gallium with injectable apatitic cements for the development of a local delivery system of this ion in osteoporotic sites. In the present paper, we describe the development of a suitable protocol for this purpose while preserving the textural, mechanical and setting properties of the cement. The ability of the gallium-doped cements to release gallium ions was investigated under in vitro conditions, and in vivo implantation of these cements was also performed in bone critical defects on a rabbit animal model.

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2. Materials and methods

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2.1. Materials

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Commercially available Na2HPO4 (Fluka), anhydrous dicalcium phosphate (DCPA –Sigma Aldrich, France), gallium nitrate hydrate (Alfa Aesar, Germany), Hydroxypropylmethylcellulose (HPMC, E4 MÒ – Colorcon-Dow chemical (Bougival, France)) were used as received. CDA was prepared by alkaline hydrolysis of DCPD using aqueous ammonia, as previously described [52]. a-TCP (alpha tricalcium phosphate) was prepared by calcination of a 2:1 M mixture of CaHPO4 and CaCO3 at 1350 °C for at least 4 h, and subsequent rapid cooling to room temperature. The obtained reaction product contained less than 5% of b-TCP. Gallium-doped b-TCP (Ca10.5 1.5xGax(PO4)7, x = 0.5) was prepared by solid state reaction as previously reported [53].

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Gallium-doped CDA. In a typical procedure, a mixture of gallium nitrate hydrate and calcium nitrate tetrahydrate was dissolved in a beaker containing 125 mL of ultrapure water, with a (Ca + Ga)/P molar ratio of 1.515 and a Ga/Ca molar ratio in the 0–0.08 range. The pH of the solution was adjusted in the 9–9.5 range by means of a concentrated solution of ammonia. The reaction mixture was then introduced in a three-neck angled round bottom flask placed in an oil bath and equipped with a dropping funnel. The temperature of the reaction mixture was raised to 50 °C and 1.089 g of diammonium hydrogen phosphate (8.25 mmol) dissolved in 125 mL of ultrapure water was added dropwise over a 5–10 min period. The mixture turned white and the pH was adjusted in the 7.5–8 range by means of a concentrated solution of ammonia. After 30 min, the obtained suspension (pH was neutral) was filtered off while hot and washed with 250 mL of ultrapure water. After repeating this procedure four times, the white waxy product was dried in an oven at 80 °C for 24 h. The gallium content of the collected aqueous fractions was measured by atomic absorption spectroscopy, to determine the amount of gallium incorporated in the isolated solid phase. Two samples were prepared containing 1.5 and 3 wt.% of gallium, respectively. Preparation of the cement samples. The composition for the solid phase of the commercially available apatitic cement reference (GraftysÒ QUICKSET noted as QS-CPC) was the following: a-TCP (Ca3(PO4)2, 78 wt.%), mixed with anhydrous dicalcium phosphate [DCPA] (CaHPO4, 10 wt.%), calcium deficient apatite (Ca10 x[ ]x(HPO4)y(PO4)6 y(OH)2 z[ ]z, 10 wt.%), hydroxypropyl methyl cellulose [HPMC] (2 wt.%). The composition of the different cement samples is reported in Table 1. For clarity, the references chosen for the CPC powders doped with gallium (Table 1, left column) indicate their gallium weight content and the nature of the Ga-doped component. Each CPC powder was milled to obtain a similar particle size distribution, and then sterilized by c-irradiation. Paste samples were prepared by mixing 6 g of the powdered preparation with 2.7 mL of a 0.5 wt.% Na2HPO4 aqueous solution for 2 min (liquid/solid ratio = 0.45 mL g 1).

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2.2. Methods

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1 H and 31P solid-state magic angle spinning (MAS) NMR experiments were performed on a Bruker Avance 300 spectrometer operating at 7.0 T (1H and 31P Larmor frequency of 300.0 and 121.5 MHz, respectively) using a 4 mm double-resonance MAS probe. 1H MAS NMR spectra were recorded at a spinning frequency of 14 kHz using a p/4 flip angle (pulse length of 2.5 ls) and a recycle delay of 3s. All 31P NMR spectra were recorded using a spinning frequency of 14 kHz and 1H SPINAL-64 decoupling [54] (RF field strength of 70 kHz) was applied during signal acquisition. 31P quantitative MAS spectra were obtained using a p/9 flip angle (pulse length of 0.8 ls) and a recycle delay of 30 s to ensure no saturation. 1D {1H}–31P CP-MAS and 2D 1H–31P heteronuclear correlation (HETCOR) spectra were recorded at different contact times (ranging from 0.25 to 12.5 ms) using a recycle delay of 1s. The 71Ga solid-state NMR experiments were performed on Bruker Avance 750 and 850 spectrometers operating at 17.6 T and 20.0 T (71 Ga Larmor frequencies of 228.8 and 259.3 MHz, respectively) using a 1.3 mm double-resonance MAS probe. 71Ga 1D MAS central transition spectra were recorded at high spinning frequencies ranging from 60 to 65 kHz using a Hahn echo sequence with a 71 Ga nutation frequency of 62.5 kHz (central transition selective p/2 pulse length of 4 ls). The echo delay was set to two rotor periods and the recycle delay was set to 3s. Under these experimental conditions, 1H decoupling was not applied since it did not improve the spectral resolution for the studied sample nor reduce the linewidth of the 71Ga resonances. 1H, 31P and 71Ga chemical shifts were

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C. Mellier et al. / Acta Biomaterialia xxx (2015) xxx–xxx Table 1 Composition of the cement samples prepared in this study.

a-TCP QS-CPC (b-TCP)-CPC 0.32 wt.%(Ga-b-TCP)-CPC 0.3 wt.%(Ga-CDA)-CPC 0.15wt.%(Ga-CDA)-CPC 0.15 wt.%(Ga-CDA + CDA)-CPC 0.075 wt.%(Ga-CDA + CDA)-CPC

234 g 204 g 204 g 234 g 234 g 234 g 234 g

b-TCP

Ca9.75Ga0.5(PO4)7

CDA

30 g

30 g 30 g 30 g

30 g

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referenced relative to TMS, a 85% H3PO4 solution and a 1.1 mol kg 1 Ga(NO3)3 D2O solution, respectively. Simulations of all spectra were performed using the dmfit software [55]. X-ray diffraction (XRD) powder patterns were recorded on a Bruker D8 Advance diffractometer (Cu Ka1, 2 radiation, 40 kV, and 30 mA) equipped with a linear Vantec detector. Energy dispersive X-ray spectroscopy (EDS) measurements were performed on a scanning electron microscope (XL40 TMP FEI) after coating the powder by a carbon layer (electron beam of 20 keV, dead time of 30%). Electron microscopy and electron diffraction measurements were performed on a Philips CM20 transmission electron microscope. Samples to be observed were first crushed in ethanol, and a drop of the solution with the small crystallites in suspension was deposited onto a carbon-coated copper grid.

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2.3. In vitro gallium release measurements from cement pellets

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2.3.1. Cell culture The RAW264.7 cell line (Ref. # TIB-71) was obtained from ATCC (LGC Standards, Molsheim, France). Cells were cultured in DMEM containing 5% fetal bovine serum (Hyclone serum, Thermo Fisher Scientific, Brebière, France). For osteoclastic differentiation experiments, RAW264.7 cells were seeded at 5000 cells/cm2 in a-MEM containing 5% Hyclone serum and effectors were added immediately [56]. RANKL (Receptor Activator of Nuclear Factor- B Ligand) was used at 20 nM. For resorption assay, cells were cultured for seven days with a renewal of the medium once every two days.

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2.3.2. In vitro release assay Cement samples were prepared as described above, and were directly introduced into silicone molds (Ambrose Mesa Mold, Prod #107 TED PELLA, INC.). After 48 h of setting time at 37 °C, pellets were washed 3 times in a-MEM medium and placed into 24-well plates. Cells were seeded on the surface of cement pellets at 20,000 cells/cm2 and were cultured as previously described. At days 2, 4 and 7, culture medium in each well was extracted and the gallium concentration was measured by ICP-MS analysis on a Thermo Scientific XSERIES 2 equipment, using a 2 wt.% nitric acid matrix for the dilution of samples.

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2.4. In vivo implantation of QS-CPC versus gallium-doped cements

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2.4.1. Animals and surgical procedures All animal handling and surgical procedures were conducted according to European Community guidelines for the care and use of laboratory animals (DE 86/609/CEE) and approved by the local Veterinary School ethical committee. The tested biomaterials were implanted bilaterally for 4 weeks at the distal end of 10 mature female New Zealand White rabbit (3–3.5 kg) femurs. A lateral arthrotomy of the knee joint was performed and a cylindrical 6 10 mm osseous critical-sized defect was created at the distal femoral end. After saline irrigation, the osseous cavity was carefully dried and filled with the tested

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3 wt% Ga-doped CDA

1.5 wt% Ga-doped CDA

30 g 30 g 15 g 15 g

15 g 15 g

DCPA

HPMC

30 g 30 g 30 g 30 g 30 g 30 g 30 g

6g 6g 6g 6g 6g 6g 6g

calcium phosphate cements. Five rabbits were implanted with QS-CPC vs 0.3 wt.%(Ga-CDA)-CPC and five ones with QS-CPC vs 0.32 wt.%(Ga-b-TCP)-CPC.

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2.4.2. Histological studies Implanted and control samples were classically prepared for qualitative histological examination on light microscopy. The femoral ends were fixed in glutaraldehyde solution and successively dehydrated in graded ethanol and acetone. Eventually the non-decalcified bone specimens were infiltrated and embedded in a glycolmethylmethacrylate resin. Undecalcified serial 7 mm sections of each sample were cut perpendicularly to the drilling axis of the implantation area using a hard tissue microtome (Reichert-Jung Supercut 2050, Austria) equipped with a D profile tungsten carbide knife. From these sections, Movat’s pentachrome staining was assessed. This bone specific staining is perfectly adapted to distinguish mineral (yellow–green), osteoid tissue (red line) and cement (gray–blue) [25].To analyze more specific tissue components, hematoxylin-eosin was performed. Samples were observed with a polarized light microscope (Axioplan2Ò, Zeiss, Germany).

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2.4.3. Statistical analysis Compressive strength measurements (N = 70), Initial setting times (N = 28) and gallium concentrations (N = 27 with or without cells) were analyzed trough an ANOVA using a post hoc Fisher test. For all comparisons, a p-value < 0.05 was considered as significant.

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3. Results and discussion

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In the present study we have explored different possible options for the introduction of gallium(III) ions into a commercially available injectable apatitic cement (GraftysÒ QUICKSET noted as QS-CPC). This includes (i) dissolution of a gallium salt in the liquid phase (ii) mixing a gallium salt with the ground solid phase (iii) chemical combination of gallium ions with one of the calcium phosphate components of the solid phase. In this context, one important point first to be considered is the thermodynamic stability of soluble gallium species in solution. In strongly acidic solutions, the solubility limit is 10 2 mol L 1 below pH 2, with gallium present as octahedral hexa-aqua complexes [57,58]. In basic medium, gallium is present as Ga(OH)4 gallate ions with a solubility limit of 10 3.3 mol L 1 at pH 10 [59,60].

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3.1. Interaction of gallium with the components of the calcium phosphate cement

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The setting reaction of the QS-CPC reference proceeds at a pH varying in the 8.5–6.8 range upon mixing a 0.5 wt.% aqueous disodium phosphate buffer with the solid phase (liquid/powder ratio: 0.45 mL g 1). Attempts to introduce gallium nitrate in this liquid phase resulted in the immediate precipitation of an amorphous white solid (absence of Bragg peaks in X-ray diffraction pattern, Fig. S1 in Supplementary data) which was identified from semi-quantitative EDS analyses and 1H solid-state NMR spectra

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as a hydrated sodium gallium phosphate, with a weaker sodium content as the amount of gallium nitrate added to the liquid phase increases (Table S1 in Supplementary data). This was confirmed by 31 P NMR spectra showing a broad resonance at 3.6 ppm, characteristic of an amorphous hydrated orthophosphate, which is shifted toward lower frequencies ( 6.0 ppm) when a larger amount of gallium nitrate is added to the liquid phase (Fig. S2 and Table S2 in Supplementary data) [53]. Moreover, the corresponding 71Ga MAS NMR spectra exhibit an intense resonance and a peak of weaker intensity associated to GaO6 and GaO4 units, respectively, both of them with 71Ga isotropic chemical shifts and lineshapes characteristic of Ga environments in a disordered orthophosphate phase (Fig. S2 and Table S2 in Supplementary data) [53]. Alternatively, replacing the phosphate buffer by a Ga(H2O)3+ 6 or a Ga(OH)4 water solution resulted in the inhibition of the setting reaction due to an inappropriate pH value of the cement paste (too acidic or too basic, respectively). The same phenomenon was observed, when solid gallium nitrate crystals were dispersed in the calcium phosphate mixture before adding the phosphate buffer. Therefore, since the two first options were found to be inappropriate, attempts to incorporate gallium in one of the individual calcium phosphate components of the QS-CPC were performed. This apatitic-type CPC is mainly composed of a-TCP [a-tricalcium phosphate (Ca3(PO4)2) – 78 wt.%], mixed with DCPA [anhydrous dicalcium phosphate CaHPO4 – 10 wt.%], CDA [calcium-deficient apatite Ca10 x []x(HPO4)y(PO4)6 y(OH)2 z[]z – 10 wt.%] and HPMC [hydroxypropyl methyl cellulose – 2 wt.%]. While the driving force of the cement setting is based on the transformation of a-TCP into CDA, doping this structure with gallium ions is very likely to affect its solubility and accordingly the kinetics of the hardening process. For this reason, this route was not considered and experiments were thus focused on the use of DCPA, CDA in addition to b-TCP. Indeed our previous studies have shown that up to 13% of a-TCP can be replaced by b-TCP without significantly affecting the main properties of the CPC, and Lopez-Heredia et al. have reported similar results for another a-TCP-based cement formulation [61]. 3.1.1. Ga-doping of b-TCP We have very recently reported that gallium can be combined to b-TCP ceramics [53]. Gallium insertion in the b-TCP structure

(i.e. Ca10.5 1.5xGax(PO4)7 with x < 0.75) was found to occur by substitution of one of the five calcium sites in the b-TCP network (M5 site) along with the concomitant decrease of the occupation of another Ca site (M4 site) in inverse proportion to the gallium content. In summary, since the amount and location of gallium loaded onto b-TCP can be fully controlled, this route seems suitable for the introduction of gallium in QS-CPC. Preparation of gallium-doped b-TCP can be performed either by sintering of a CaHPO4/CaCO3/Ga2O3 mixture with a suitable stoichiometry, or calcination of gallium-doped calcium-deficient apatites (vide infra).

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3.1.2. Ga-doping of calcium-deficient apatites (CDA) Gallium-doped CDAs were synthesized by co-precipitation of a mixture of gallium nitrate hydrate and calcium nitrate tetrahydrate with diammonium hydrogen phosphate [(Ca + Ga)/P molar ratio = 1.515)] in the 9–9.5 pH range. For initial Ca/Ga molar ratios less than 0.08, quantitative incorporation of gallium in the isolated solid phase is observed (up to 4.5 Ga wt%), since no residual gallium species are present in the supernatant. The electron and X-ray diffraction powder patterns of the obtained nanoparticles are characteristic of a nanocrystalline calcium deficient apatite (Fig. 1). Such nanocrystalline calcium deficient apatites obtained by precipitation are known to possess a peculiar structure which consists of a nearly stoichiometric apatitic crystalline core and a disordered hydrated surface layer [62–65]. This hydrated surface layer, which involves labile ionic species with non-apatitic chemical environments, confers specific physical–chemical properties to these nano-sized CDA such as capabilities to reversibly exchange ions and adsorb organic molecules and proteins [63,65]. To investigate the nature of the gallium species into the obtained CDA nanoparticles, a detailed solid-state NMR structural characterization was performed. As shown in Fig. 2, the 31P MAS and CP-MAS spectra of the Ga-doped CDA nanoparticles exhibit a narrow peak at 2.9 ppm, characteristic of stoichiometric hydroxyapatite and assigned to the core of the nanocrystals, and an additional broad resonance centered at 0.6 ppm which corresponds to the disordered surface layer. This broad resonance is shifted upfield with respect to the one observed in undoped nanocrystalline CDA (at 3 ppm), while the position of the narrow peak remains unchanged, [64] indicating that gallium ions are only incorporated in the disordered surface layer of the nanoparticles. The assignment of the broad line at 0.5 ppm to the disordered

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Fig. 1. (a) TEM image of 3 wt.% gallium-doped CDA nanoparticles and the corresponding (b) electron and (c) X-ray diffraction powder patterns. In (b, c), the most intense reflections characteristic of the hexagonal hydroxyapatite structure are indicated (P63/m space group).


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Fig. 2. (a) 31P quantitative MAS NMR spectrum of a 3 wt.% gallium-doped CDA. (b, c) {1H}–31P CP-MAS spectra of the same sample recorded with contact times of (b) 0.5 ms and (c) 10 ms. The 31P MAS spectra of an undoped CDA sample recorded under the same experimental conditions are shown in (d)–(f). In (a)–(f), the best fits of the experimental spectra considering two contributions associated to the ordered core (blue line) and the disordered surface layer (green line) of the nanoparticles are shown as dashed red lines.

Fig. 3. (a) 2D 1H–31P HETCOR MAS spectra of a 3 wt.% gallium-doped CDA, recorded using a magnetization transfer time of 0.25 ms. (b)–(d) 31P MAS spectra extracted from the 2D map at the 1H frequencies in the vertical dimension of (b) 0, (c) 6.0 and (d) 12.0 ppm characteristic of OH-, H2O and HPO4 groups, respectively, with a fourfold magnification for spectra (c, d).

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surface layer of the Ga-doped CDA nanoparticles is further supported by two-dimensional (2D) 1H–31P heteronuclear correlation (HECTOR) MAS NMR spectra, in which the 31P MAS lineshape is correlated with the 1H MAS spectrum. Indeed, the 1H–31P HETCOR spectrum recorded with a short magnetization transfer time of 0.25 ms (Fig. 3) shows an intense correlation peak between the narrow 31P resonance at 2.9 ppm and the 1H resonance at 0 ppm, characteristic of the OH groups of the hydroxyapatite structure. In contrast, the broad 31P resonance at 0.6 ppm mainly exhibits correlations with the 1H resonances associated to non-apatitic environments, i.e. HPO24 groups and H2O molecules, as observed for undoped CDA nanoparticles for which the broad 31 P resonance at 3 ppm shows correlation with similar 1H non-apatitic resonances [64,66,67]. It should be noted that additional 2D 31P–31P proton-driven magnetization exchange MAS experiments indicates that the disordered and ordered regions belong to the same nanocrystal (Fig. S3 in Supplementary data), thereby confirming the results of previous 1H EXSY experiments [64]. These observations thus clearly indicate that the Ga-doped CDA nanoparticles consist of an ordered hydroxyapatite core with very few structural defects and a disordered outer shell mainly

containing HPO24 and PO34 non-apatitic environments, structural water molecules and incorporating the Ga ions. Finally, 71Ga MAS NMR spectra of the Ga-doped CDAs were recorded at high magnetic field (17.6 and 20.0 T) to probe the Ga environments in the disordered surface layer of the nanocrystals. The obtained spectra (Fig. 4) showed three gallium resonances with asymmetric lineshapes characteristic of a distribution of the quadrupolar interaction: a major one (ca. 67%) corresponding to GaO6 environment and two other signals of weaker intensities assigned to GaO5 and GaO4 units (Table 2), indicating that most of the Ga ions are incorporated in the disordered surface layer as GaO6 octahedral units. In conclusion, since the amount and location of gallium loaded onto CDA can be fully controlled, this route seems suitable for the introduction of gallium in QS-CPC.

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3.1.3. Ga-doping of DCPA Attempts to prepare gallium-doped DCPA were performed by suspending the calcium phosphate in a gallium aqueous solution (5 10 3 mol L 1 – S/L = 10 g L 1) for 2 days. At the end of the reaction, the residual gallium concentration in the liquid phase was under the detection limit (atomic absorption) and the

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Fig. 4. Experimental (black lines) 71Ga central transition MAS NMR spectra of a 3 wt.% gallium-doped CDA, recorded at magnetic fields of 20.0 (top) and 17.6 T (bottom) with a spinning frequency of 65 kHz. Fits of the experimental spectra considering a Gaussian distribution of 71Ga isotropic chemical shift and a Czjzek distribution [61] of the 71Ga quadrupolar coupling parameters (Gaussian isotropic model) [62] are shown as dashed red lines. Individual contribution associated to GaO6, GaO5 and GaO4 units are shown as blue lines.

Table 2 Ga average isotropic chemical shifts (dISO ± 2), average quadrupolar coupling constant (CQ ± 0.1) and relative intensities (±4%) of the GaO6, GaO5 and GaO4 resonances determined by fitting simultaneously the 71Ga spectra recorded at 17.6 and 20.0 T. 71

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Resonance

dISO (ppm)

CQ (MHz)

I (%)

GaO6 GaO5 GaO4

-7 48 135

8.9 9.0 8.2

67 14 19

obtained solid was studied by solid-state NMR (Fig. 5). In addition to the two intense resonances (at 1.5 and 0.2 ppm) typical of pure DCPA, the 31P MAS NMR spectrum of the isolated solid phase exhibited a broad peak at about 6 ppm characteristic of a disordered orthophosphate phase (Table S3 in Supplementary data). Similarly, a broad line at about 6 ppm assigned to hydrogen-bonded water molecules and two intense peaks (at 16 and 13.3 ppm) associated to pure DCPA were observed in the 1H MAS NMR spectrum of the solid. The corresponding 71Ga MAS NMR spectrum showed an intense resonance assigned to GaO6 environments and a peak of weaker intensity associated to GaO4 units, both of them with 71Ga isotropic chemical shifts and lineshapes typical of a disordered orthophosphate phase. Excepted very small variation of the chemical shifts, the overall 31P, 1H and 71 Ga NMR signatures of this disordered phase were thus strongly similar to those of the amorphous solid previously obtained when adding gallium nitrate in the aqueous disodium phosphate buffer. Therefore, this indicates that precipitation of an amorphous hydrated gallium orthophosphate, likely containing some amount of calcium, takes place onto the surface of the DCPA particles during the reaction. This is the result of the pH of the reaction medium (initial value: 4.11 – final value: 4.69) for which the stability of soluble gallium species is expected to be low. Therefore, since a mixture of phases is obtained in an uncontrolled manner, this option was considered as not suitable for the introduction of gallium in QS-CPC.

Fig. 5. (a) 1H and (b) 31P MAS NMR spectra of the solid obtained by suspending DCPA in a gallium aqueous solution for 2 days. The 1H and 31P resonances characteristic of pure DCPA are indicated by crosses. A magnification of the 31P spectrum along the vertical axis is also shown in (b), highlighting the 31P broad peak characteristic of a disordered orthophosphate phase. (c) 71Ga central transition MAS NMR spectrum of the solid phase revealing the presence of GaO6 and GaO4 units in a disordered orthophosphate phase.

3.2. Effect of the gallium doping on the cement main properties

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The different strategies investigated for the controlled incorporation of gallium in QS-CPC are summarized in Scheme 1, including a short description of the reason why some options failed. Taking account of these experimental results, the selected options for the introduction of gallium in the cement formulation were thus the following: replacement of 13% of a-TCP by gallium-doped b-TCP (Ca10.5 1.5xGax(PO4)7 with x = 0.5), full or half replacement of CDA by gallium doped CDAs. For the latter option, the gallium weight content in the cement was also varied (see Fig. 6). The initial setting time (Gillmore needle standard – ASTM C 266-89), and

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Scheme 1. Summary of the strategies investigated for the incorporation of gallium in the QS-CPC formulation.

Fig. 6. (a) Initial setting time and (b) compressive strengths after 72 h for the cement samples prepared in this study (# and ⁄p < 0.05).

Fig. 7. SEM micrographs of CPC fracture surface (a) QS-CPC reference (b) 0.32 wt.%(Ga-b-TCP)-CPC and (c) 0.3 wt.%(Ga-CDA)-CPC.

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mechanical strength (ASTM F 451method) of the resulting cements were investigated and compared with the QS-CPC, used as a reference. Replacement of 13% of a-TCP by b-TCP leads to a ca. twofold increase of the initial setting time that is still acceptable for a practical use. However, this makes no difference whether b-TCP is doped or not with gallium, which suggests a limited Ga3+ release since gallium is part of the b-TCP crystal network (entries 1–3). On the other hand, replacement of CDA by gallium-doped CDA also lengthened the setting time, the higher the gallium loading

the higher the variation (entries 1, 4–7). This observation suggests the release of some gallium species which might alter the setting reaction. Interestingly, slightly higher compressive strengths were obtained upon addition of gallium combined to CDA. Finally, for a given total gallium loading, replacement of only half the amount of CDA with gallium-doped CDA resulted in no significant difference (entries 6–7) when compared to the case of a full replacement. Comparative SEM observation of hardened cement blocks corresponding to entries 1, 3 and 4 (Fig. 7) showed no significant difference between the QS-CPC reference and the sample containing


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Ga-doped b-TCP, in terms of crystal size of the formed CDA upon setting. On the contrary, the presence of Ga-doped CDA in the cement composition led to the formation of significantly much larger CDA crystals during the setting process. This might result from the slower rate of the setting reaction observed for these samples together with a texture of higher fluidity, compared to the case of the undoped cement. To investigate a potential effect of gallium on the a-TCP to CDA transformation in cured cements (after 72 h), analysis of the composition of the cement samples was performed by means of 31P solid-state NMR. Thus two samples corresponding to the highest gallium loading (0.3 wt.%(Ga-CDA)-CPC, 0.32 wt.%(Ga-b-TCP)-CPC) were compared to their respective undoped analogs (QS-CPC, (b-TCP)-CPC). As a first example, the 31P quantitative MAS spectrum and CP-MAS spectra (recorded with a contact time of 5 ms) of the solid component alone (i.e. before preparation of the cement paste) for the two undoped references are depicted in Fig. 8. While the quantitative 31P MAS spectra were dominated by the resonances of a-TCP (the main constituent of the mixtures), the 31P CP MAS spectrum gives clear evidence of the presence of DCPA and CDA through the resonances at 0.2 and 1.5 ppm, and 2.9 ppm, respectively. In (b-TCP)-CPC, supplementary resonances corresponding to b-TCP are also present. Typical simulations of the 31P quantitative MAS spectra using the individual sub-spectra of the constituents of the mixture, from which the relative P content of each phase could be determined, are also shown in Fig. 8. Then, the four cement pastes were prepared and the samples were quenched after 72 h in acetone to stop hydration of the TCP particles. The quantitative 31P MAS spectra of the samples were recorded (Fig. 9), and the a-TCP to CDA transformation was measured from 31P NMR spectra: QS-CPC (74%), (b-TCP)-CPC (75%),

0.3 wt.%(Ga-CDA)-CPC (72%), 0.32 wt.%(Ga-b-TCP)-CPC (75%). This suggests that, while the replacement of a fraction of the a-TCP by b-TCP or Ga-doped b-TCP as well as the replacement of CDA by Ga-doped CDA in the powder mixture lengthened the setting time of the cement paste, this does not affect significantly the rate of a-TCP to CDA transformation determined after 72 h from the analysis of quantitative 31P NMR data.

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3.3. Measurement and in vitro effect of gallium release from cement pellets

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The ability of the gallium-doped cements to release gallium ions was monitored versus time (2–7 days) under in vitro conditions, in the absence or presence of murine RAW 264.7 cells seeded on the surface of cement pellets and stimulated with 20 nM RANKL during 2, 4 and 7 days as mentioned in the M&M (Fig. 10). In all cases, soluble gallium species were detected by ICP-MS analysis in the culture medium at relatively low concentration, corresponding to a release of less than 0.001% of the total amount of gallium loaded in the cement, probably due to a process only occurring at the surface because of the limited porosity of the materials. In addition, the precipitation of part of the gallium species once released from the CPC cannot be excluded. Interestingly, the gallium release was found to increase in direct relation with the initial gallium-loading in the cement, while for the higher loading, the amount of gallium in solution was roughly similar when gallium was combined to CDA compared with b-TCP. As expected, an increase of the gallium release was observed over time in the presence of RAW cells in sharp contrast to the situation where no cell was added, for which no significant variation occurred. This gradual release is very likely due to the osteoclastic resorption activity that increased with the maturation of RAW 264.7 cells.

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Fig. 8. 31P quantitative MAS and {1H}–31P CP-MAS (contact time of 5 ms) spectra of the (a) QS-CPC and (b) (b-TCP)-CPC powder mixtures. In (a, b), the CP-MAS spectrum is shown below the quantitative MAS spectrum and the 31P resonance of DCPA and CDA are indicated by crosses and asterisks, respectively. The simulations (dashed red lines) of the 31P quantitative MAS spectra (black lines) using the individual sub-spectra of the constituents of the mixture are shown in (c) and (d) for the QS-CPC and (b-TCP)-CPC references, respectively. In (c, d), the green, purple, blue and brown lines correspond to a-TCP, CDA, DCPA and b-TCP, respectively.


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Fig. 9. 31P quantitative MAS spectra (black lines) of the (a) QS-CPC, (b) (b-TCP)-CPC, (c) 0.3 wt.%(Ga-CDA)-CPC and (d) 0.32 wt.%(Ga-b-TCP)-CPC cured cements (after 72 h). The dashed red lines correspond to simulations of the 31P spectra using the individual sub-spectra of the constituents of the initial mixture. The green, purple, blue, brown, orange and pink lines correspond to a-TCP, CDA, DCPA, b-TCP, Ga-doped CDA and Ga-doped b-TCP, respectively.

Fig. 10. In vitro gallium release measurements from cement pellets (a) incubated without cells and (b) cultured with RAW 264.7 cells (# and *p < 0.05).

Fig. 11. Movat staining of cement explants: (a) QS-CPC, (b) 0.32 wt.%(Ga-b-TCP)-CPC and (c) 0.3 wt.%(Ga-CDA)-CPC. Green: mineralized bone, red lines: osteoid tissue with osteoblasts, gray: unresorbed biocements.


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Hence, these results strongly suggest that the release of gallium from CPC might occur from both chemical desorption/dissolution and resorption processes.

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3.4. Histological studies

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Two gallium-doped cements (0.32 wt.%(Ga-b-TCP)-CPC and 0.3 wt.%(Ga-CDA)-CPC) and the undoped reference (QS-CPC) were implanted in bone critical defects on a rabbit animal model. For all implanted materials no adverse foreign body reaction was observed. Four weeks after implantation, new bone formation occurred in close contact with all cement surfaces and without an intervening layer of fibrous tissue. Newly formed bone was well mineralized, as indicated by stained sections. Well-mineralized newly formed bone that reached the implanted material, spread along its surface, and in some rare cases, penetrated into the periphery around fragmented or early degraded pieces of cement (Fig. 11). Stained sections showed that body fluids were able to diffuse between the cement crystals, whereas newly formed bone did not penetrate deeply inside the cement. The amount of resorbed biomaterial was quite low, and as a consequence the gallium release, so that no difference was observed between QS-CPC and the gallium containing formulations.

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4. Conclusion

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This paper reports that gallium(III) ions, which are known for their antiresorptive and antimicrobial properties, can be combined with an apatitic calcium phosphate cement, up to 0.3 wt.% with respect to the solid phase. A limited number of options were found to be appropriate for introducing gallium into the cement formulation, while keeping suitable setting times. This consisted either in the replacement of 13% of a-TCP by gallium-doped b-TCP, or partial or full replacement of CDA by gallium-doped CDA, with in this latter case slightly improved mechanical properties at the end of the hardening process. The cement blocks obtained following these two routes were found to release low amounts of gallium when soaked in a culture medium and this effect was significantly more pronounced in the presence of murine RAW 264.7 cells seeded on the surface. Importantly, these experiments clearly indicate that in vivo implantation of the reported gallium-doped cements in bone tissues is very likely to result in the local delivery of gallium species, although the dose of gallium expected to be released cannot be assessed at this stage. Very interestingly, in vivo implantation of the two types of gallium-doped cements in bone critical defects on a rabbit animal model, showed an excellent interface between the implant surface and newly formed bone and no adverse effect occurred. However, due to the low rate of CPCs resorption, longer-term implantations need to be performed in the next future, preferably in specific animal models presenting either osteopenia [68] or bone tumors [41], to investigate whether the combined release of both calcium and gallium ions by CPCs might improve bone regeneration.

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5. Disclosure

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The authors of this publication have research support from Graftys SA. The terms of this arrangement have been reviewed and approved by both CNRS and the University of Nantes in accordance with their policy on objectivity in research.

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Acknowledgements

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E. Veron and M. Allix are acknowledged for EDS and TEM analysis. The ICMN laboratory (Orléans, France) is thanked for

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providing TEM access. This work was partially supported by ANR (BiotecS 2008 program – grant ANR-08-BIOT-008-02), and the Graftys company. Financial support from the TGIR RMN THC FR3050 for conducting the research is gratefully acknowledged.

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Appendix A. Figures with essential color discrimination

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Certain figures in this article, particularly Figs. 1–6, 8–11, are difficult to interpret in black and white. The full color images can be found in the on-line version, at http://dx.doi.org/10.1016/j.actbio.2015.05.027.

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Appendix B. Supplementary data

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Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.actbio.2015.05. 027.

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References

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Nanomaterials: the next step in injectable bone cements.

Thermoreversible and Injectable ABC Polypeptoid Hydrogels: Controlling the Hydrogel Properties through Molecular Design.

The properties of four dental cements.

Physical properties of different orthodontic cements.

The mechanical properties of bone cements.

Injectable fillers: review of material and properties.

Physicochemical and biological properties of a novel injectable polyurethane system for root canal filling.

or fast setting tricalcium silicate cements.

The design and evaluation of field trials of injectable contraceptives.

In-vitro Comparison of the Antimicrobial Properties of Glass Ionomer Cements with Zinc Phosphate Cements.

Enhanced mechanical properties of a novel, injectable, fiber-reinforced brushite cement.

Silver nanoparticles in resin luting cements: Antibacterial and physiochemical properties.

Injectable bioadhesive hydrogels with innate antibacterial properties.

Design and characterization of an injectable tendon hydrogel: a novel scaffold for guided tissue regeneration in the musculoskeletal system.

Polyethylene glycol improves elution properties of polymethyl methacrylate bone cements.

Design of Injectable Materials to Improve Stem Cell Transplantation.

Thio-urethanes improve properties of dual-cured composite cements.

Silver-Doped Calcium Phosphate Bone Cements with Antibacterial Properties.

Pre-apatitic mineral deposition in Bacterionema matruchotii.

Design and evaluation of a nanoenhanced anti-infective calcium phosphate bone cements.

A novel liposomal drug delivery system for PMMA bone cements.

Design, synthesis, and anticancer properties of novel benzophenone-conjugated coumarin analogs.

Design, synthesis and biological activity of a novel Rutin analogue with improved lipid soluble properties.

Effect of addition of nano-hydroxyapatite on physico-chemical and antibiofilm properties of calcium silicate cements.